• PB2 : many avian influenza viruses can infect mouse cells but cannot replicate, frequently because amino acid residue 627 of the PB2 protein of the viral polymerase differs between avian and mammalian influenza viruses. In the avian virus, this residue is usually glutamic acid, whereas in mammalian influenza virus it is lysine^ref, suggesting that PB2 residue 627 might be important in determining species range. In experimentally infected mice, a glutamic acid–to–lysine mutation at this position in the PB2 protein (E627K) of H₅N₁ virus results in increased virulence and the ability to invade extrarespiratory organs^ref. It is notable that both H₅N₁ virus from human patients (Hong Kong in 1997, Vietnam in 2004 and Thailand and Indonesia in 2005, Turkey in 2006 cluster, Indonesia in 2006 North Sumatra cluster ?)^ref and H₇N₇ virus from a fatal human case in the Netherlands^ref possess a lysine at this site, suggesting rapid evolution within humans of a virus originating in poultry. Strikingly, though, and worryingly, lysine is the PB2 residue in H₅N₁ viruses isolated from wild waterfowl in mid-2005 from Qinghai Lake, in China^{ref1, ref2}. Similarly, wild and domestic cats and a domestic dog were also fatally infected with H5N1 with PB2 E627K. E627K, which allows the H₅N₁ to grow more efficiently at lower temperatures, 33°C (since the body temperature of birds is 41°C and of humans is 37°C)^ref : human-to-human transmission is more likely through droplets coughed from the nose and throat than from infections further down the respiratory tract
• PB1
• PB1-F2 (87 amino acids, 11-kDa) is encoded by a  frameshifted region of the influenza A virus PB1 gene, probably through a ribosomal scanning mechanism : unusual features include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2^ref. It creates variably sized pores in planar lipid membranes, so the basis for PB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis^ref
• PA
• hemagglutinin (HA) : 15 variants from H₁ to H₁₅ (a virus possesses one HA and one NA subtype, apparently in any combination). They bind to sialic acid (SA). Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids^ref. Avian viruses hemagglutinin preferentially bind to sialic acid (SA)-a-2,3-Gal–terminated saccharides on intestinal epithelial cells, whereas human viruses are specific for SA-a-2,6-Gal–terminated saccharides which are predominant on non-ciliated epithelial cells of human trachea^ref. Avian viruses can anyway infect humans because SA-a-2,3-Gal–terminated saccharides are found on a small number of ciliated tracheobronchial cells^{ref1, ref2}. To define the receptor preferences of a number of human and avian H₁ and H₃ viruses, including the 1918 H₁N₁ pandemic strains, their hemagglutinins were analyzed using a glycan array which contains 200 carbohydrates and glycoproteins, not only revealing clear differentiation of receptor preferences for a2-3 and/or a2-6 sialic acid linkage, but also detecting fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the 2 1918 HA variants, the South Carolina (SC) HA (with Asp¹⁹⁰, Asp²²⁵) bound exclusively a2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly²²⁵), had mixed a2-6/a2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp¹⁹⁰Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only 2 positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H₅N₁ avian influenza. A number of HA receptor binding studies have been reported previously, but these have primarily employed cell-based assays to probe sialic acid specificities among different influenza viruses. Such assays involve enzymatic removal of endogenous sialic acid from red blood cells using sialidases, followed by resialylation with linkage-specific sialyltransferases. While such assays approximate the natural binding of a virus to the host cell receptors, the results are subject to variation based on the quality of the cell preparations and degree of enzymatic modification, both of which are difficult to control.  Use of whole viruses also introduces the uncertain effects of the viral neuraminidase on the binding assays. In other studies, competitive binding of viruses to synthetic natural analogs was analyzed, but again whole viruses were used here. Recombinant HA protein  was used as a probe to circumvent these problems. The array technology, and the ease with which the chip can be customized for display of appropriate glycans, enhances the ability to probe pathogen-specific glycan interactions. In this application, the array could detect fine differences in HA specificity, allowing systematic investigation of the residues responsible for receptor specificity. The array results were surprising in a number of respects. A distinct binding profile was observed for each HA that could not be obtained by the usual hemadsorption or hemagglutination assays. With this powerful glycan microarray technology, it is now possible to map the fine specificity of emerging influenza viruses and to revisit and complete the analyses on earlier human, pig and bird isolates. Changes in receptor specificity can now be quickly monitored and correlated with mutations in the receptor binding site to aid in prediction of new pandemics or epidemics. In-depth cellular studies will also now be needed to ascertain the range, levels and distribution of different carbohydrates on lung epithelial tissue and human airways, especially since HA specificities for sialylated sugars can be assessed not only for a2-3 or a2-6 linkages, but also can now include preferences for GalNAc versus GlcNAc at position 3 of sialylated sugars as well as for additional substituents, such as sulfate, fucose and extra sialic acid moieties^ref. Lung type-I cell membrane-associated glycoprotein (T1A-2) could be a receptor. HA selectively inhibits LPS-induced transcription of IL-12 p35^ref. The HA molecule has a large ectodomain of 500 aa. A posttranslational cleavage by host-derived enzymes generates 2 polypeptides that remain linked by a disulfide bond. The larger N-terminal fragment (HA₁, 320–330 aa) forms a membrane-distal globular domain that contains the receptor-binding site and most determinants recognized by virus-neutralizing antibodies. The smaller C-terminal portion (HA₂, 180 aa, excluding transmembrane and cytoplasmic domain) forms a stemlike structure that anchors the globular domain to the cellular or viral membrane. 16 HA subtypes have been identified among influenza A viruses^ref; 3 of these (H₁, H₂, H₃) have been associated with classic influenza isolates, and 3 (H₅, H₇, H₉) have been associated with recent sporadic human isolates^ref. Influenza B viruses possess only 1 HA subtype. Although the degree of sequence diversity between subtypes is great, particularly in the HA₁ polypeptides (34%–59% homology between subtypes), more conserved regions are found in HA₂ (51%–80% homology between subtypes). The most notable region of conservation is the sequence around the cleavage site, particularly the HA₂ N-terminal 11 aa, termed fusion peptide, which is conserved among all influenza A subtypes and differs only by 2 conservative aa replacements in influenza B virus. Part of this region is exposed as a surface loop in the HA precursor molecule (HA₀)^ref. However, when HA₀ is cleaved into HA₁/HA₂, the newly generated terminals separate, and the hydrophobic fusion peptide becomes tucked into a cavity of the stem^ref. As most HA subtypes are cleaved by extracellular enzymes, this surface loop may be accessible to antibody, at least temporarily, on HA₀ expressed in the plasma membrane of infected host cells^ref. In poultry, whether infection is localized or systemic depends on the amino acid sequence at the cleavage site of the precursor hemagglutinin. This cleavage is required for the hemagglutinin to become fully functional. Low pathogenicity influenza viruses require extracellular proteases limited to the respiratory and gastrointestinal tracts to cleave the precursor hemagglutinin, whereas highly pathogenic avian influenza viruses have changes in the cleavage site that allow the precursor hemagglutinin to be processed by ubiquitous intracellular proteases, resulting in fatal systemic infection^ref. In mammals, the viral factors determining systemic infection are less clear, although cleavability of the precursor hemagglutinin plays an important role^ref. The index case in the Turkish cluster had H₅ S227N and PB2 E627K mutations^ref.
• nucleoprotein (NP)
• neuraminidase (NA) : 9 variants from N₁ to N₉. Like the respective hemagglutinins, neuraminidases from avian influenza viruses have a preference for SA-a-2,3-Gal–terminated saccharides, whereas those from many human influenza viruses prefer SA-a-2,6-Gal–terminated saccharides^ref. NA is thought to promote virus entry, and thereby enhances infection efficiency^ref. 3 currently accepted roles for this NA in promoting influenza virulence are: 1. NA cleaves newly formed virus particles from the host cell membrane. Without NA, newly formed virus would remain attached to the cell within which it was produced. 2. NA prevents newly released virus particles from aggregating to each other, preventing clumping that would reduce dissemination. 3. NA promotes viral penetration of sialic acid-rich mucin that bathes and protects respiratory epithelium through which the virus must spread and replicate. 2 further, albeit hypothetical, functions of NA together could cause disruption the mucosa-IgA axis, creating localized partial immunosuppressed state, enhancing both influenza infection itself and secondary bacterial pneumonia: 4. IgA provides primary immunoglobulin defense of mucosal surfaces. The hinge region of IgA is normally sialylated. IgA denuded of sialic acid is recognized, bound, and cleared by hepatic asialoglycoprotein receptor (ASGPR). Thus, IgA exposed to free NA would be so denuded and have increased hepatic clearance. 5. NA removes sialic acid moieties from mucosa-residing g/d T cells or IgA producing B cells. Previous work indicates desialylation of these lymphocytes' outer cell membrane results in altered homing, to bone marrow, away from mucosa^ref.
• matrix protein 1 (M1)
• matrix protein 2 (M2) / ion channel protein : M2 forms tetramers that exhibit pH-inducible proton transport activity. It regulates the pH of the viral core after virus uptake into the host cell's endosomal compartment during initiation of infection and subsequently of vesicles that transport the viral transmembrane proteins to the cell surface during the late stage of infection. M2 tetramers are expressed at high density in the plasma membrane of infected cells and are well accessible to M2e-specific antibodies in this location, but only a few copies become incorporated into the envelope of mature infectious virus particles^{ref1, ref2}. M2 has a small, nonglycosylated ectodomain (M2e) of 23 amino acids (aa), not counting the posttranslationally removed N-terminal Met. This region has shown only limited variation among human influenza A viruses. This remarkable degree of structural conservation of M2e is attributable mainly to its genetic relation with M1, the most conserved protein of influenza A viruses with which it shares coding sequences. Thus, aa residues 1–9 of M2e and M1 are encoded by the same nucleotides in the same reading frame and aa 10–23 of M2e and 239–252 of M1 in a different reading frame.
• segment 8 encodes :
• nonstructural protein 1 (NS1) : the NS1 protein is synthesized in infected cells but not incorporated into virus particles. Rather, this small, multifunctional protein participates in both protein-RNA and protein-protein interactions during infection. Its amino-terminal RNA-binding domain binds to and sequesters double-stranded RNA (dsRNA) with low affinity^ref, evading PKR-mediated responses and enabling the virus to disarm the host cell type IFN defense system in influenza A virus-infected lung epithelial cells^ref, but the significance of this activity during viral infection is controversial^{ref1, ref2}. The NS1 protein also binds and inhibits the function of 2 cellular proteins (30 kDa subunit of cleavage and polyadenylation specific factor 4 (CPSF4) and influenza virus NS1A binding protein (IVNS1ABP)) that are required for the modification of the 3' ends of cellular pre-mRNAs, to inhibit binding to the RNA substrate, 3' end cleavage, polyadenylation of host pre-mRNA and the nuclear export of cellular, and not viral, mRNAs^ref. Consequently, the production of a key component of the host antiviral response, IFN-b mRNA, is substantially reduced, although not eliminated^ref. The NS1 sequences that participate in binding to either these 2 cellular proteins or dsRNA are not part of the putative new virulence determinant in H₅N₁ NS1. The multimerization domain of the NS1 protein (but not the effector domain) is sufficient to induce apoptosis in MDCK and HeLa cells^ref. Anyway, in IFN-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses^ref. Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits pro-inflammatory cytokine release^ref. NS1 also may help influenza viruses to replicate in IFN-treated cultured cells, as has been reported for H₅N₁ virus isolates from 1997^ref
• nonstructural protein 2 (NS2)
NS1 and NS2 share 10 amino terminal residues, including the terminal methionine, and the NS2 protein is derived by mRNA splicing from segment 8 RNA. NS1 inhibits transport of mRNA from the infected cell nucleus and acts as an inhibitor of interferon, whereas NS2 functions as a nuclear export protein. The first large-scale sequencing of AIV including 2,196 AIV genes and 169 complete genomes, including 7000 avian influenza viruses, was reported in Jan 2006. The researchers used a collection of samples of 11 000 influenza viruses, including 7000 avian influenza viruses, assembled by St. Jude's Dr. Robert Webster over a period of 30 years. They sequenced a diverse sampling of 336 avian influenza viruses from this collection including isolates from ducks, gulls, shorebirds and poultry collected in North American, Eurasian, and Australasian countries, primarily during the years 1976 to 2004. The avian versions of NS1 and NS2 seem to allow the virus to do much more damage to a cell than the human versions. People infected with the H₅N₁ bird flu virus in Viet Nam and Thailand had the "avian" version of the influenza virus, as did the victims of the 1918 influenza pandemic, which killed tens of millions of people globally. But the human influenza viruses that cause the normal seasonal human misery, and those that caused the less deadly 1957 and 1968 human influenza pandemics, do not carry the avian genes^ref

• Influenzavirus A
• H₁ infects humans and Sus scrofa and has healthy reservoir in water Avesspp.
• H₁N₁ subtype
• Influenza A virus (A/Brevig_Mission/1/1918(H1N1)) : the most complete set of flu sequences came from a lung biopsy taken in 1997 from the frozen remains of a flu victim who died in Teller Mission (now called Brevig Mission), Alaska, in November of 1918. Other flu DNA segments were obtained from formalin-fixed paraffin-imbedded tissues archived by the Armed Forces Institute of Pathology. These were taken from two soldiers, one who died of the flu at Fort Jackson, South Carolina, and another at Camp Upton, New York, both in September of 1918. Finding these 1918 virus segments at the NCBI is most easily accomplished using the taxonomy database.
• Influenza A virus (A/New_York/1/18(H1N1))
• Influenza A virus (A/South Carolina/1/18 (H1N1))
=> Great Pandemic of deadly Spanish flu (a.k.a. the "Spanish lady", "The Naples Soldier" in Spain, and a variety of other names around the world) : the site of origin has never been clearly established.
• a new review of contemporary medical and lay literature has found previously-overlooked epidemiological evidence that suggests that the pandemic began in a sparsely populated region of southwestern Kansas^ref. In March 1918 the first wave of illness with fever and severe respiratory symptoms in Fort Riley, Kansas, and other military training camps throughout the USA, did not raise alarm bells : attention was focused on World War I in its final stages, and it took some months, and simultaneous severe outbreaks around the globe, before the world began to realise that this influenza epidemic was different
• other have found evidence of early outbreaks of respiratory disease in France and the UK in the years 1915 to 1917 : certain of these earlier focal outbreaks--called epidemic bronchitis rather than influenza--occurred during the winter months when influenza was known to be in circulation, and presented with a particular heliotrope cyanosis that was so prominent in the clinical diagnosis in the world pandemic outbreak of 1918-1919 (the Great Pandemic). The outbreaks in army camps at Etaples in France and Aldershot in the UK in 1916-1917 caused very high mortality in 25-35 year olds. Increased deaths from bronchopneumonia and influenza were also recorded in England
On the balance of probability the Great Pandemic was not initiated in Spain in 1918 but in another European country on the Western Front in the winter of 1916 or 1917 and World War I was a contributor^ref. Cases had cropped up over the spring and summer in other countries, too, from Norway to India, China to Costa Rica, but in Spain, in late spring of 1918 suddenly 8 million people were down with the bug. And as the summer of 1918 turned to fall, the epidemic lost its mildness: people started to die. It infected an estimated 20-50% of the worldwide population on every continent (from Norway to India, China to Costa Rica) except Antarctica. In Geneva, Switzerland, the Spanish flu epidemic affected > 50% of the population. The mortality was higher among those aged between 20-49 years and among men^ref. Though globally the victims of influenza lived primarily in urban areas, it was Iran's rural regions that suffered the most casualties. In addition, contrary to the prevailing notion that the 1918 influenza targeted the young and healthy, famine, opium consumption, malaria, and anemia were fundamentally responsible for the high mortality in Iran^ref. Historians note the 1918 Cedar Rapids Swine Show brought
together sick and healthy pigs. When the show ended, newly infected pigs were returned to farms throughout the Midwest, contributing to the spread of the deadly virus. Spanish flu was probably imported to Norway across the North Sea from Britain. From the middle of June 1918, Spanish flu swept across the country in 3 different waves: the summer epidemic of 1918, the autumn epidemic of 1918 and the winter epidemic of 1918-19. In Norway 13,000-15,000 died, most of them during the autumn of 1918 and mostly from pneumonia or pulmonary complications^ref. The influenza pandemic of 1918-20 is recognized as having generally taken place in 3 waves, starting in the northern spring and summer of 1918. This pattern of 3 waves, however, was not universal: in some locations influenza seems to have persisted into or returned in 1920. The recorded statistics of influenza morbidity and mortality are likely to be a significant understatement. Limitations of these data can include nonregistration, missing records, misdiagnosis, and nonmedical certification, and may also vary greatly between locations. Further research has seen the consistent upward revision of the estimated global mortality of the pandemic, which a 1920s calculation put in 21,642,274 million. A 1991 paper revised the mortality as being in the range 24.7-39.3 million. A work suggests that it was of the order of 50 million. However, it must be acknowledged that even this vast figure may be substantially lower than the real toll, perhaps as much as 100% understated : 100 million dead^ref (1-2% of those infected), more than in the preceding 4 years of carnage in the Great War of 1914-18.
• USA : 20 million Americans became sick and > 500,000 died, with a 195,000 deaths peak in October 1918. About 57,000 American soldiers (40% of US soldiers sent in Europe) died from influenza during WWI vs. about 53,500 died in battle : 50% were aged 20-40 years old. World population was then only 28% what it is today, and most deaths occurred in a 16 week period, from late September 1918 (when cases of the flu started looking abnormally high) to early January 1919.
• UK : 250,000 deaths
On July 1919 cases returned to about normal. It combined the highly unusual characteristics of high pathogenicity, the ability to transmit between humans, and high mortality among young people^ref. The excess death rate was 166 per 100 000 among people older than 65 but 546 per 100 000 among younger people—a hundred times higher than the rate in the 1968-9 pandemic. This is the only catastrophic flu pandemic with high case fatality among younger people known in all human history^ref. By fitting a deterministic susceptible-exposed-infectious-recovered (SEIR) model to pneumonia and influenza death epidemic curves from 45 US cities, the median value of reproductive number for 1918 influenza is < 3. The estimated proportion of the population with A/H₁N₁ immunity before September 1918 implies a median basic reproductive number < 4. These results strongly suggest that the reproductive number for 1918 pandemic influenza is not large relative to many other infectious diseases. In theory, a similar novel influenza subtype could be controlled. But because influenza is frequently transmitted before a specific diagnosis is possible and there is a dearth of global antiviral and vaccine stores, aggressive transmission reducing measures will probably be required^ref. The only footprints the virus left behind in the human population were the protective anti-viral antibodies in the blood of those who survived the 1918 outbreak. From these, virologists could deduce the class of influenza strains the virus belonged to, make some general inferences about its relatedness to other influenza strains in both humans and other animals and make a rough estimate of the time it might have entered the human population. But to make these deductions more precise molecular evidence was needed. No 'living' sample of the virus has ever been found, and the Washington team's achievement began with detective work in the Army medical archives by one of the authors, Jeffery K. Taubenberger. They retrieved 74 slides of lung tissue samples taken from victims of the 1918 epidemic, 13 of which seemed to be worth subjecting to molecular analysis for viral genetic material - which is RNA in the case of influenza virus. Their first published report, in 1997, that fragments of influenza virus RNA had been detected in one of the samples, then brought help from an entirely unexpected source. As a young man in the 1950s, pathologist Johan Hultin had realized that frozen virus might be preserved in the bodies of influenza victims who had been buried in the far north, in the deep-frozen permafrost of the Alaskan tundra. Hultin had travelled to the Alaskan settlement of Brevig Mission, where over 80% of the adult population had perished in the 1918 epidemic and been buried in a mass grave. He had collected some frozen tissue but had - probably fortunately given its virulence - failed to isolate live virus. Now retired, Hultin returned to Brevig Mission and with the cooperation of the present-day villagers collected some new samples. The complete base sequence for the viral HA^ref1, NA^ref, MA^ref, and NS^ref genes is nowadays available : H₁^ref and NP^ref from wild waterfowls captured in 1917 are more closely related to that of modern avian viruses than it is to that of the pandemic virus, suggesting (i) that there was little drift in avian sequences over the past 85 years and (ii) that the 1918 pandemic virus did not acquire its HA directly from a bird. Read one way, the sequence data suggest that the virus entered the human population sometime between 1905 and 1915 and then underwent changes that turned it into the virulent pandemic strain of 1918, but if one assumes a slightly higher mutation rate for the virus, and takes all the existing circumstantial evidence into account, the virus could well have entered humans directly from birds in or around 1918. The crystal structure of the uncleaved precursor of the major surface antigen of the extinct 1918 virus was determined at 3.0Å resolution after re-assembly of the HA gene from viral RNA-fragments preserved in 1918 formalin-fixed lung tissues. A narrow, avian-like receptor binding site, two novel histidine patches and a less exposed surface loop at the cleavage site that activates viral membrane fusion reveals structural features primarily found in avian viruses, that may have contributed to the extraordinarily high infectivity and mortality rates observed during 1918^{ref1, ref2}. The virus has been isolated in 2000 from the bodies of coal miners victims that had been buried in the eternal frost on Spitsbergen, Norway^ref. Traces of the virus was also found in the brains of the dead, which may eventually give a link between the influenza and "sleepy sickness" / encephalitis lethargica (EL) / epidemic encephalitis A / von Economo's disease. 1918 viral HA and NA genes assembled by reverse genetics and inserted into a modern influenza virus cause a flu-like lung disease characterized by severe bleeding in lab mice. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Only HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic. Because of the obvious hazards, the work was at first conducted in a BSL4 lab at the National Microbiological Laboratory in Winnipeg, Canada. After demonstration that the souped-up virus was susceptible to oseltamivir, the research was transferred to a BSL3 laboratory at the University of Wisconsin^ref. In addition to reverse genetic engineering of influenza, Dr. Kawaoka has NIAID funding to perform reverse genetic engineering of Ebola virus. Similarly, Dr. Katze of the University of Washington in Seattle has proposed to conduct aerosol challenge of primates with influenza containing up to 5 of the 1918 genes at BSL-3. (These grants may be viewed in the NIH CRISP database). As was noted in the Sunshine Project's recent report on IBCs, even the 1st reported creation of a 1918 chimera and subsequent experiments at the USDA BSL-3ag facility in Athens, GA were not reviewed by any IBC. Thus, it is unclear how USDA arrived at its determination of BSL-3ag containment (not BSL-4) for its experiments with 1918 constructs. To wit, the University of Washington is presently trying to convince USDA to release 1918 constructs to it, despite the fact that UW-Seattle does not have a BSL-3ag facility (that's not an endorsement of USDA's BSL-3ag designation -- it is an illustration of how biosafety levels are being dragged down). Unfortunately, several years after the 1918 construct experiments began, the formal federal advice for containment in pandemic influenza experiments remains BSL-2. An unmistakable shift in the age distribution of epidemic deaths occurred during the 1917/1918 influenza season in New York City : the timing, magnitude, and age distribution of this mortality shift provide strong evidence that an early wave of the pandemic virus was present in New York City during February-April 1918^ref. Analysis of 209 complete genomes of A(H₁N₁) virus, encompassing a total of 2,821,103 nucleotides, showed that it originated in birds (all 8 of the genome segments from the 1918 virus differ in important ways from other human flu sequences, suggesting that none of the genome came from a strain that had previously infected people) and shares genetic mutations with the bird flu virus now circulating in Asia^{ref1, ref2} : the suggestion that the virus had the potential to jump between humans without first combining reassorting with a human virus made it even more of a threat^ref. The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors1, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus have presented and it has been proposed that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of 10 amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H₅N₁ viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans^ref. The characterization of the recovered 1918 virus in tissue culture and mice reveals at least 2 unique qualities^ref :
• this virus is able to replicate and form plaques on tissue-culture monolayers in the absence of the protease trypsin. Normally, a protease such as trypsin is required to activate the hemagglutinin in order to initiate the infection of tissue culture, but the 1918 virus can activate its own hemagglutinin through the action of neuraminidase, either directly or indirectly (possibly by neuraminidase's binding of a host protease). The exact mechanism by which the neuraminidase takes on the protease activity has not been determined
• the 1918 virus is 100 times as lethal in mice as any other human influenza virus; the median lethal dose (LD₅₀, or 103.5 to 3.75 median egg infectious doses [EID₅₀]) is low, and the virus replicates rapidly so that high titers (>10⁷ EID₅₀ per milliliter) are found in the lungs of infected mice. High virus inocula result in the death of mice as early as 3 days after they have been infected. The vigorous release of cytokines in mice infected with the 1918 influenza virus is associated with rapid onset of pulmonary disease and death : compounds that block the action of specific cytokines can now be evaluated as therapeutics that might help to reduce the mortality associated with pandemic influenza.
Taubenberger's team has already identified 25 changes in the protein sequences of the 1918 strain that have been present in subsequent human flu viruses. These mutations are likely to be particularly important. One such change, in the polymerase gene PB2, was found in the virus isolated from the only human fatality in a 2003 outbreak of H₇N₇ bird flu in the Netherlands. When Terrence Tumpey at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and his co-workers used a re-created strain to infect mice they found it was extremely virulent, and after 4 days had generated 39,000 times more virus particles in the animals' lungs than a modern flu strain. Replacing the haemagglutinin gene, which helps the virus to enter cells, with a modern one made it unable to kill mice. Replacing all 3 of the polymerase genes, which allow the virus to replicate, significantly reduced its virulence^{ref1, ref2}. Using an enhanced biosafety level-3 lab for the work was inadequate. If the researchers were going to do the work at all, they should have used level 4, the strictest biosafety condition, he says. This requires experimenters to wear full body suits. In 2003, he points out, a SARS virus escaped accidentally from a level-3 lab in Singapore, and in 2004 two further escapes occurred from such labs in Beijing. Enhanced level 3, which requires upper body suits and respirators, is safe enough. Disgruntled employees aren't a concern either, because he is the only one who works with the virus. The few researchers with access to the lab undergo extensive background checks, and retina and fingerprint scans are used to prevent any unauthorized entry to the lab. Even if the virus did escape, it wouldn't have the same consequences as the 1918 pandemic. Most people now have some immunity to the 1918 virus because subsequent human flu viruses are in part derived from it. And, in mice, regular flu vaccines and drugs are at least partly effective against an infection with reconstructed viruses that contain some of the genes from 1918 flu. The other potential threat comes from the availability of the full genome sequence, which has been put on the GenBank database — a condition of the paper's publication. Anyone can order DNA to be made to a certain sequence. There are currently no governmental controls on what sequences can be used, although some DNA synthesis companies now screen their orders for pathogenic sequences. If someone wants to reconstruct the virus, the technology is available. Although Nature did not seek advice on whether to publish the work, he has done so for previous flu-virulence and pathogen genome papers. The benefits clearly outweigh the risks. The US National Science Advisory Board for Biosecurity (NSABB) reached a similar conclusion about both studies, after calling an emergency meeting last week to consider the risks. But, concerned about public fears, it asked the authors of both papers to add a passage to the manuscripts stating that the work is important for public health and was conducted safely. But government bureaucracies and committees may push to avoid perceived risks, at the potential expense of benefits to public security. There can be no absolute guarantee of safety. We are aware that all technological advances could be misuse. But what we are trying to understand is what happened in nature and how to prevent another pandemic. In this case, nature is the bioterrorist
Modern histopathological analysis of autopsy samples from human influenza cases from 1918 revealed significant damage to the lungs with acute, focal bronchitis and alveolitis associated with massive pulmonary oedema, haemorrhage and rapid destruction of the respiratory epithelium. The contribution of the host immune response leading to this severe pathology remains largely unknown. Mice infected with the reconstructed 1918 influenza virus displayed an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology. Mice infected with a virus containing all 8 genes from the pandemic virus showed marked activation of pro-inflammatory and cell-death pathways by 24 h after infection that remained unabated until death on day 5. This was in contrast with smaller host immune responses as measured at the genomic level, accompanied by less severe disease pathology and delays in death in mice infected with influenza viruses containing only subsets of 1918 genes. The results indicate a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza virus requires the use of the fully reconstructed virus. With recent concerns about the introduction of highly pathogenic avian influenza viruses into humans and their potential to cause a worldwide pandemic with disastrous health and economic consequences, a comprehensive understanding of the global host response to the 1918 virus is crucial. Moreover, understanding the contribution of host immune responses to virulent influenza virus infections is an important starting point for the identification of prognostic indicators and the development of novel antiviral therapies^ref
Therapy : recombinant viruses possessing the 1918 NA or both the 1918 HA and 1918 NA were inhibited effectively in both tissue culture and mice by the NA inhibitors, zanamivir and oseltamivir. A recombinant virus possessing the 1918 M segment was inhibited effectively both in tissue culture and in vivo by the M2 ion-channel inhibitors amantadine and rimantadine^ref.
Re-analysis of the influenza pandemic of 1918 has given reassurance about a rather low reproductive number (R₀), a prolonged herald wave of virus and that the skewed mortality towards the young adult could be a singularly unique event dependent upon previous infection history, perhaps not to be repeated in a future pandemic. Over 99% of those who contracted the virus survived, in spite of the absence of antivirals, vaccine and antibiotics for the secondary bacteria infections which probably accounted for one-third of the 50million deaths^ref.
Only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. 2 amino acid mutations that cause a switch in receptor binding preference from the human a-2,6 to the avian a-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual a-2,6 and a-2,3 specificity suggests that a predominant human a-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals^ref.
• 1976 pandemic
• the 1977 USSR isolate (A/USSR/90/77 H₁N₁) was the 1st H₁N₁ identified since 1958. Its HA is most closely related to A/Lepine/48. Relatives of this strain subsequently became quite widespread. By the single criterion of sequence stability/evolution, there are 2 other examples :
• A/Alma Ata/84. Its HA is uniquely closely related to A/swine/Iowa/15/30 and A/swine/29/37
• the pair of A/Mongolia/153/88 and A/Mongolia/111/91. The HA of the pair is most closely related to A/NWS/33, A/WSN/33, A/Puerto Rico/8/34, A/Cambridge/39.
Unlike A/USSR/90/77, there were no further isolates closely related to these strains. This fact suggests that, if they were "escapees," they were localized in time and area. However, in light of the seriousness of the discussion about lab-escaped pathogens, it is important to note that there are alternate, null hypotheses that should be considered 1st :
• one type of null hypotheses is that lab mix ups, or cross-contamination, resulted in the sequencing of an old strain instead of a newly isolated strain (or, conceivably, the submission of the incorrect sequence!)
• a second type of null hypothesis is that the strains were vaccine-derived revertants.
The Mongolia isolates were likely derived from a vaccine (A/Puerto Rico/8/34 x A/USSR/90/77) in use in the Mongolian population at the time^ref. Live vaccine-derived virulent strains are (unfortunately) not unfamiliar to virologists (e.g., poliovirus, porcine reproductive and respiratory syndrome virus^ref)
=> pandemic in 1977 (Russian influenza)
• swine fever : Influenza as a disease of pigs was 1st recognized during the Spanish influenza pandemic of 1918­1919. Veterinarian J S Koen was the 1st to describe the illness, observing frequent outbreaks of influenza in families followed immediately by illness in their swine herds, and vice versa [1]. Influenza virus was 1st isolated from pigs in 1930 by Shope and Lewis [2], with the virus isolated from humans several years later [3]. The 1st isolation of a swine influenza virus from a human occurred in 1974 [4], confirming speculation that swine-origin influenza viruses could infect humans. In the second place, influenza viruses regularly circulate in swine populations and include H1N1, H3N2, H1N2, H1N3 most commonly, with almost 25% of > 114 000 swine serum samples in the US being positive for one of the serotypes (see Choi et al. Arch Virol 2002 Jun;

147(6): 1209-20). As such, it is well controlled in swine populations, even though it can cause concern on particular farms that don't manage well for its occurrence or at particular times. Then, thirdly, swine flu viruses have been known to infect humans -- 50 cases were turned up from an extensive review of the literature. Of the non-military cases, 19 occurred in the United States, 6 in Czechoslovakia, 4 in the Netherlands, 3 in Russia, and 1 each in Canada and Hong Kong. Most (61%) of the cases studied had reported an exposure to swine and the median age was 24 years (see Myers below). The most recognizable H1N1 transmission event involved 12 soldiers at Fort Dix in 1976, one of whom died. Contact with swine was never established. Another important transmission resulted in the death of a pregnant woman, who was exposed to pigs, in 1988 in Wisconsin. CDC receives about one human influenza isolate each year that tests positive for a swine influenza virus. H1N1 and H3N2 swine flu viruses are endemic in US pig populations. Swine flu outbreaks in pigs typically occur in late fall and winter months. Vaccines are available to be given to pigs to prevent swine influenza, but there is no vaccine to protect humans from swine flu. Seasonal influenza vaccines are likely to partially protect against swine H3N2 viruses, but not the H1N1 subtype. Sporadic human infections due to influenza viruses of swine origin have been described previously since 1950's, mostly in young persons (younger than 25 years) in contact with pigs^{ref1, ref2, ref3}. Human to human transmission has been reported before; however in these cases transmission was limited to one generation^ref. Unlike the non-zoonotic swine fevers it is not a disease that comes under the European Union's harmonised Animal Disease Notification System and there are no routine European surveillance data. In the past, CDC has received reports of approximately one human swine influenza virus infection every 1-2 years in the USA^{ref1, ref2}. However, during December 2005-January 2009, 12 cases of human infection
with swine influenza were reported; 5 of these 12 cases occurred in patients who had direct exposure to pigs, 6 in patients reported being near pigs, and the exposure in one case was unknown^{ref1, ref2, ref3}. On the contrary of Europe, in the United States, novel influenza A virus infections in humans, including swine influenza infections, have been nationally notifiable conditions since 2007. The symptoms and pathogenesis of influenza in pigs show remarkable similarities with those of seasonal influenza in humans, but the epidemiology is different. Part of this is due to the structure of the swine industry and the extremely rapid turnover of the swine population, with the constant introduction of immunologically naive animals into swine herds. In swine-dense regions in particular, most pigs show serological evidence of having been infected with influenza by the end of the 6-month-long fattening period, and many of them have undergone simultaneous or consecutive infections with 2 or even 3 swine influenza subtypes^ref. Unlike human viruses in temperate climates, swine influenza viruses circulate at comparable levels year round. Also, the viruses in Europe differ significantly in their antigenic and genetic make-up from those circulating in North America, even though they consist of the same H and N subtypes, and hence findings in the United States should not necessarily be extrapolated to Europe^ref
• in 1988, an H1N1 swine flu virus was found in a previously healthy 32-year-old pregnant woman who died 8 days after she was hospitalized for pneumonia, according to the CDC. 4 days before she got sick she had visited a swine exhibit at a county fair where a flu-like illness was widespread among the pigs. Follow-up studies showed that 76 percent of swine exhibitors had antibodies to the swine flu virus, though no illnesses were reported. However, researchers found that one to 3 health care workers who had contact with the woman experienced mild flu symptoms with antibody evidence of swine flu exposure.
• in September 2008, researchers from the CDC and public health officials from Wisconsin published a case report in Emerging Infectious Diseases on a healthy 17-year-old boy who had mild respiratory symptoms in December 2005, 3 days after helping his brother-in-law butcher pigs. At an outpatient clinic a few days later, health care workers collected nasal wash specimens, which tested positive for influenza A and were forwarded to the Wisconsin State Laboratory of Hygiene^ref. Though further testing isolated influenza A, the virus didn't match human H3 or H1 subtypes or the H5 avian subtype. CDC investigators sequenced the virus, identifying it as a swine influenza A (H1N1) triple reassortant virus, A/Wisconsin/87/2005 H1N1. The report said that triple reassortant H1N1 subtypes are the predominant genotype in North American pigs. There are 4 main influenza type A virus subtypes that have been isolated in pigs: H1N1, H1N2, H3N2, and H3N1. However, most of the recently isolated influenza viruses from pigs have been H3N2 and H1N1 viruses^ref.
• In December 2007, researchers reported that a new swine flu subtype found recently in Missouri pigs [H2N3] combined genes from avian and swine flu viruses, could cause experimentally induced infections in mice, and was transmissible in pigs and ferrets. The findings, which appeared in Proceedings of the National Academy of Sciences (PNAS), bolstered the theory that pigs can serve as a mixing vessel for flu viruses and a possible source for a human pandemic strain.
• in Mid-October 2008 a patient in Texas got sick : A/H1N1 was identified during routine influenza surveillance^{ref1, ref2, ref3}.

in November 2008 a human case of swine influenza A/H1 (phylogenetically close to the human isolate A/Switzerland/8808/2002 of swine origin^ref) in a 50 year old woman from a village near Teruel (Aragon, in the north east of Spain) and recovered fully. The case worked on a family swine farm and had direct and close exposure to
pigs. No other family members or co-workers reported flu-like symptoms before or after this case and no symptoms were observed in the pigs^ref.
• the 2009 pandemic : so far, the exact source of the outbreak remains a mystery. Un-confirmed rumours point to La Gloria, a dusty town in the southern Mexican state of Veracruz, where it is believed that 5-year-old Edgar Hernández became infected with the A (H1N1) virus in late March. Some local residents in La Gloria have blamed the nearby US-owned industrial pig farms for the outbreak of the disease^ref
• USA : on 17 Apr 2009, CDC determined that 2 cases of febrile respiratory illness occurring in 2 unvaccinated children (a 10-yrs boy from San Diego county and a 9-yrs old girl from Imperial county) in southern California were caused by infection with a swine influenza A (H1N1) virus. The 10-yrs old boyrecovered after symptomatic treatment : his mother had respiratory symptoms without fever in the 1st few days of April 2009, and a brother aged 8 years had a respiratory illness 2 weeks before illness onset in the patient and had a 2nd illness with cough, fever, and rhinorrhea on 11 Apr 2009. However, no respiratory specimens were collected from either the mother or brother during their acute illnesses. Public health officials are conducting case and contact investigations to determine whether illness has occurred among other relatives and contacts in California, and during the family's travel to Dallas, Texas on 3 Apr 2009. The 9-yrs old girl recovered after amoxicillin/clavulanate : she had attended an agricultural fair where pigs were exhibited approximately 4 weeks before illness onset. She reported that she did not see pigs at the fair and went only to the amusement section of the fair. The patient's brother aged 13 years had influenza-like symptoms on 1 Apr 2009, and a male cousin aged 13 years living in the home had influenza-like symptoms on 25 Mar 2009, 3 days before onset of the patient's symptoms. The brother and cousin were not tested for influenza at the time of their illnesses. The viruses from the 2 cases are closely related genetically, resistant to amantadine and rimantadine, and contain a unique combination of gene segments that previously has not been reported among swine or human influenza viruses in the United States or elsewhere. Neither child had contact with pigs; the source of the infection is unknown^ref. The majority of their genes, including the hemagglutinin (HA) gene, are similar to those of swine influenza viruses that have circulated among US pigs since approximately 1999; however, 2 genes coding for the neuraminidase (NA) and matrix (M) proteins are similar to corresponding genes of swine influenza viruses of the Eurasian lineage 1st seen in Thailand^ref : the swine flu virus contains segments from 4 different viruses: some North American swine, some North American avian, one human influenza, and 2 Eurasian swine^ref. On April 23, 2009 5 new cases that recovered, including 2 new clusters : 2 16-year-old boys in Guadalupe County, San Antonio, Texas, who attended the same school and fall ill on Apr 10 and 13, respectively, and a father and daughter from San Diego County, California. In addition a 5th new case occurred in the vaccinated 54-yo father of the affected 9-yo girl from Imperial County^ref.  A woman aged 41 years with an autoimmune illness who resided in Imperial County developed fever, headache, sore throat, diarrhea, vomiting, and myalgias on [12 Apr 2009]. She was hospitalized on [15 Apr 2009]. She recovered and was discharged on [22 Apr 2009]. A respiratory specimen obtained [16 Apr 2009] was found to be influenza A positive by RT-PCR at the San Diego Country Health Department Laboratory, but could not be further subtyped. The woman had not been vaccinated against seasonal influenza viruses during the 2008--09 season. Three household contacts of the woman reported no recent respiratory illness^ref.  On Apr 24 about 75 students at the private St Francis Preparatory School in Queens, New York, have fallen ill with flu-like symptoms^ref. No. of laboratory confirmed cases
• California: 7 cases
• Texas: 2 cases
• Kansas: 2 cases : 2 adults residing in the same household in Dickinson County tested positive on Apr 25. One of the patients had recently traveled to Mexico, flying in and out of Wichita. Both persons work in Saline County ^ref
• Ohio : 1
• New York City : 28 cases
• Minnesota : a suspected case
Total count: 20 cases
• Mexico has reported more than 1324  cases and 81 deaths^ref (149 suspected^ref) since Apr 13 in as many as 19 of Mexico's 32 states. Mexico initially asked Canada's Public Health Authority (PHAC) to assist in finding the source of the illnesses^ref
• The 1st death occurred in southern Oaxaca state, in the south, on [13 Apr 2009], but Mexico didn't send the 1st of 14 mucous samples to CDC until [18 Apr 2009]
• Distrito Federal (Mexico City)(south central Mexico) involved 854 cases and 59 deaths
• San Luis Potosi (about 400 miles north of Mexico City), with 24 cases and 3 deaths
• 2 in Baja California Norte, near San Diego, California
• 4 cases with no deaths in Mexicali (on the border with Calexico in Imperial county California).
The PHAC report said the disease outbreak struck some healthcare workers and that most patients were previously healthy young adults between the ages of 25 and 44. Mexican officials detected some influenza A/H1N1 and influenza B viruses, but have apparently ruled out H5N1 virus involvement. The infection has now affected employees of the main public hospitals in the capital Mexico City, including the Juarez Hospital, the General Hospital and the National Institute of Respiratory Diseases, and the hospitals in the Tlalpan area, where the National Institute of Cardiology, the National Institute of Nurtition and the Manuel Gea Gonzalez, as well as the Polanco Red Cross, are located^ref. Schools were closed and all public events suspended in the capital until further notice -- including more than 500 concerts and other gatherings in the metropolis of 20 million.^ref. Mexico has enough Tamiflu to treat 1 million people, and 500 000 doses of  flu vaccines. No surveillance yet but the SIV (swine influenza virus) is endemic and controlled by vaccination here in the US and probably in Mexico. Mexico's pork production is just like that in the US, Canada and Europe -- very intensive. CDC's laboratory analyzed 14 samples from severely ill Mexican patients and found that 7 of them had the same swine flu mix as the virus that infected the US patients^ref; Canada's national laboratory has confirmed swine flu A/H1N1 in 18 isolates from Mexican patients, 12 of which were genetically identical to the swine flu viruses from California^ref.
• Canada : a man from Cornwall, Ontario, who returned from Mexico with a mysterious illness is believed to be one of a handful of people in Ontario who may be linked to the respiratory ailment, provincial officials say. The 47 year old man was airlifted to an Ottawa hospital in late March and admitted into the hospital's intensive care unit, a hospital official said Thursday. He spent 11 nights in hospital before being released on 9 Apr 2009^ref. The new cases appear to have somewhat more vomiting and diarrhea than is usually seen in flu, which mostly causes coughing, fever, sore throat and muscle aches.
• Nova Scotia : 4 cases in students  ranging in age from 12 to 17 or 18 in the Windsor area are recovering on Apr 26^ref
• British Columbia : 2 suspected cases^ref
• UK : a male British Airways cabin crew member is undergoing precautionary tests in a London hospital after falling ill with "flu-like" symptoms on a flight from Mexico City. He was taken to Northwick Park Hospital, in Harrow after landing at Heathrow airport at 1400 BST (1300 GMT) on Saturday [25 Apr 2009]^ref. 2 people, a man and a woman admitted to a Scottish hospital after returning from Mexico and currently recovering, were confirmed as the 1st British cases of swine flu. 7 other people who had been in contact with them were now displaying mild symptoms^ref. At 4 schools in the UK where pupils have confirmed infection, prophylactic oseltamivir has been issued to hundreds of fellow students. Although this policy follows Health Protection Agency advice, for a group of 1000 people 16 times as much antiviral was needed to prevent influenza infection as to treat it. By this measure, the UK has stockpiles of oseltamivir sufficient to treat 30 million people (about half the population) but to prevent infection in only 1·9 million.
• New Zealand : A group of 25 staff and students from Rangitoto College on Auckland's North Shore are being tested for the deadly swine flu after returning from a

school trip to Mexico. 18 people in the group of 25 staff and students have reported influenza-like illnesses^ref. Northcote College having recently returned from Mexico, while Pinehurst and Westlake Girls High also had pupils in the United States and South America^ref.
• Israel : a man who had recently visited Mexico had been hospitalized while authorities try to determine whether he had the disease^ref.
• French : 4 possible cases of swine flu in 2 regions are currently under investigation. All recently returned from Mexico^ref.
• Spain : 3 people who just returned from Mexico were under observation in hospitals in the northern Basque region, in south eastern Albacete and the Mediterranean port city of Valencia^ref. 1st confirmed case on Apr 27, returning from Mexico^ref
Prior infection with an H₁N₁ swine influenza virus partially protects pigs against a low pathogenic H₅N₁ avian influenza virus^ref
Laboratory examinatins : relative lymphopenia (< 20%) is a high diagnostic marker, as for seasonal flu^ref
Prevention : anti-H₁N₁ vaccines. Only 12% of Germans say they will have H₁N₁ vaccine after row blows up over safety of adjuvants^ref. Delays in vaccination of 30 days or more reduced the effectiveness of vaccination in lowering the attack rate. However, pre-existing immunity in 15% or more of the population kept the attack rates low, even if the whole population was not vaccinated or vaccination was delayed. School closure was effective in reducing the attack rate, especially if applied early in the outbreak, but this is not necessary if vaccine is available early or if pre-existing immunity is strong^ref. Previous vaccination of children with any of four seasonal trivalent, inactivated influenza vaccines (TIV) or with live, attenuated influenza vaccine (LAIV) did not elicit a cross-reactive antibody response to the novel influenza A (H₁N₁) virus. Among adults, vaccination with seasonal TIV resulted in a twofold increase in cross-reactive antibody response to the novel influenza A (H₁N₁) virus among those aged 18-64 years, compared with a twelvefold to nineteenfold increase in cross-reactive antibody response to the seasonal H₁N₁ strain; no increase in cross-reactive antibody response to the novel influenza A (H₁N₁) virus was observed among adults aged >60 years^ref.
Prognosis : mortality rate of infected dialysis patients (confirmed, probable and suspected cases) was around 5%^ref.
Web resources :
• CDC recommendations for infection control, treatment, and chemoprophylaxis
• Pig Flu Map
• H₁N₂ subtype appears to have resulted from the reassortment of the genes of the circulating influenza A(H₁N₁) (including HA) and A(H₃N₂) (including NA) subtypes
• H₁N₇ subtype^{ref1, ref2}
• H₁N₉ subtype
• H₂ infects humans and has healthy reservoir in water Aves spp.
• H₂N₁ subtype
• H₂N₂subtype(Asiatic flu, 1957-1958) :
Epidemiology :
• first identified in China in late February 1957; it spread to the USA by June; it caused 1 million deaths, 70,000 in the USA. The virus initially retained a binding preference for "avian" SA-a-2,3-Gal–terminated sialosaccharides but switched affinity to "human" SA-a-2,6-Gal–terminated saccharides during subsequent influenza seasons in the human population^ref
• in late January 2004 it was found in Pennsylvania, USA : the state typically finds bird flu at about 40% of the markets
• on Apr 13, 2005, the WHO said > 5,000 vials of a 1957-58 pandemic H₂N₂ flu strain sent through DHL and FedEx to > 3,747 laboratories in 19 countries (61 outside USA and Canada. Other countries where laboratories received the samples are listed by the WHO as Bermuda, Belgium, Brazil, Canada (41), Chile, France, Germany, Hong Kong Special Administrative Region of China, Israel, Italy, Japan, Lebanon, Mexico, Saudi Arabia, Singapore, South Korea, Taiwan, and UK (1); H₂N₂ samples were sent to 3747 labs under CAP auspices and to about another 2700 labs certified by other organizations)^ref by the College of American Pathologists (CAP), which obtained the samples from Meridian Bioscience Inc. in Cincinnati (which in turn received the virus from another company in 2000), for routine testing from September 2004 until early April 2005 should be destroyed within 1-2 days either by incineration or by chemicals. An additional 2750 laboratories, all in the United States, received the samples as part of other certification processes and were asked to destroy them. On March 25 the National Microbiology Laboratory in Winnipeg, Manitoba, identified it as the 1957 pandemic strain and reported to the Public Health Agency of Canada (PHAC). This information was passed to WHO and the United States Department of Health and Human Services (HHS) on 8 Apr 2005. Given the concerns that the virus could be used in bio-terrorism, letters were sent to the laboratories before the mistake was made public. On 8 Apr 2005, the CAP, at the request of the US government, contacted all laboratories participating in the proficiency testing and asked them to destroy all samples containing A/H₂N₂ virus. On 12 Apr2005 the CAP further asked all the laboratories to send confirmation of the destruction, and to investigate and notify national authorities of any respiratory disease seen in laboratory staff. WHO has the addresses of all laboratories involved and has passed these on to the relevant ministries of health, requesting their collaboration. The European Early Warning and Response System (EWRS)^ref today issued a level 2 alert to the competent public health authorities in the EU, with the information currently available from WHO. The European Commission is in permanent contact with the member states concerned and WHO and is following the situation closely. The European Influenza Surveillance Scheme (EISS) has today issued an urgent questionnaire for all laboratories participating in the Community Network of Reference Laboratories for Human Influenza in Europe^ref, with guidance. All laboratories in this network can detect the A/H₂N₂ virus, but only 38% (12/32) of them currently have the reagents available to identify the H₂ subtype of the virus^ref. EISS is working with laboratories to improve the detection of potential pandemic viruses, including A/H₂N₂ viruses^ref. Based on virus detection, there have so far been no reports of any A/H₂N₂ infections in laboratory staff associated with this sample distribution. When proper biosafety precautions are taken, the risk of laboratory-acquired influenza infections is greatly reduced and the likelihood of any infections in these staff and the general public is low. WHO recommends that all proficiency panel specimens containing A/H₂N₂ be destroyed immediately, and that biosafety procedures for influenza viruses that are no longer circulating in humans be reviewed. Because the virus has not been included in flu vaccines since 1968, anyone born after that would not be immune to that strain. Virus samples are distributed to laboratories so lab workers can test their ability to identify samples, but they are normally strains in current or recent circulation. The panel samples usually include only strains of influenza virus that are relatively benign : this was an unwise and unfortunate decision. The risk is relatively low that a lab worker will get sick, but there was a concern because of the large number of labs that received it. If someone does get infected, the risk of severe illness is high and this virus has shown to be fully transmissible. The 1957 flu virus has for years been a BSL2 virus, but many countries have upgraded it to a BSL3 because so many people have no immunity to it. Neither the college nor the company was aware CDC was considering whether to reclassify the strain as too deadly to ship. A mechanism is being established to require anyone shipping pathogens to notify the CDC about what strains of virus are involved. Seroarchaeology suggest that H₂N₂ serotype influenza virus was the predominant human influenza virus just prior to 1900. In 1900 the predominant serotype became H₃N₈. Phylogenetic analysis suggests that a serotype H₁N₁ virus of avian origin then became predominant, infecting both humans and pigs, and spreading from America to Europe with troop movements. This virus was probably responsible for the Spanish influenza pandemic of 1918. The Asian influenza pandemic of 1957 was caused by an H₂N₂ serotype virus, which carried 3 genes (PB1, H₂ and N₂) derived from feral waterfowl and the remaining 5 genes from the human H₂N₂ influenza virus. The previously predominant H₁N₁ strain disappeared simultaneously with the appearance of the pandemic H₂N₂ strain, a circumstance not yet adequately explained. The virus responsible for the Hong Kong influenza H₃N₂ pandemic of 1968 probably acquired two genes (PB1 and H₃), again from wild ducks, while retaining the six other genes of the circulating human influenza virus. Coincident with the appearance of the H₃N₂ virus, the H₂N₂ virus disappeared, again a circumstance not adequately explained. Later, in 1977, a Russian H₁N₁ serotype strain that had been present in the 1950s reappeared and has remained in circulation together with H₃N₂ serotype virus since that time^ref. (Webster RG and Kawaoka Y, Semiminars in Virology 5, 103-111, 1994) As a consequence of these events the majority of the global human population has not been exposed to H₂N₂ serotype virus and lacks immunity to infection. Hence the urgent need to recall or destroy the samples of H₂N₂ serotype virus apparently distributed in error as a diagnostic standard for human influenza A virus. An unresolved issue is whether antibodies to the N₂ component of influenza virus might provide some degree of protection sufficient ameliorate the  effects of H₂N₂ virus infection. 13 of the 19 countries that received samples had confirmed their labs had destroyed the virus : however, labs in Lebanon, Mexico and Chile never received the specimen, even though they were on the distribution list. It was possible the shipments had never been sent out at all, but none could not be sure : WHO has requested an investigation into the missing kits. Previously unaccounted for samples sent to Mexico and South Korea also have been destroyed. South Korean officials had previously reported to WHO that they had destroyed half the samples they had been sent but hadn't confirmed that they had also destroyed the other half : the destruction of all the samples sent to South Korea has now been confirmed. A missing shipment to Mexico has been tracked down in a warehouse and has also been destroyedThe world's last missing sample of the killer influenza virus was found at Beirut's International Airport and officials are waiting for orders from the U.N. World Health Organization on whether to destroy in Beirut or send it back to them. Among the countries that have confirmed destruction of all samples by their labs are Canada, Hong Kong, Belgium, France, Germany and Bermuda. Other countries with labs that received the kits were Saudi Arabia, Brazil, Israel and Japan; they have not yet sent confirmation but received instructions to destroy the virus. As of 3 May 2005, all samples of H₂N₂ influenza virus that were sent to thousands of laboratories in 18 countries have been accounted for and destroyed : the same day CDC and NIH recommended that labs use Biosafety Level 3 (BSL-3) instead of BSL-2 precautions when working with the virus
• 
  
• H₂N₃ subtype
• H₂N₈ subtype
• H₂N₉ subtype
• H₃ infects humans, Sus scrofa and Equus caballus, and has healthy reservoir in water Aves spp.. Based on a crystallographic model, the receptor-binding site of the H₃ subtype includes conserved residues Tyr98, His193, Glu190, Trp53, and Leu194^ref. 2 other conserved residues at positions 226 and 228 within the binding pocket determine host range specificity^ref. Leu226 and Ser228 selectively bind to a2,6 sialosides found on human and swine cells, while Gln226 and Gly228 bind to the a2,3 sialosides found predominantly on avian cells^{ref1, ref2, ref3}.
• H₃N₁ subtype
• H₃N₂ subtype (Hong Kong flu, 1968-1969) : was first detected in Hong Kong in early 1968 and spread to the USA later that year. It caused 1 milion deaths, approximately 34,000 in USA. This virus is still in circulation today. Antigenic evolution of human influenza A (H₃N₂) virus from 1968 to 2003. The relative positions of strains (colored shapes) and antisera (open shapes) were adjusted such that the distances between strains and antisera in the map represent the corresponding hemagglutination inhibition (HI) measurements with the least error. The periphery of each shape denotes a 0.5 unit increase in the total error and is a measure of confidence in the placement of the strain or antiserum. Strain color represents the antigenic cluster to which the strain belongs; antisera are not colored. Clusters were identified by a k-means clustering algorithm and named after the first vaccine strain in the cluster. 2 letters refer to the location of isolation: HK, Hong Kong, China; VI, Victoria, Australia; TX, Texas, United States; BK, Bangkok, Thailand; SI, Sichuan, China; BE, Beijing, China; WU, Wuhan, China; SY, Sydney, Australia; and FU, Fujian, China) and the 2 digits refer to year of isolation. The grid represents one unit of antigenic distance (i.e., a two-fold dilution in HI titer).


+ salicylates (+ EBV ?) => Reye's syndrome
Therapy : adamantane derivatives, such as amantadine and rimantadine, have been used successfully for the prevention and treatment of influenza A virus infection for more than 30 years^{ref1, ref2, ref3}. These drugs, known as M2 channel blockers, inhibit influenza A virus replication by blocking the M2 protein ion channel thereby preventing fusion of the virus and host-cell membranes and the release of viral RNA into the cytoplasm of infected cells^ref. The prophylactic effect of these drugs varies between 80% and 90%, and the drugs can reduce the duration of illness by about 1.5 days if given within 48 h of infection^{ref1, ref2} (Oxford JS, Galbraith A. Anti influenza virus activity of amantadine. Viral Chemother 1984; 1: 169-252). Human and animal studies have shown the frequent occurrence of amantadine-resistant influenza viruses after exposure to the drug, and drug-resistant viruses can be transmitted from one person to another without apparent loss of pathogenicity    ^{, ref5}. Additionally, complete cross-resistance between amantadine and rimantadine has been shown^ref. The genetic basis for resistance to these drugs has been well characterised and is associated with an aminoacid substitution at position 26, 27, 30, 31, or 34 in the transmembrane region of the M2 protein^ref (Hay AJ, Zambon MC, Wolstenholme AJ, Skehel JJ, Smith MH. Molecular basis of resistance of influenza A viruses to amantadine. J Antimicrob Chemother 1986; 8 (suppl B): 19-29). Most drug-resistant influenza viruses contain 1 of these aminoacid changes, but variants with dual mutations have also been described^{ref1, ref2}. Resistance to adamantanes has been reported predominantly in people in semi-closed settings (eg, nursing home facilities, paediatric wards, and family households) where antiviral treatment was used^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}. Since 1991, drug-susceptibility surveillance has been undertaken routinely in the characterisation of influenza virus isolates submitted to the WHO Collaborating Center for Influenza at the US Centers for Disease Control and Prevention. Previous surveillance studies have identified a low incidence of resistance to amantadine and rimantadine (approximately 1%) among circulating influenza viruses. However, 10 years have elapsed since the last comprehensive global study of resistance to these drugs was published^{ref1, ref2, ref3, ref4}. Furthermore, influenza A (H₅N₁) viruses isolated from both human beings and avian sources in southeast Asia since 2003 have an S31N aminoacid substitution in the M2 protein and, thus, are resistant to amantadine and rimantadine^{ref1, ref2}. Results of a surveillance study for resistance to adamantanes among circulating influenza viruses collected worldwide between Oct 1, 1994, and Mar 31, 2005, revealed that during the decade of surveillance a significant increase in drug resistance was noted, from 0.4% in 1994–1995 to 12.3% in 2003–2004. This increase in the proportion of resistant viruses was weighted heavily by those obtained from Asia with 61% of resistant viruses isolated since 2003 being from people in Asia. This study reveals an alarming increase in the incidence of amantadine-resistant and rimantadine-resistant H₃N₂influenza A viruses over the past decade. The study, which assessed > 7000 influenza A viruses obtained worldwide, is the largest and most comprehensive report on adamantane resistance to date. The last major surveillance study by Ziegler and co-workers^ref showed a drug-resistance frequency of 0.8% among H₁N₁ and H₃N₂ viruses and identified only 16 drug-resistant viruses internationally over a 4-year period. This low reported incidence accords with findings from previous studies^{ref1, ref2, ref3}. These reports had shown that isolation of drug-resistant influenza viruses was a rare event, with a frequency of 1–2%. We have shown an escalating trend in circulating drug-resistant viruses in recent years. This trend was first observed among viruses isolated in Asia starting from 2000 and then in other regions of the world, including North America in 2004. Viruses collected in 2004 from South Korea, Taiwan, Hong Kong, and China show drug-resistance frequencies of 15%, 23%, 70%, and 74%, respectively. The first 6 months (October, 2004, to March, 2005) of the 2005 influenza season in the USA have already shown a significant increase in the incidence of adamantane resistance from 2% in 2004 to 15% in 2005 among isolates obtained from a comparable geographic and demographic distribution. Of particular note from the 10-year study period is the fact that of all resistant viruses, more than 84% were identified since the 2003 influenza season, indicating a distinct spike in drug resistance among circulating H₃N₂ influenza viruses throughout the world. This trend was recorded with no fundamental changes in the surveillance methods over the study period other than an expansion in the number of viruses being submitted over time. Up to 30% of individuals who receive amantadine or rimantadine for the treatment of influenza virus infection can excrete viruses resistant to these drugs^{ref1, ref2, ref3, ref4, ref5}. Clinical and epidemiological data that indicate whether resistant isolates were recovered as a result of adamantane treatment or exposure were not available from countries outside the USA making it difficult to fully assess the effect of adamantane use on the observed trend. However, of 92 US patients from whom drug-resistant viruses were isolated since 2004, only 2 (2%), who were from nursing home facilities, were known to have received adamantane treatment before virus collection. None of the other drug-resistant isolates obtained in the USA since 2004 were from patients documented to be on adamantane therapy, residents from semi-closed settings, nor in contact with patients on antiviral therapy. The trend of a rising incidence of resistant viruses isolated from Asian countries compared with the lower incidence in the Americas during the period studied might be a consequence of differences in the procurement of amantadine and rimantadine in these countries. In the USA, adamantanes are licensed as anti-influenza drugs that are prescribed for influenza or influenza-like illness only by licensed physicians. However, in China, Russia, and some other countries, amantadine or rimantadine are available in over-the-counter formulations and are included in various cold remedies that do not need a prescription^{ref1, ref2}. Additionally, adamantanes are now broadly available as generic drugs, making it difficult to obtain or compare quantitative data on their distribution and clinical use. The lack of these data and their effect on increased resistance to these drugs is a limitation of our study. Alternatively, drug-resistant mutations in the M2 gene could have occurred spontaneously, as was previously reported for some influenza H₁N₁ viruses (A/PR/8/34, A/WSN/33) that were resistant to adamantanes before these drugs were developed. Such a spontaneous mutation in the M2 gene may have been accompanied by the virus antigenic drift that allowed the widespread of recent influenza H₃ viruses over the world. Such an assumption could explain the fact that the high increase in the proportion of drug-resistant isolates was observed for influenza A (H₃) but not for influenza A (H₁) viruses. The possibility that further genetic and antigenic evolution of influenza A (H₃N₂) viruses could result in the disappearance of this mutation in the M2 protein, reverting back to the drug-sensitive phenotype, should not be excluded. Epidemic and pandemic strains of influenza often have been identified first in Asia^ref. Many influenza experts have speculated that variants of influenza A (H₅N₁) viruses, which are currently circulating in avian species throughout southeast Asia and causing severe disease and mortality in human beings, have potential to cause the next influenza pandemic^{ref1, ref2, ref3, ref4, ref5, ref6}. Since 2003 all human and most avian influenza A (H₅N₁) isolates tested are resistant to amantadine and rimantadine^{ref1, ref2}. Data reported here on the high incidence of drug-resistant H₃N₂ viruses circulating in the same geographic region as H₅N₁ viruses suggest that greater caution is needed in the use of amantadine and rimantadine. With the increasing rates of resistance shown here, amantadine and rimantadine will probably no longer be effective for treatment or prophylaxis in the event of a pandemic outbreak of influenza. These findings have broad implications for agencies and governments planning to stockpile these drugs as a front-line arsenal against epidemic or pandemic strains of influenza. With the establishment and increasing incidence of amantadine-resistant and rimantadine-resistant viruses throughout the world, careful assessment of the ability to rely on such drugs for treatment and prophylaxis before and during a pandemic or seasonal epidemic is warranted. These data raise concerns about the increasing incidence of adamantane-resistant influenza A viruses circulating throughout the world and draw attention to the importance of tracking the emergence and worldwide spread of drug-resistant viruses^ref.
• H₃N₄ subtype
• H₃N₅ subtype
• H₃N₈ subtype/ A/equine 2 : Canis familiaris
The Canadian Food Inspection Agency (CFIA) in 2005 quarantined a turkey layer farm in Abbotsford, British Columbia based on preliminary results from the British Columbia Ministry of Agriculture, Food and Fisheries (BCMAFF) indicating the presence of the H₃ influenza virus in the flock. The turkey farm is near a swine farm that recently experienced an H₃ influenza infection, and the virus is suspected to have originated from swine. Transmission of this influenza strain between swine and turkeys has been documented previously.
• H₄ infects Phocidae and Sus scrofa and has healthy reservoir in water Aves spp.
• H₄N₆ subtype
• H₄N₈ subtype
Epidemiology : on Nov 2005 10 ducks of a farm in Osaka, Japan, have been infected with the H₄ strain of AI, which has yet to be transmitted to humans. Experts are conducting more tests on 47 other ducks of the farm, after initial tests showed that some of them might catch the disease. This case seems to be unrelated to the outbreak of low-pathogenic H₅N₂, which has been simmering in Ibaraki since April 2005, probably caused by an illegal vaccine. In contrast to the H₅ and H₇ avian influenza strains, H₄ is not an OIE-notifiable animal influenza (NAI) strain. However, if found highly pathogenic by means of the
prescribed pathogenicity tests, it will become notifiable
• H₅ infects humans and has healthy reservoir in water Aves spp.. Both low- and high-pathogenic strains exist, depending at least partly on the amino acid sequence at the cleavage site in HA1/HA2. Detection of an H₅ subtype of influenza A in a human should always arouse a very high level of suspicion that the case was caused by the H₅N₁ strain of avian influenza, particularly in a country with ongoing outbreaks of H₅N₁ in poultry. H₅N₁ is the only strain within the H₅ subtype that has ever been shown to cause disease in humans. The next influenza pandemic: lessons from Hong Kong^ref.

Epidemiology :
• The Netherlands : on Mon Mar 15, antibodies against H₅ LPAI were detected also in ducks at a farm in Lopik in the central province of Utrecht : although the virus could not be isolated, the government decided not to take any risk and thus ordered the culling of all 800 ducks. On Mar 19 some 1000 birds (442 ducks, 531 chickens, 5 turkeys, 9 geese, and 77 doves) were culled at a farm in Steenbergen (in the southern province of North Brabant), which had links to a farm in Lopik. The source may have been 2 shipments of a total of 2500 ducks from France : 5 other farms, which also received ducks from the 2 shipments, are still under investigation. On Mar 20 99 birds (34 ducks, 35 chickens, 3 turkeys, 3 geese, and 24 doves) were culled in a farm located at Liempde, province of North-Brabant
• Canada : on 18 Nov 2005 a duck on a farm in Abbotsford in the Fraser Valley east of Vancouver, south western British Columbia tested positive by RT-PCR. On Jun 2006 4 domestic goose (purchased about 6 weeks before from a local co-operative) died in western Prince Edward Island and one tested positive for an H₅ avian influenza virus^ref
Subtypes :
• H₅N₁ subtype

First isolated from Sterna hirundo (common terns) in South Africa in 1961^ref. it was probably transmitted by Odontophoridae infected with H₉N₂, Anser spp. infected with H₅N₁, and Anas spp. infected with H₆N₁ from Continental China to Gallus gallus in Hong Kong markets, and then to humans. Researchers introduced the H₅N₁ virus into the airways of 3 cats and 3 other cats were fed an infected chick. Finally, 2 cats were exposed to the virus by being placed in the same cage as the 1st 3 cats. The cats soon showed signs of disease: raised body temperature, decreased activity, and labored breathing. All developed severe lung disease. One cat died after 6 days of infection. The study authors also tested the effect of another type of influenza virus, H₃N₂, which most commonly causes flu in humans. Cats exposed in the same way to this virus did not develop disease. During H₅N₁ virus outbreaks, domestic cats are at risk of disease or death from H₅N₁ virus infection, either due to feeding on infected poultry or wild birds, or due to contact with infected cats. The role of cats in the spread of H₅N₁ virus between poultry farms, and from poultry to humans, needs to be re-assessed. Cats may form an opportunity for this avian virus to adapt to mammals, thereby increasing the risk of a human influenza pandemic^ref. Anyway it could not be passed between humans. Due to Asp⁹²=>Glu in NS1, virus is resistant to IFNs and TNF-a.
Epidemiology (see also under veterinary medicine : avian influenza). One of the 2 strains, or clades, of HA that emerged since 2003 made people sick in Vietnam, Cambodia and Thailand in 2003 and 2004, while the other caused the disease in people in Indonesia in 2005. Recent avian and human isolates are predominantly genotype Z viruses but additional avian isolates of genotype V and Z/W reassortants are also circulating. The hemagglutinin (HA) gene has evolved considerably since 2003 into distinct clades (1, 1’ and 2). Human and avian isolates from
Vietnam, Thailand, Cambodia, Malaysia and Laos are genotype Z and have a clade 1 HA. Viruses with clade 2 HA belong to genotypes Z, V or Z/W reassortants and are responsible for the recent outbreaks of avian influenza in Indonesia, China, Japan, South Korea, Vietnam and Eastern Europe andhuman cases in Indonesia. Clade 2 HA can be further subdivided into at least 6 subclades (2A-F) with distinct amino acid motifs and the potentialfor different antigenic profiles. Positive selection analysis of all H₅N₁ HA showed that amino acids corresponding to two major antigenic sites are under selective pressure (Garten, R. Analysis of H₅N₁ Influenza Viruses from Humans and Birds Between 2003 and 2005 Reveals an Increase in the Spectrum of Viruses with Pandemic Potential. ICEID2006 Abstract^ref)

Proteomics :
• the hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H₅N₁ influenza virus, is more related to the 1918 and other human H₁ HAs than to a 1997 duck H₅ HA. Glycan microarray analysis of this Viet04 HA reveals an avian a2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H₁ serotype HAs to human a2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H₂ and H₃ HAs to human receptor specificity, when inserted onto the Viet04 H₅ HA framework, permitted binding to a natural human a2-6 glycan that suggests a path for this H₅N₁ virus to gain a foothold in the human population^ref. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H₅N₁ viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Some H₅N₁ viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H₅N₁ viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H₅N₁ virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H₅N₁ field isolates^ref.
• NS1 protein : the carboxyl terminus of the vast majority of avian H₅N₁ viruses contains a sequence motif, Glu-Ser-Glu-Val (ESEV). These residues are predicted to mediate binding to proteins bearing a region called a PDZ domain. The multitude of human proteins that contain a PDZ domain function in diverse cellular signaling pathways including those that regulate protein traffic within the cell and those that maintain cell morphology and organization. Another PDZ-binding sequence, Glu-Pro-Glu-Val (EPEV), was identified at the carboxyl terminus of the NS1 proteins of all the virulent H₅N₁ viruses isolated from humans. In contrast, the carboxyl terminus of the NS1 proteins of low-virulence human influenza A usually contains a different sequence, Arg-Ser-Lys-Val (RSKV), which is not a PDZ-binding motif. It it has already been established that this carboxyl-terminal sequence is not required for the virulence of previously isolated H₅N₁ viruses in ferrets^ref. An analysis of the virulence of H₅N₁ viruses isolated in 2004 identified the human isolate A/Vietnam/1203/04 as the most pathogenic isolate. The NS1 protein encoded by this virus is truncated and consequently lacks the suspect carboxyl-terminal ESEV/EPEV motif^ref. The genetic basis underlying the virulence and host range of 2 H₅N₁ influenza viruses in chickens was investigated. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/ 2/96 (GS/GD/2/96) is unable to replicate in chickens. These 2 H₅N₁ viruses differ in sequence by only 5 amino acids mapping to the PA, NP, M1, and NS1 genes. Reverse genetics was used to create 4 single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining 7 gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these 2 NS1 genes resides at position 149. A recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs^ref. This is a significant piece of research indicating that a single amino acid substitution in the NS1 protein of H₅N₁ avian influenza virus disables the interferon response in infected chickens, a factor contributing to the pathogenicity of the virus. This observation does not necessarily confer enhanced vaccine potential. An NS1-truncated, live-attenuated influenza vaccine is effective in pigs. The modified live-virus (MLV) vaccine completely protected against homologous and partially protected against heterosubtypic virus challenge. Importantly, MLV-vaccinated pigs can be differentiated from naturally infected animals. These results suggest that NS1-truncated MLV vaccines represent a new generation of swine influenza vaccines with great potential for use in other hosts, such as horses, dogs, birds, and humans^ref

a2-6-linked sialic acids receptors were common in the nose and throat, while a2-6-linked sialic acids -- H₅N₁'s preferred site - are found only in type 2 alveolar cells^ref. The Dutch team also found binding to alveolar macrophage and non-ciliated bronchiolar cells in human LRT and that this pattern was most closely mirrored in cat and ferret tissues^ref
Transmission :
• direct contact with infected poultry and their feces or dust/soil contaminated with feces. Birds that survive infection excrete virus for at least 10 days, orally and in faeces, thus facilitating further spread at live poultry markets and by migratory birds. Environmental resistance :
• in water, the virus can survive 2 min at 100°C, 30 min at 60°C, for up to 4 days at 22°C and > 30 days at 0°C
• in bird feces, H₅N₁ virus can survive for at least 35 days at low temperature (4°C or 39°F). At a much higher temperature (37°C or 98.6°F), H₅N₁ viruses have been shown to survive, in fecal samples, for 6 days (Lu et al., 2003. Avian Dis 47:1015-1021)
• if exposed to sunlight : 40-48 hrs
• in glycerin : 1 year
• if exposed to UV light, ether, chloroform, acetone, oxydizing agents, halogens : immediately inactivated
• in frozen material the virus survives indefinitely (years in temperatures as low as -70°C (-94 F)
• it can be killed if meat is cooked properly (70°C) but not at pH = 4.0
Human infections via poultry products -- for example, meat or table eggs -- have never been reported : anyway WHO emphasizes the importance of good hygiene practices during handling of poultry products, including hand washing and prevention of cross-contamination. Some migratory birds leave Siberia and stop by Japan and the 2 Koreas before arriving in Taiwan. Others travel along the Chinese coast and Kinmen before arriving in Taiwan. In 2003, about 100 000 ornamental birds, mainly psittacines (parrots, cockatoo, budgerigars) have been imported from Pakistan, China, and Indonesia to EU : people who have seen the animal holding facilites at London-Heathrow, New York-Kennedy and Amsterdam-Schipol describe warehouses in which every type of bird and other exotic animal are kept cheek by jowl in conditions resembling those in the Guangdong food markets, awaiting trans-shipment. There, poultry can come into close contact with wild-caught birds. The tendency to hide or smuggle especially valuable birds, such as fighting cocks, can also help maintain the virus in the environment or contribute to its further geographical spread. An Oct 2004 laboratory study of domestic ducks infected with several 2004 H₅N₁ viruses shows that, when compared with infections caused by viruses from 2003, domestic ducks are shedding more virus for longer periods^ref. The majority are doing so without showing symptoms of illness. The quantities of virus excreted by healthy-looking ducks approach those excreted by diseased -- and visibly very ill -- chickens. This suggests that domestic ducks might now be acting as a "silent" reservoir for the H₅N₁ virus, which is highly pathogenic for chickens. To date, no evidence has linked human H₅N₁ cases to exposure to asymptomatic domestic ducks. However, the laboratory findings come at a time when some human cases could not be traced to contact with diseased or dead poultry. The concern is greatest in rural areas of affected countries, where free-ranging ducks and chickens often mingle, frequently sharing the same water supplies. The new findings expand on recent evidence, based on characterization of H₅N₁ viruses from southern China, that domestic ducks have played a central role in generating and maintaining H₅N₁, in its highly pathogenic form, in parts of Asia^ref. It might also explain the recurrence of the disease in countries like Thailand, where stamping-out policy without vaccination has been applied. In view of the said findings, the following public health recommendations for affected countries should be applied :
• investigation of human cases should now include possible exposure to apparently healthy domestic ducks, as well as to diseased or dead poultry, as a risk factor for infection.
• advice for people living in an affected area should be revised to include precautions for apparently healthy ducks similar to those for visibly ill poultry.
• ducks should not be kept as pets or allowed to enter households.
• water supplies for human use should not be drawn from open ponds used by domestic ducks and should be stored in ways that prevent contact with ducks.
• once prepared and properly cooked, duck meat and eggs do not pose a risk to human health.
• significant exposure can occur during home slaughtering and preparation prior to cooking, and these risks need to be addressed.
Available data would tend to indicate that human infection is associated with handling and preparing diseased birds prior to cooking. So far, there are no detailed descriptions of the route of infection for human cases of avian influenza nor detailed descriptions of the exact kind of "close poultry contact" human cases have had so far. Such information will help a lot in better designing prevention campaigns addressing high-risk behaviors in at-risk populations. However, from the information available, it is clear that the large majority of human cases have been in rural settings, linked to "handling" diseased poultry. Here, "handling" probably covers a series of high-risk activities such as slaughtering, defeathering, eviscerating, and preparing raw poultry for cooking. The virus is inactivated at temperatures achieved in usual cooking (at least 70°C at the centre of the product), and consumption of properly cooked poultry is not a high-risk activity.  If this were the case, we would see small family clusters of people having shared the same dish. What we do seem to find is mostly individual cases of people who participated in the above-mentioned high-risk activities. They have of course also consumed the poultry afterwards. It would seem strange if in all these cases, they had been the only ones eating the contaminated bird. Normally a chicken dish would be shared among several persons. Along the (often very short) food chain in rural Asia -- between slaughter of an infected bird all the way to consuming it in a cooked dish -- the high-risk activities are the ones in the first phases of the process as described above and not the actual eating of the cooked dish. However, there have been reports of a few cases potentially linked to consumption of raw poultry ingredients (e.g. raw blood-based dishes). It should therefore be reiterated that consumption of any raw poultry ingredients must be considered a high-risk activity and banned. Slaughtering, defeathering, and eviscerating poultry are very messy activities that cannot be done "safely" unless in full protective gear; therefore such activities under traditional Asian rural settings, with obviously diseased birds, must be stopped. If properly explained and enforced by rural populations in affected areas, this would go a long way towards stopping the current pattern of human infections^ref. For unknown reasons, very few cases have been detected in presumed high-risk groups, such as commercial poultry workers, workers at live poultry markets, cullers, veterinarians, and health staff caring for patients without adequate protective equipment. Also lacking is an explanation for the puzzling concentration of cases in previously healthy children and young adults. Paradoxically poultry industry workers and poultry cullers in Southeast Asia have rarely, if ever, exhibited over signs and symptoms oft avian influenza virus infection; human disease being associated mainly with domestic situations and food preparation. All cases of avian influenza in humans have been associated with food preparation and consumption of diseased poultry, or proximity to poultry and pet birds. So far, no human cases have been attributed to contact with free-living species of birds
• limited human-to-human transmission of a HPAI virus is not entirely unexpected, as this has been documented to have occurred on 2 occasions in the recent past (in Hong Kong in 1997 and in the Netherlands in 2003)
• wild birds : reports of their role as the cause of new bird flu outbreaks occur almost daily , but at the present time, there is little evidence available to support such statements. One of the few pieces of published data that addresses the question of H₅N₁ isolation from wild birds is from the work of colleagues in Hong Kong. H₅N₁ was isolated from clinically healthy birds in Penfold Park during the 2002 HPAI outbreak in Hong Kong SAR. In addition to the isolation of H₅N₁ from sick and dying birds at the park, the virus was isolated from apparently healthy birds, include 2 Canada geese (Branta canadensis), one bar-headed goose (Anser indicus) and 2 other geese of unspecified Anser species (Ellis et al., 2004. Avian Pathol 33:492-505). [Initially, the SAR authorities reported to the OIE that the affected population in the Penford Park included resident waterfowl (ducks, geese, and swans) and wild little egrets (Egretta garzetta); no reference was made to tests in clinically unaffected birds. It should be noted that, although the Canada goose and the bar-headed goose are migratory species, these birds were in a captive situation, and so the question of whether they are capable of migration remains unanswered. A further point illustrating the lack of data on the role of wild birds in HPAI H₅N_A transmission is the outbreak in Novosibirsk, Russia. The source of the outbreak has been attributed to wild birds^ref. But, in some of the limited information available on the nature of the Novosibirsk HPAI H₅N₁ virus, as provided by Russia to OIE^ref, the 4 isolates of H₅N₁ from domestic poultry in 2 regions of Novosibirsk are similar, but one sample, which is from a wild duck, clearly has a different PCR electrophoreogram pattern. While other data not included in the report may show that the virus in wild birds is related to those isolated from affected poultry in the Novosibirsk region, the available data suggest that such is not the case, and certainly no data that shows the wild birds were the vector of transmission has been made available at the present time. Movement of birds, including annual migration, is only one of several possible means of dissemination of the HPAI H₅N₁ virus. In many of the areas of recent outbreaks, there is a thriving trade of live birds and poultry products. Some of the areas such as Qinghai in China and Hovsgol, Mongolia are tourist destinations. There is no evidence of sustained human to human transmission at the present time. But because the influenza virus can survive in poultry droppings for up to 2 weeks, movement of people and contaminated farm equipment can rapidly spread the virus from one locale to another. Although much has been made of the recent pattern of spread as indicative of avian migration, many ornithologists have indicated that the spread of H₅N₁ does not fit with known behavior of the bird species in that area of the world^ref): It should be noted that the same pattern of spread can just as easily be seen as from the major routes of human transportation. Outbreaks of HPAI caused by H₅N₁ viruses were reported almost simultaneously in 8 neighboring Asian countries between December 2003 and January 2004, with a 9th reporting in August 2004, suggesting that the viruses had spread recently and rapidly. However, they had been detected widely in the region in domestic waterfowl and terrestrial poultry for several years before this, and the absence of widespread disease in the region before 2003, apart from localized outbreaks in the Hong Kong Special Autonomous Region (SAR), is perplexing. Possible explanations include limited virus excretion by domestic waterfowl infected with H₅N₁, the confusion of avian influenza with other serious endemic diseases, the unsanctioned use of vaccines, and the under-reporting of disease as a result of limited surveillance. There is some evidence that the excretion of the viruses by domestic ducks had increased by early 2004, and there is circumstantial evidence that they can be transmitted by wild birds. The migratory birds from which viruses have been isolated were usually sick or dead, suggesting that they would have had limited potential for carrying the viruses over long distances unless subclinical infections were prevalent. However, there is strong circumstantial evidence that wild birds can become infected from domestic poultry and potentially can exchange viruses when they share the same environment. Nevertheless, there is little reason to believe that wild birds have played a more significant role in spreading disease than trade through live bird markets and movement of domestic waterfowl. Asian H₅N₁ viruses were 1st detected in domestic geese in southern China in 1996. By 2000, their host range had extended to domestic ducks, which played a key role in the genesis of the 2003/04 outbreaks. The epidemic was not due to the introduction and spread of a single virus but was caused by multiple viruses which were genotypically linked to the Goose/GD/96 lineage via the HA gene. The H₅N₁ viruses isolated from China, including the Hong Kong SAR, between 1999 and 2004 had a range of genotypes and considerable variability within genotypes. The rising incidence and widespread reporting of disease in 2003/04 can probably be attributed to the increasing spread of the viruses from existing reservoirs of infection in domestic waterfowl and live bird markets leading to greater environmental contamination. When countries in the region started to report disease in December 2003, others were alerted to the risk and disease surveillance and reporting improved. The H₅N₁ viruses have reportedly been eliminated from 3 of the 9 countries that reported disease in 2003/04, but they could be extremely difficult to eradicate from the remaining countries, owing to the existence of populations and, possibly, production and marketing sectors, in which apparently normal birds harbor the viruses^ref. During early stages of the outbreak, it was argued that the pattern of spread strongly suggested that the virus was carried by people smuggling poultry, a practice reportedly widespread in southeast Asia, rather than by migratory birds. Though there were reports of mass die-offs of rare birds in zoos in Thailand, regular monitoring of migratory birds in Thailand did not reveal the virus. In regions with big outbreaks in poultry, local wild birds were affected; the question remained as to whether their infection did not originate from the domestic birds. Useful information on waterbird populations worldwide can be found on the web-site of Wetland International; the organization has recently published the drafted 4th edition (2005) of "Waterbird population estimates"^ref. Outbreaks of HPAI, which originate in poultry upon transmission of low pathogenic viruses (LPAI) from wild birds, have occurred relatively frequently in the last decade. During our ongoing surveillance studies in wild birds, we isolated several influenza A viruses of hemagglutinin subtype H₅ and H₇ that contain various neuraminidase subtypes. For each of the recorded H₅ and H₇ HPAI outbreaks in Europe since 1997, our collection contained closely related virus isolates recovered from wild birds, as determined by sequencing and phylogenetic analyses of the hemagglutinin gene and antigenic characterization of the hemagglutinin glycoprotein. The minor genetic and antigenic diversity between the viruses recovered from wild birds and those causing HPAI outbreaks indicates that influenza A virus surveillance studies in wild birds can help generate prototypic vaccine candidates and design and evaluate diagnostic tests, before outbreaks occur in animals and humans. Wild birds, predominantly ducks, geese, and shorebirds, form the reservoir of influenza A viruses in nature^ref. Influenza A viruses are subtyped on the basis of the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, expressed on the surface of virus particles. To date, 16 HA and 9 NA subtypes have been detected in wild birds and poultry throughout the world^{ref1, ref2}. Viruses containing HA of subtypes H₅ and H₇ may become highly pathogenic after introduction in poultry and cause outbreaks of HPAI^ref. The switch from a low pathogenic avian influenza (LPAI) phenotype, common in wild birds and poultry, to the HPAI phenotype is achieved by the introduction of basic amino acid residues into the HA0 cleavage site^ref. HPAI isolates have been obtained primarily from commercially raised birds such as chickens, turkeys, quail, guinea fowl, and ostriches^ref. In the last decade, the frequency of detected HPAI outbreaks has increased, with outbreaks of avian influenza A viruses of subtype H₅N₂ in Mexico (1994); Italy (1997) and Texas (2004); H₅N₁ in Hong Kong (1997) and Southeast Asia (ongoing since 1997); H₇N₃ in Australia and Pakistan (1994); H₇N₄ in Australia (1997); H₇N₁ in Italy (1999); H₇N₃ in Chile (2002) and Canada (2003); and H₇N₇ in the Netherlands (2003)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}. Influenza A viruses of subtypes H₅ and H₇ have been frequently detected in mammals^ref. H₇N₇ viruses have been endemic in horses for some time^ref, were transmitted from seals to humans in the USA in 1980^{ref1, ref2}, and were isolated from humans in the United Kingdom in 1996^ref and the Netherlands in 2003^{ref1, ref2}. H₇N₂ and H₇N₃ influenza A viruses were isolated from humans in the USA in 2003^ref and Canada in 2004, respectively. HPAI H₅N₁ viruses circulating in Southeast Asia since 2003 have been detected in at least 108 human cases of respiratory illness, of which 54 were fatal^ref. In addition, these H₅N₁ influenza A viruses have been detected in pigs, cats, leopards, and tigers in Southeast Asia. As a consequence of the relatively frequent zoonoses caused by influenza A viruses of subtypes H₅ and H₇, these virus subtypes are given high priority with respect to pandemic preparedness. Wild birds harbor the LPAI ancestral viruses of HPAI strains of poultry (and mammals). In this influenza A virus surveillance studies in wild birds in northern Europe, numerous influenza A viruses of subtype H₅ and H₇ in Mallards (Anas platyrhynchos) were detected. For each of the HPAI outbreaks that occurred in Europe since 1997, we have found close LPAI relatives in Mallards. Influenza A virus surveillance in wild birds provides opportunities for pandemic preparation; the prototype influenza A viruses obtained from wild birds may guide production of vaccines as well as reagents to develop and validate diagnostic tests. The body of the paper contains a map of migration routes of waterfowl in northern Europe, phylogenetic analysis and antigenic characterization of some 172 samples isolated during the period 1999-2002 in the Netherlands and Sweden. The authors conclude that: Because HPAI outbreaks in poultry find their origin in LPAI viruses present in waterfowl, influenza A virus surveillance in wild birds could function as an early warning system for HPAI outbreaks and as a means to keep panels of reference reagents, required for diagnostic purposes and vaccine production, up-to-date. Wild bird surveillance would also be relevant for HPAI viruses that represent pandemic threats. Minor antigenic and genetic diversity were observed among the HA genes of Mallard influenza A virus isolates and those of HPAI virus strains. This finding implies that the influenza A virus isolates obtained during wild bird surveillance studies may also be prototypic vaccine candidates for human or veterinary use. Limited numbers of prototype vaccine strains, representing both the American and Eurasian genetic lineages of influenza A virus, could be generated to cover a wide range of HPAI strains. Such vaccine seed strains can be produced well ahead of outbreaks in poultry, other animals, or humans. The disadvantage of the minor antigenic differences between the vaccine strain and the epidemic strains will likely be compensated by the immediate availability of the vaccine. An additional advantage of the use of LPAI strains from wild birds as prototype vaccine strains is that they do not contain a basic cleavage site in the HA gene. Before HPAI strains can be used as vaccine candidates, the basic amino acid residues in the HA gene need to be removed by using reverse genetics technology; this would result in an extra modification step, which would consume precious time. Moreover, these vaccine strains can be generated only after an outbreak of HPAI has started^ref. Concern has shifted to the possibility that the spread of influenza virus from waterfowl, where transmission probably occurs predominantly by an enteric route, to domestic (terrestrial) poultry, where transmission may occur predominantly by a respiratory route, a circumstance that may promote the evolution of viruses with enhanced virulence for mammals and a propensity to spread by a respiratory route. There is some circumstantial evidence that such a process may be occurring. The human species has lived more or less in harmony with waterfowl for millennia, but the changed dynamic is the vast increase in recent years of the production and global trading of poultry as a source of food for an ever-expanding human population. It is correct to query the relevance of the spread of avian influenza viruses by migratory birds as a factor in spread of human disease. It is more likely that a novel pandemic virus would spread along trade and communications routes rather than via the migratory pathways of free-living birds. The evidence so far is equivocal. Outbreaks of disease have occurred along migratory routes, but the majority of migrating birds have proven to be free of H₅N₁ virus, and the minority of birds exhibiting disease may be victims rather than carriers of disease (the "dead birds do not migrate" hypothesis). The most westerly H₅N₁-positive bird (recovered in Finland) carried a low-pathogenicity -- possibly indigenous -- virus. The current spate of accounts of detection of H₅N₁-positive free-living birds is not particularly helpful and requires critical evaluation. If HPAI enters a commercial flock, the effects are usually obvious and quickly identified; contact between humans and sick birds is likely to be less intense. Deaths in backyard poultry are rarely investigated by veterinarians. If an epidemic disease enters a village, and birds in a single holding start dying, other owners may rush to slaughter or sell their birds before they have the opportunity to die, but perhaps not before they are infected. Such a situation might enhance human exposures and spread. I am not trying to imply that strict controls or culling should be imposed on village poultry; apart from the logistical problems that would occur, village poultry are too important to those who own them for drastic actions to either be supportable or even feasible. Birds would be hidden or sent elsewhere. What is needed is a heat-stable AI vaccine for village poultry and the means to deliver it. Considering the reported widespread infection with H₅N₁ in SE Asia and the recent findings in Central Asia attributed to migratory birds, Australia and NZ are indeed an enigma: 2 islands endowed with wild aquatic birds, on the migration zone of several flyways in and out of SE Asia, and yet untouched by the subtype ravaging this continent. Since the emergence of the AIV Goose/GD/96 in the province of Guangdong in 1996, it is estimated that approximately 27 million migratory birds have visited Australian shores, approximately 3 million birds per year (according to Wetlands International 2002, Waterbirds population estimates 3rd Ed., Global Series 2002 no 12). Another intriguing aspect is that while both countries are on the migration route of several flyways, Australia has experienced 5 outbreaks in chicken flocks but NZ has experienced none. In the 5 outbreaks in Australia, all in intensive poultry units, the H₇ subtype was involved, and in all 5 outbreaks migratory/aquatic birds were considered the source. This conclusion was largely based on (1) the premise that aquatic wild birds are a significant reservoir of AIV and (2) anecdotal evidence that suggested presence of aquatic wild birds in the vicinity of an infected farm or inhabiting a body of water that supplied water to the infected farm. AIV of the H₇ subtypes were never reported in Australia or NZ in wild waterfowl before, during, or after the outbreaks^{ref1, ref2, ref3}. Several other AIV subtypes were found in wild waterfowl during surveys over the years, but none of those was ever found in the infected chicken flocks. During the last outbreak in Australia (1997), the same AIV subtype which was found in chicken (but of lower virulence) was isolated from farmed emus adjacent to the infected biosecured index chicken shed. The emu as a possible source of infection was largely ignored in favour of the water from the river being the source of the infection in the chicken, despite the fact that it was chlorinated (albeit with fluctuating levels of chlorine). Considering the repeated outbreaks in Australia with H₇ and the uniqueness of emus to Australia, this population should perhaps receive more consideration as a potential reservoir of AIV infection in Australia. While it is acknowledged that absence of evidence is not necessarily evidence of absence of infection with H₇ subtype in Australian and NZ aquatic wild birds, it is highly significant that the H₇ subtypes isolated from the AI outbreaks in Australia between 1985 to 1994 were all phylogenetically delineated from H₇ subtypes found in other regions of the globe and the Australian H₇ subtype formed a separate sublineage^ref (Aust Vet J 2004; 82(6), Jun). Would this delineation persist for such a long period if migratory birds were responsible for the AI outbreaks in Australia? Unlike Europe, Australian and NZ Anatides are considered nomadic within their respective countries and generally do not migrate internationally or intercontinentally. While the European data may point to a strong association between migratory birds and outbreaks in domestic poultry, this has not been consistently the case, for example, in North America, where the infections were, in significant numbers of outbreaks, related to local bird markets. When the spread of the current epidemic in SE Asia occurred, migratory waterfowl were almost instantaneously blamed as the source, although the timing and distribution of several new outbreaks did not fit any known migratory pattern for any species including terrestrial birds (Melville DS & KF Shortridge. Reflection and reaction. Lancet Infect Dis 2004; 4: May). The presence of H₅N₁ in live bird markets as early as 1999 (Hong Kong in geese) and 2001 in Vietnam in Geese imported from China^ref, provided a credible alternative explanation for H₅N₁ outbreaks in domestic poultry. The paper by Nguyen et al (2005) identified the domestic duck as being the major reservoir of the AIV pool in nature and the live bird markets in Asian countries is a suitable environment for reassortment and transmission. Perhaps the Australia and NZ scenario provides another reason to examine the possible association between migratory birds and outbreaks in domestic poultry with an open mind. Requiring a thorough examination of the evidence that links migratory birds or other wild waterfowl and AI outbreaks in domestic poultry is not aimed to weaken or to question the concept of biosecurity. The promotion of the concept of biosecurity and exclusion of wild birds from poultry enterprises should be viewed as a tool to reduce disease risks rather than as an undisputed epidemiological association and acceptance of the direct role of wild birds in all AI outbreaks on poultry farms. DEFRA decided that the UK will not follow the Dutch example of moving all domestic poultry indoors, on the grounds that no H₇N₇ virus appeared in the UK notwithstanding the frequent movement of free-living birds between northern Europe and the UK. A comprehensive review paper titled "Potential risk of HPAI spreading through wild water bird migration", including maps, has been prepared by FAO's avian influenza task force (Rome and Bangkok) and published in issue No 33 of FAO-AIDE-NEWS update on the avian influenza situation, as of 1 Sep 2005. There seems to be no dispute about clinically unaffected aquatic birds, both sedentary as well as migratory, being the reservoir of avian influenza viruses (especially LPAI) that might mutate into HPAI when introduced into susceptible populations of domestic poultry. There is an ongoing debate about the role of migratory birds in the dissemination of the highly pathogenic H₅N₁ strain which has been the causal agent of the pandemic in southeastern and central Asia, and particularly about their potential to spread the agent to other parts of the globe. The debate is related to the observations about the potential of the said strain to clinically affect -- and kill -- several species of migratory avians. All parties seem to agree about the urgent need for enhanced virological surveillance in healthy, free-roaming avians, within and outside the affected areas.
• carnivores can become infected, after consuming infected poultry that succumbed to the disease. :
• Canis familiaris : to date no H₅N₁ clinical cases of dogs have been reported but in an unpublished study carried out in 2005 by the National Institute of Animal Health in Bangkok, researchers tested 629 village dogs and 111 cats in the Suphan Buri district of central Thailand. Out of these, 160 dogs and 8 cats had antibodies to H₅N₁, indicating that they were infected with the virus or had been infected in the past.  The cutoff point in this study is 1:40. The scientists are still debating what the real cutoff point for dogs. They are doing further studies using other methods. Currently they are in doubt about the high percentage of seroreactivity in the dogs. This study was done during the 2nd quarter of 2005 in an area that has a large poultry outbreak. A possible explanation is that the dogs and cats eat sick poultry. While there was a report of a dead cat with H₅ in the 1st round outbreak, there is no report in dogs. Most are asymptomatic.In conclusion, it is likely that dogs can be infected by H₅ but the possibility of spreading the disease to humans is unlikely. However, the role of spreading disease among dogs or to other poultry deserved further study. An equine virus has recently shown up in dogs. This inter-species re-assortment is not uncommon for type A influenza viruses..
• Sus scrofa are known "mixing vessels" for different influenza virus subtypes and therefore present a risk for avian influenza virus re-assorting with a human influenza virus into a strain more apt to infect humans. Regarding the present H₅N₁ subtype, studies conducted in pigs in Vietnam yielded 8 seropositive animals out of the 3000 pigs tested. None of the animals had any clinical signs and it was not possible to isolate any virus
• ruminants are susceptible to influenza viruses but so far mainly H₃N₈ have been identified. Regular vaccination is carried out.
• mice can be infected experimentally but their role in natural transmission has not been established.
Pathogenesis : domestic cats were inoculated with H₅N₁ virus intratracheally (n = 3), by feeding on virus-infected chicks (n = 3), or by horizontal transmission (n = 2) and examined by virological and pathological assays. In all cats, virus replicated not only in the respiratory tract but also in multiple extra-respiratory tissues (digestive tracts, liver, kidney, heart, brain, and lymph nodes). Virus antigen expression in these tissues was associated with severe necrosis and inflammation 7 days after inoculation. In cats fed on virus-infected chicks only, virus-associated ganglioneuritis also occurred in the submucosal and myenteric plexi of the small intestine, suggesting direct infection from the intestinal lumen. All cats excreted virus not only via the respiratory tract but also via the digestive tract. This study in cats demonstrates that H₅N₁ virus infection causes systemic disease and spreads by potentially novel routes within and between mammalian hosts^ref. The susceptibility of carnivores to avian influenza A virus was first recorded in a paper published in the China Journal of Veterinary Science in 2003, demonstrating that tigers had died of influenza A virus infection in China in 2002. Subsequently in 2004 during the H₅N₁ avian influenza outbreak in Thailand considerable mortality occurred among zoo tigers and leopards probably as a result of ingestion of diseased poultry. More recently, deaths of chickens and dogs were reported in a rural village (Bozkurt) in Turkey, although the cause was not determined. In 1997, avian influenza virus H₅N₁ was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H₅N₁ infection prompted us to compare the chemokine responses between influenza virus-infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H₅N₁) (H₅N₁/97), its precursor A/Quail/Hong Kong/G1/97 (H₉N₂) (H₉N₂/G1), and human influenza virus A/Hong Kong/54/98 (H₁N₁) (H₁N₁/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H₅N₁/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus-infected adult MDMs. The strong induction of chemokines and their receptors by AIV, particularly in adult MDMs, may account for the severity of H₅N₁ disease^ref.
Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, virological and immunological studies were performed in 18 individuals with H₅N₁ and 8 individuals infected with human influenza virus subtypes. Influenza H₅N₁ infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H₅N₁ cases and was associated with higher pharyngeal viral loads. Low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels were observed in H₅N₁-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H₅N₁ viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. High viral load, and the resulting intense inflammatory responses, are central to influenza H₅N₁ pathogenesis. The focus of clinical management should be on preventing this intense cytokine response by early diagnosis and effective antiviral treatment. The authors conclude that their observations point to a central role for high viral burden in the pathogenesis of human H₅N₁ disease and suggest that timely suppression of viral replication should remain the mainstay for treatment of H₅N₁ influenza. This is the most unequivocal outcome of their investigation. Detection of virus RNA in blood and gastrointestinal canal are other interesting features of their data. Also their genetic characterization of virus isolated from their patients are consistent with other data demonstrating the importance of the viral polymerase complex in determining H₅N₁ influenza virus virulence in mammals (see: Salomon R., et al., The polymerase complex genes contribute to the high virulence of the human H₅N₁ influenza virus isolate A/Viet Nam/1203/04. J.Exp. 203, 689-697, 2006). To what extent, however, their observations are specific for H₅N₁ influenza is debatable. Their 2 groups of patients were not precisely comparable, the seasonal influenza patients being hospitalized at an earlier stage in the disease process and possibly from urban rather than rural communities. Of greater relevance may be the genetic constitution of their patients, since most humans are vulnerable to seasonal influenza, whereas few contract avian influenza. Furthermore, detrimental chemokine and cytokine cascades can be an accompaniment of other respiratory virus infections, such as severe
respiratory syncytial virus (RSV) infections in infancy.
Symptoms & signs : incubation period ranging from 2 to 8 days and possibly as long as 17 days. Initial symptoms include a high fever, usually with a fever higher than 38°C (100.4°F), and influenza-like symptoms (acute pharyngitis, rhinorrhea, myalgia), dyspnea (100%; 1-5 days after symptom onset). Diarrhea, vomiting, abdominal pain, chest pain, and bleeding from the nose and gums have also been reported as early symptoms in some patients. Diarrhea and vomiting were not reported in Thailand, while diarrhea occurred in 80% of patients from Vietnam : in contrast with the cases reported from Hong Kong, gastrointestinal symptoms were not prominent features. Mild-to-moderate elevations in hepatic transaminases were found in some patients. All patients have developed interstitial pneumonia with chest radiograph changes, and usually very early on the illness. On present evidence, difficulty in breathing develops around 5 days following the 1st symptoms. Respiratory distress, a hoarse voice, and a crackling sound when inhaling are commonly seen. There is hemoptysis. Another common feature is multiorgan dysfunction, notably involving the kidney and heart. All patients had respiratory failure and required intubation a median of 7 days (range: 4-10 days) after onset of illness : there is clinically diagnosed acute respiratory distress syndrome (ARDS) with compatible chest radiograph changes. 2 patients had a pneumothorax. 3 patients required inotropic support for decreased cardiac function; 2 patients had renal impairment as a later manifestation
Influenza H₅N₁ infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H₅N₁ cases and was associated with higher pharyngeal viral loads. Low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels were observed in H₅N₁-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H₅N₁ viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. High viral load, and the resulting intense inflammatory responses, are central to influenza H₅N₁ pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment^ref
Laboratory examinations :
• the 5 laboratories and experts which have been officially recognised to deal with HPAI by WHO are the following:
• Dr D.J. Alexander, Veterinary Laboratories Agency (VLA) Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK
• Dr A.J. Della-Porta, CSIRO, Australian Animal Health Laboratory, Division of Animal Health, Institute of Animal Production and Processing, Ryrie Street, Private Bag 24, Geelong, Victoria 3220, Australia
• Prof. Erhard F. Kaleta, Institut fur Geflugelkrankheiten der Justus-Liebig-Universitat Giessen, Frankfurter Strasse 91, 35392 Giessen, Germany
• Dr. B. Panigrahy, National Veterinary Services Laboratories, P.O. Box 844, Ames, IA 50010, USA
• Dr. Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia, Via Romea 14/A, 35020 Legnaro, Padova, Italy. Capua entered H₅N₁ sequence data from 2 recently infected countries, Nigeria^ref and Italy^ref, into the GenBank database the same day. She also rejected an offer by the World Health Organization (WHO) to join the special secured section at the Influenza Sequence Database at Los Alamos National Laboratory in New Mexico, a select circle of 15 labs that share bird flu sequences on a password-protected Web site created by the WHO Global Influenza Programme in 2004. Capua's lab is a reference center for the U.N. Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (OIE), and officials at those agencies say they support her call. But some scientists say sharing data instantly is complicated by the need for credit, and WHO argues that without some form of confidentiality, some countries would not submit samples at all^ref.
• OIE's 6 reference experts and laboratories for HPAI are the following:
• Dr Ortrud Werner, National Reference Laboratory for Highly pathogenic avian influenza and Newcastle disease, Institute of Diagnostic Virology, Federal Research Centre for Virus Diseases of Animals (BFAV) Insel Riems, Boddenblick 5a, 17493 Greifswald - Insel Riems, GERMANY. Tel: (41) 383.517.152 Fax: (41) 383.517.151
• Dr Ian Brown, VLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UNITED KINGDOM. Tel: (44.1932) 34.11.11 Fax: (44.1932) 34.70.46
• Dr Paul W. Selleck, CSIRO, Australian Animal Health Laboratory (AAHL), 5 Portarlington Road, Private Bag 24, Geelong 3220, Victoria. AUSTRALIA. Tel: (61.3) 52.27.50.00 Fax: (61.3) 52.27.55.55
• Dr B. Panigrahy, National Veterinary Services Laboratories, P.O. Box 844, Ames, IA 50010, UNITED STATES OF AMERICA. Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
• Dr Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia, Via Romea 14/A, 35020 Legnaro, Padova, ITALY. Tel: (39.049) 808.43.69 Fax: (39.049) 808.43.60
• Dr H. Kida, Graduate School of Veterinary Medicine, Hokkaido University, Department of Disease Control, Kita-18, Nishi-9, Kita-ku, Sapporo 060-0818. JAPAN. Tel: (81.11) 706.52.07 Fax: (81.11) 706.52.73
• lymphopenia (<1000/mL) with normal or decreased WBCs, thrombocytopenia (75 500/mL).
• virus isolation in tissue culture : specimens should be collected from suspected cases before or soon after medicines such as oseltamivir are administered : nasal and throat swabs from Vietnamese patients who responded well to oseltamivir were unable to detect the virus 48-to-72 hours after beginning treatment and diagnostic swabs done 2 days or more after the start of treatment may reduce the possibility of detecting the virus. False-negatives can also occur because replication of the virus in these parts of the upper respiratory system may be low at the time the patient was tested : the median interval from the onset of illness to the detection of viral RNA was 5.5 days (range : 2-15)^ref
• viral antigens identified by IFA : while other kits developed in the US, like Directgene, can distinguish between Influenza A or B, scientists working at Shantou University and Xiamen University in mainland China announced on Dec 7 that they had developed a new diagnostic kit able to spot AI subtypes in 30-60 minutes with sensitivity > 90%. It uses direct and IFA, an ELISA, and a colloidal gold-based immunochromatographic dipstick to test for viral antigens and antibodies. The testing kits can use nasal pharyngeal aspirate, throat swab, virus isolate, and serum for detection or confirmation of the infection^ref
• RT-PCR : China has developed a "real-time" RT-PCR method to test for avian influenza A (H₅N₁) virus in 4 hours, much shorter than the 21 days taken by the internationally-accepted detection method recommended by the OIE. Anyway it needs to be carefully controlled with parallel testing by the slower method before it can be pronounced free of false positives and false negatives. The diagnostic test was designed at the Winnipeg lab in Canada using genetic sequencing information from samples of the virus that circulated in Vietnam in the 1st quarter of 2004. Genetic sequences from more recent versions of the H₅N₁ virus are needed to produce more up-to-date primers used in PCR testing. At least part of the problem behind the out-of-date test stems from the fact that Vietnamese laboratories have had limited success in isolating and growing stocks of the circulating viruses in 2005 : for reasons that are not clear, the virus is not growing well in the cell culture medium that was used in the past, so that's been impeding the collection of new sequence information. The National Institute for Hygiene and Epidemiology has been using the Canadian test as a screening tool. All specimens from suspected H₅N₁ cases were tested using the Canadian assay. Samples that tested positive were then re-tested using primers designed at the U.S. Centers for Disease Control in Atlanta. If the 2nd test came back positive as well, the case was considered confirmed. But samples that tested negative during the screening phase were not subjected to the 2nd test. The primers designed in Winnipeg are aimed at 3 different genetic targets on the H₅N₁ virus, including the HA gene. The primers may still be sensitive enough to catch most or all H₅N₁ cases, but that needs to be confirmed. Until we know more about the whole sequence, we won't know really whether they've been missing a lot of cases or not. Still, It's possible the out-of-date primers have generated false negative tests. The Winnipeg lab will now try to design more sensitive primers. Failure to isolate infectious virus may be dependent on the amount of virus present in an infected individual, whereas failure of RT-PCR diagnostic testing can be presumed to be sequence-mismatching as a consequence of genetic drift. But there may be a real decline in the number of cases of avian influenza in humans, rather than a divergence of sequence. Earlier in 2005, Japanese scientists retested a number of specimens that technicians at a lab in Ho Chi Minh City had determined were negative and found that several of the rejected cases were actually positive.
• TaqMan^® Influenza A/H₅ Detection Kit Version 1.0 (Applied Biosystems)
It is nearly 8 months since the WHO last saw data on isolates from infected poultry in Asia. And from the dozens of patients who caught the deadly H₅N₁ strain this year, the WHO has managed to obtain just 6 samples. Affected countries are failing, or refusing, to share their human samples with the WHO's influenza programme in Geneva. The UN Food and Agricultural Organization (FAO) set up a network of labs to collect animal samples in 2004, but it has not received any for months, and the FAO hasn't been sharing what it does have. Such lack of cooperation is a key concern as anxiety about a possible pandemic increases. Human cases are beginning to appear in clusters, which suggests that people are transmitting the virus, older people are falling ill, and milder cases are being reported. Taken together, these trends suggest that the virus is becoming less virulent and more infectious — 2 characteristics typical of pandemic flu strains. With so few samples to work on, it is impossible to judge how worried to be. Of the 6 human samples that the WHO has received from Vietnam, several contain a mutated version of H₅N₁. But that is not enough to indicate a broader change in the strain. It is also impossible for the agency to link this mutation of the virus to possible changes in how pathogenic and transmissible it is in humans. That would require molecular information on hundreds of viruses, and full clinical data on the cases from which they come. Such studies aren't happening. Early signals that the virus is mutating might be picked up from viruses circulating in poultry. The FAO and the World Organisation for Animal Health (OIE) should be collecting samples, but a recent FAO check reveals that the agency has not been receiving any. The WHO's flu programme was last given access to a sample in October 2004, so it has no idea how the virus is changing in birds. Some countries don't have the resources to collect, conserve and securely transport samples^ref, but things that should be happening are not : samples sometimes sit in labs, lacking authorization for export. Countries are wary of sharing viruses with outside laboratories because they fear losing control over information. Authorities in Vietnam are very sensitive as to what they tell the people. They don't want outside groups making pronouncements and these getting into the press without being vetted by the ministries of health and agriculture. Scientists in countries with avian flu often want to work on virus samples first, he adds. They want to get credit for their work, and to use the data to develop their own vaccines. There is a "time lag" in sharing what samples there are with the WHO. But the FAO and OIE are in a difficult position : some countries have provided samples but stipulated that the information can't be shared with the wider community. But the FAO argues it has no recent samples to share. There has been "complacency" at national levels : FAO has now instructed its regional networks to redouble their efforts to acquire isolates. And the FAO and OIE are drafting a standard 'material transfer' agreement to clarify the conditions of use of flu samples, and the intellectual-property rights of the countries that provide them. Meanwhile, the WHO has begun soliciting poultry samples directly from affected countries. Vietnam has recently agreed to ship a large number of poultry samples direct to the WHO flu centre at the US Centers for Disease Control and Prevention in Atlanta, Georgia. There is no refusal to share human samples by Viet Nam or any other country with avian influenza cases : Vietnam has provided > 100 clinical samples in 2005. What influenza experts have expressed concern about is the limited number of viruses derived from these samples. Criticism of the FAO "is not appropriate as FAO does not receive any animal virus samples; instead, FAO, in collaboration with the World Organisation for Animal health (OIE), relies on the joint FAO/OIE network of national and international reference laboratories for the handling of such samples^ref
Therapy : a group of 55 influenza A(H₅N₁) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however 2 strains demonstrated increased IC₅₀ values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H₅N₁) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H₅N₁) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H₅N₁) virus infections^ref.
• oseltamivir phosphate^ref : there is only one option for extinguishing an emerging human pandemic: rapid identification of cases, and treatment of patients and all their contacts with oseltamivir. Modelling studies suggest this should work if action is taken quickly enough^ref. The models predict that there will be only a short window of opportunity in which to act: up to 30 days after a new case is detected. But the Vietnamese health ministry often takes 2-4 weeks to declare cases, according to the WHO. This leaves just a few days to intervene, making it very unlikely that we could stop a virus. Without fast reporting and detection of cases, the world is gambling away the small (and unproven) chance we have of stopping an emerging pandemic in its tracks. Incomplete evidence suggests that there may be a shift in the epidemiology of the disease : more clusters are being seen than last year, older people are now coming down with the diseases, and more cases are milder. Taken together, these characteristics could indicate that the virus is becoming less virulent and more infectious, he says, which could signal the start of a pandemic. The WHO has offered assistance to Vietnam, but this hasn't been accepted fully. The affected countries lack trust in the international agencies, including the WHO. A stochastic influenza simulation model for rural South East Asia was used to investigate the effectiveness of targeted antiviral prophylaxis, quarantine, and pre-vaccination in containing an emerging influenza strain at the source. If the basic reproductive number (R₀) were below 1.60, simulations show that a prepared response with targeted antivirals would have a high probability of containment. In this case, an antiviral agent stockpile on the order of 100,000-1 million courses would be sufficient. If pre-vaccination occurs, then targeted antiviral prophylaxis could be effective for containing strains with an R₀ as high as 2.1. Combinations of targeted antiviral prophylaxis, pre-vaccination and quarantine could contain strains with an R₀ as high as 2.4^ref.  Anyway his model should consider that Tamiflu may be ineffective in treating the new strain (already effective in just 50% of patients in Thailand !), a higher-than-expected R₀ value (up to 3 in previous flu pandemics (B. Cooper et al., poster presented at Influenza Vaccines for the World, Lisbon, 24 to 26 May 2005)), and the possibility of a paucity of antiviral drug/vaccines in areas without facilities to produce generics. Such a worst-casescenario model provides valuable information for resource planning, for example, the number of ventilators, the amount of intensive care, and even funeral facilities that will be required^ref. For R₀ <= 1.5, quarantine levels as low as 50% of people being restricted to the household and neighborhood cluster could possibly contain outbreaks. For R₀ <= 1.6, quarantine levels of 60% or higher could be effective. With an R₀ as high as 2.4, we would need a 90% effective quarantine for containment. For larger values of R₀, containment would not be possible with quarantine measures alone. If we consider an R₀ of 2.4 and total viral resistance to oseltamivir to be the worst-case scenario, then only an extremely tight household and neighborhood cluster quarantine could effectively contain the spread of the virus. Other forms of quarantine also could be effective. The effectiveness of the household and neighborhood cluster quarantine started 14 days after the first case at different values of R₀. Outbreaks that result in a cumulative incidence of  1 case per 1000 are considered contained (horizontal line). The cumulative incidence per 1000 does not always decrease monotonically with increasing intervention coverage for small differences due to stochastic variability


2 recent studies^{ref1, ref2} concluded that containment might work, under certain assumptions. First, the strain must be only moderately transmissible, with a basic reproductive number < 1.8. In other words, this means that containment is likely to work only if each individual infected with the pandemic strain infects an average of < 1.8 additional individuals. If such a strain emerged, the models suggest, containment would require that several further conditions also be met: the emerging pandemic is detected within the first 20 cases^ref or 7–21 days^ref; thereafter, antiviral prophylaxis is delivered efficiently to a geographic area (e.g., to an area with a ten kilometer radius) around 90% of clinical cases within 2 days of symptom onset; the strain remains susceptible to oseltamivir (a growing concern following reports of partially and fully resistant strains among recent H₅N₁ cases^{ref1, ref2}); and adequate antiviral supplies (between 100,000 and 3 million courses) are available. Even if these conditions are met and the next introduction of a pandemic-capable strain is contained, a containment policy alone is unlikely to prevent a pandemic entirely. If a single introduction of a pandemic-capable strain is expected, multiple introductions should also be expected. Each containment effort would likely be more difficult than the last as manpower, antiviral stockpiles, and other scarce resources become depleted. Even if each successive containment effort is no more difficult than its predecessor, the chance of at least one failure increases with the number of introductions. At best, a containment policy will only postpone the emergence of a pandemic, “buying time” to prepare for its effects. Genetic evidence of multiple reassortment events or mutations early in the 1968 H₃N₂ pandemic^ref is consistent with the possibility of multiple introductions, but does not demonstrate this conclusively. We are not aware of such evidence for other pandemics, none of which were contained. However, one might expect that if multiple introductions did occur but none were contained, the evidence for such introductions might be difficult to detect because of depletion of susceptible hosts by the first uncontained introduction, competitive exclusion by the most transmissible of the introduced strains, or other factors. The benefits of containment decrease if we face a growing hazard of introduction, regardless of whether this growth is abrupt or gradual^ref
Another simulation model of influenza transmission in Southeast Asia evaluated the potential effectiveness of targeted mass prophylactic use of antiviral drugs as a containment strategy. Other interventions aimed at reducing population contact rates are also examined as reinforcements to an antiviral-based containment policy. Elimination of a nascent pandemic may be feasible using a combination of geographically targeted prophylaxis and social distancing measures, if the basic R₀ of the new virus is below 1.8. A stockpile of 3 million courses of antiviral drugs should be sufficient for elimination. Policy effectiveness depends critically on how quickly clinical cases are diagnosed and the speed with which antiviral drugs can be distributed^ref. While this analysis is encouraging, Thailand may not have been the best model. Thailand has been relatively successful in controlling avian disease, and the majority of the human cases and deaths have occurred in Viet Nam. There have been no human cases or deaths in Thailand since the end of 2004, compared with 60 cases and 18 deaths during the same period in Viet Nam^{ref1, ref2}.
A large-scale epidemic simulation to examine intervention options should initial containment of a novel influenza outbreak fail, using Great Britain and the USA as examples. Border restrictions and/or internal travel restrictions are unlikely to delay spread by more than 2–3 weeks unless more than 99% effective. School closure during the peak of a pandemic can reduce peak attack rates by up to 40%, but has little impact on overall attack rates, whereas case isolation or household quarantine could have a significant impact, if feasible. Treatment of clinical cases can reduce transmission, but only if antivirals are given within a day of symptoms starting. Given enough drugs for 50% of the population, household-based prophylaxis coupled with reactive school closure could reduce clinical attack rates by 40–50%. More widespread prophylaxis would be even more logistically challenging but might reduce attack rates by over 75%. Vaccine stockpiled in advance of a pandemic could significantly reduce attack rates even if of low efficacy. Estimates of policy effectiveness will change if the characteristics of a future pandemic strain differ substantially from those seen in past pandemics^ref.
A large-scale stochastic simulation model to investigate the spread of a pandemic strain of influenza virus through the U.S. population of 281 million individuals for R₀ (the basic reproductive number) from 1.6 to 2.4. The impact that a variety of levels and combinations of influenza antiviral agents, vaccines, and modified social mobility (including school closure and travel restrictions) have on the timing and magnitude of this spread was modeled : in a highly mobile population, restricting travel after an outbreak is detected is likely to delay slightly the time course of the outbreak without impacting the eventual number ill. For R₀ < 1.9, the rapid production and distribution of vaccines, even if poorly matched to circulating strains, could significantly slow disease spread and limit the number ill to <10% of the population, particularly if children are preferentially vaccinated. Alternatively, the aggressive deployment of several million courses of influenza antiviral agents in a targeted prophylaxis strategy may contain a nascent outbreak with low R₀, provided adequate contact tracing and distribution capacities exist. For higher R₀, multiple strategies in combination (involving both social and medical interventions) will be required to achieve similar limits on illness rates^ref.
Sensitivity to oseltamivir of the NA of H₅N₁ viruses isolated in 2004 and 2005 is about 10-fold higher as compared to earlier H₅N₁ viruses or to currently circulating H₁N₁ viruses. Three-dimensional modelling of the N1 predicted that Glu²⁴⁸Gly and Tyr²⁵²His changes could account for increased sensitivity. Genetic variation in the absence of any drug selective pressure may result in significant variations of sensitivity to anti-NA drugs. Although the clinical relevance of a ten-fold increase in sensitivity of the NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly even in the absence of treatment underline the need for a continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential^ref.
• zanamivir is not used because it is an inhaler and patients in the recent Asian outbreak were too ill to inhale even though laboratory tests show it has some effects on H₅N₁. Manufacturers should think about producing an injectable form. Drugs that are administered intravenously can be better absorbed in patients who have stomach and acidity problems. We don't have to worry about absorption, injections take drugs right in. But if the patient takes them orally, maybe some amounts won't be absorbed or some may be destroyed by stomach acids. Intravenous zanamivir would also ensure faster onset, which would be critical in patients who are seriously ill. Orally taken drugs take 3-4 hours to reach maximum blood concentration and 3-4 hours is very critical in severe cases. But injectable zanamivir takes only 30 minutes to reach maximum blood concentration, this is a huge difference. With an intravenous antiviral, doctors can also vary the doses. Only Germany is storing zanamivir up in large quantities as part of its protection against a possible pandemic, although other European countries and the USA have some stocks. Drug companies have been slow to develop antivirals that could be used to fight bird flu. Tamiflu can be stored in bulk and has a long shelf-life, but Relenza is relatively difficult to stockpile
• A(H₅N₁) is resistant to adamantane derivatives
• empiric broad-spectrum antibiotics for community-acquired pneumonia while the cause of illness was under investigation
• systemic steroids for respiratory distress
• in the event of pandemic influenza, only limited supplies of vaccine may be available. Stochastic epidemic simulations, genetic algorithms (GA), and random mutation hill climbing (RMHC) were used to find optimal vaccine distributions to minimize the number of illnesses or deaths in the population, given limited quantities of vaccine. Due to the non-linearity, complexity and stochasticity of the epidemic process, it is not possible to solve for optimal vaccine distributions mathematically. However, we use GA and RMHC to find near optimal vaccine distributions. An influenza pandemic that has age-specific illness attack rates similar to the Asian pandemic in 1957-1958 caused by influenza A(H₂N₂), as well as a distribution similar to the Hong Kong pandemic in 1968-1969 caused by influenza A(H₃N₂), was modeled. It was found that the optimal vaccine distributions given that the number of doses is limited over the range of 10-90% of the population. While GA and RMHC work well in finding optimal vaccine distributions, GA is significantly more efficient than RMHC. The optimal vaccine distribution found by GA and RMHC is up to 84% more effective than random mass vaccination in the mid range of vaccine availability. GA is generalizable to the optimization of stochastic model parameters for other infectious diseases and population structures^ref. Stockpiles that cover 20%–25% of the population would be sufficient to treat most of the clinical cases and could lead to 50% to 77% reductions in hospitalizations. Substantial reductions in hospitalization could be achieved with smaller antiviral stockpiles if drugs are reserved for persons at high risk^ref
The traditional constituent residues around the active site of NA family are highly conserved in the H₅N₁-NA. However, a partially lipophilic pocket composed by Ala²⁴⁸ and Thr²⁴⁹ in N9-NA becomes a hydrophilic pocket because the 2 residues in the H₅N₁-NA are replaced by hydrophilic residues Ser²²⁷ and Asn²²⁸, respectively. On the other hand, two hydrophilic residues Asn³⁴⁷ and Asn³⁴⁸ in the N₉-NA are replaced by 2 lipophilic residues Ala³²³ and Tyr³²⁴ in the H₅N₁-NA, respectively, leading to the formation of a new lipophilic pocket. This kind of subtle variation not only destroys the original lipophilic environment but also changes the complement interaction between the H₅N₁-NA and DANA^ref.
Prevention :
• TriGene : tests carried out at the Harbin Veterinary Research Institute in China confirm the efficacy of against the H₅N₁ virus. The result shows complete elimination of the H₅N₁ avian flu virus in ten minutes at dilutions of 1:50 - 1:500 and 99.99% reduction at 1:800^ref
• culling, or "stamping out," of infected flocks remains the preferred option for controlling H₅N₁ outbreaks in poultry. However, the present outbreaks in poultry are historically unprecedented in their scale, geographical spread, and devastating economic consequences for both the poultry industry and rural farmers
• targeted vaccination of healthy poultry can be used as a complementary tool for achieving the rapid reduction of the risk posed by the H₅N₁ virus in its avian host -- an objective which supports both elimination of the disease in poultry and prevention of further human cases and deaths
• cullers and transporters should be provided with appropriate personal protective equipment: protective clothing, preferably coveralls plus an impermeable apron or surgical gowns with long cuffed sleeves plus an impermeable apron; heavy duty rubber work gloves that may be disinfected; N95 respirator masks are preferred (US NIOSH certified N-95, European CE P2, or comparable national/regional standards applicable to the country of manufacture); higher-level particulate respirators may also be used (in the successful containment of the outbreak of avian influenza in the Netherlands in 2003, N95 or equivalent respiratory protection was used); standard well-fitted surgical masks should be used if N95 respirators are not available; goggles;  rubber or polyurethane boots that can be disinfected or protective foot covers that can be discarded. All persons who have been in close contact with the infected animals should wash their hands frequently. Cullers and transporters should disinfect their hands after the operation. Environmental clean-up should be carried out in areas of culling, using the same protective measures as above. All persons exposed to infected chickens or to farms under suspicion should be under close monitoring by local health authorities. It is recommended that oseltamivir be readily available for the treatment of suspected avian influenza A (H₅N₁) virus respiratory infections in cullers and farm workers involved in the mass culling. They should also be vaccinated with the current WHO-recommended influenza vaccine (i.e. the current triple vaccine comprising A(H₁N₁), A(H₃N₂), and B serotype strains) as soon as possible prior to anticipated risk exposure (2 weeks are required to develop preventive immunity by vaccination) to avoid simultaneous infection by human influenza and avian influenza and to minimize the possibility of a re-assortment of the virus's genes. Additional health monitoring of chicken cullers, others involved in the process, and their family members should be carried out. These individuals should report any relevant health problems (respiratory complaints, flu-like illnesses, or eye infections) to a health care facility. Persons at high risk for severe complications of influenza (e.g. immunocompromised, over 60 years old, or with known chronic heart or lung disease) should avoid working with affected chickens. Serological surveillance of exposed animal workers and veterinarians is encouraged. In liaison with designated laboratories, full blood and post mortem specimens (intestinal contents, anal and oro-nasal swabs, trachea, lung, intestine, spleen, kidney, brain, liver and heart) of animals (including pigs) should be collected for investigation of new viral isolates. In Japan workers in biological hazard suits gathered, killed, and wrapped in plastic all fowl in the area where the H₅N₂ infection occurred, then buried them in special pits using bulldozers with special air-filtration equipment. But while the USA and other industrialized countries have such equipment, poor countries in Southeast Asia do not, and have been sending out workers with no protection into large flocks of chickens. The WHO estimates that thousands of these workers have been exposed to the disease as a result. The authorities in Vietnam have acknowledged that farmers sold close to 800,000 chickens as food that were supposed to be destroyed; the Vietnamese government has offered as little as 10 cents on the dollar in compensation to farmers who destroy their chickens, and foreign governments have not yet offered to help the impoverished country pay more. Some farms have reported that the flu is killing up to 95% of the chickens even before the remainder are culled, and there have even been reports in Bangkok of deaths of domesticated ostriches, which are raised for their meat and are exceptionally sturdy birds with a very high level of natural immunity to most bird diseases
• management of flight passengers with symptoms of possible avian influenza :
• on a conveyance :
• if flight crew members or other personnel are concerned that a passenger traveling from an area in which avian influenza cases have been reported may be ill with a fever or respiratory illness, they should keep the sick person separated from close contact with others as much as possible. A surgical mask can reduce the number of droplets coughed into the air. Ask the sick person to wear a mask if one is available, provided the person can tolerate it (that is, if the sick person does not have such severe difficulty breathing that he or she cannot use a mask). If a surgical mask is not available, provide tissues and ask him or her to cover the mouth and nose when coughing. When a sick person is unable to wear a surgical mask, personnel should wear surgical masks when working directly with that person.
• personnel should wear disposable gloves for direct contact with blood or body fluids of any passenger. (However, gloves are not intended to replace proper hand hygiene). Immediately after activities involving contact with body fluids, gloves should be carefully removed and discarded and hands should be cleaned. Gloves must never be washed or reused.
• the captain of an airliner bound for the United States is required by law to report the illness to the nearest U. S. Quarantine Station prior to arrival or as soon as illness is noted. Quarantine officials will arrange for appropriate medical assistance to be available when the airplane lands and will notify state and local health departments and the appropriate CDC Headquarters officials. Quarantine officials will work with the airline and local and state health departments to assist with medical transportation of the patient upon arrival, disease control and containment measures, passenger and crew notification and surveillance activities, and airline disinfection procedures.
• on arrival :
• for Transportation Security Administration (TSA), Bureau of Customs and Border Protection (BCBP), and other personnel interacting with passengers arriving from areas with avian influenza, CDC does not recommend protective measures beyond those already in use for interacting with the general public. As with all infectious illnesses, the first line of defense is careful hand hygiene. As a general practice, personnel should wash hands frequently with soap and water or use an alcohol-based rub if hands are not visibly soiled. Personnel who have to detain or assist a passenger who appears to have a respiratory illness, and who may have traveled from an area with avian influenza, should try to keep him or her separated from the other passengers as much as possible and immediately contact the appropriate authorities, such as the U.S. Quarantine Station with local jurisdiction, and Emergency Medical Services (EMS). In the interim, provide the ill passenger with a surgical mask if one is available to reduce the number of droplets coughed into the air and if the passenger can tolerate a mask. If a surgical mask is not available, provide tissues and ask the sick person to cover his or her mouth and nose when coughing. When an ill passenger is unable to wear a surgical mask, personnel should wear surgical masks when working directly with the sick person. Personnel should wear disposable gloves if touching blood or body fluids. (However, gloves are not intended to replace proper hand hygiene.) Immediately after activities involving contact with body fluids, gloves should be carefully removed and discarded and hands should be cleaned. Gloves must never be washed or reused.
• management of ill crew : flight crew members and ground personnel who become ill and who believe they have been exposed to avian influenza should take the following precautions:
• if illness onset occurs while traveling away from home, notify employer for assistance with locating a health care provider. Let employer know you are concerned about possible exposure to avian influenza, and ask about your health-care options.
• if illness onset occurs while outside the United States, the U.S. embassy or consulate can also provide names and addresses of local physicians. Do not travel while sick, and limit your contact with others as much as possible to help prevent the spread of any infectious illness. If a visit is planned to a doctor's office, clinic, or emergency room, tell the healthcare provider in advance about your possible exposure so that arrangements can be made, if necessary, to prevent transmission to others in the health-care setting.
• if illness onset occurs after return home, contact your healthcare provider and tell him or her what your symptoms are and the countries you have visited before going to the doctor's office or emergency room. Precautions can then be taken, if necessary, to prevent transmission to others in the healthcare setting.
• inactivated or subunit vaccines. The principal aim of vaccination of those involved in culling of avian influenza A (H₅N₁) virus-infected poultry, using the current human (H₁N₁/H₃N₂) vaccine, is not only to provide some limited cross-protection, but rather to reduce the potential for simultaneous infection of susceptible individuals by both avian and human strains of influenza virus. The intention is rather to reduce the potential for generation of novel strains by reassortment of genome subunits between human and avian virus, producing viruses that may be the precursors of human epidemic and pandemic influenza viruses.
• the European Union (EU) responded initially to the outbreaks in these countries by banning imports of poultry meat and eggs. The Standing Committee for Food Chain and Animal Health has now agreed to extend the ban on imports into the EU from Thailand of poultry meat and meat products, wild and farmed feathered game and eggs, for 6 months until 15 Aug 2004. This ban is subject to continuous review with a view to amend it if the situation allows this. No specific ban has been placed on imports of these products from other affected countries (including China, Cambodia, Japan) as importation of these products is not allowed for other reasons. The ban does not apply to Thai poultry meat products from birds slaughtered before 1 Jan 2004 or to products which have been heated to over 70°C, as these pose no risk for disease introduction. The EU has also banned the import of live pet birds from Asian countries affected by avian flu. These birds were already subject to a 30-day quarantine period on entering EU member states, and compulsory testing for avian influenza, but in the light of the recent developments, imports have been suspended entirely as a precautionary measure. Imports of unprocessed feathers are also banned. In 2003, almost 100 000 pet birds (most of them psittacines: parrots, cockatoos, and budgerigars) were imported from Asia; mainly from China, Pakistan, and Indonesia
• travel advice issued by the health ministries of various EU member states has included the following, based on original recommendations from the WHO :
• travellers should avoid contact with live poultry, live poultry markets, and farms. It is known that large amounts of the avian flu virus are excreted in infected bird droppings and that the virus can persist in the environment for a long time, therefore contact with surfaces with which bird or animal droppings have also had contact should be avoided.
• because of the good virus viability, hygiene measures such as frequent washing of hands should be strictly observed, including after any contact with animals.
• poultry meat or products and eggs should only be consumed in the affected areas if they have been thoroughly cooked, i.e. have reached an internal temperature > 72°C
Recommendation to treat asymptomatic cases with oseltamivir demands vigorous discussion in the scientific community. 2 pertinent pieces of information may be: 1) lack of transmission from such individuals in Hong Kong and 2) relatively frequent asymptomatic infection in the Netherlands. Rather than treating all such patients, WHO and national governments should consider studying the duration during which such individuals carry the virus in their respiratory tract and the quantity of virus found, as well as whether any of them develop symptoms after a period of asymptomatic carriage and the risk of transmission to others, if any. This would enable a more well thought out response in the future. Instead, treating all asymptomatic cases means that such data cannot be obtained. In addition, one must consider the risk of selecting for oseltamivir-resistant strains by treating those who may not require treatment, as well as the impact on the limited supply of this drug should many asymptomatic individuals be treated based on this recommendation. It is unclear whether such treatment decreases the length of asymptomatic carriage and/or whether it would have an effect in lessening person-to-person spread without the selection of resistant strains. 28-59% of healthcare worker influenza was subclinical^ref : this data, in a human influenza outbreak, suggests that asymptomatic infection could be common. How that relates to HPAI is unclear at this time.
The HPAI A (H₅N₁) virus requires Biosafety Level (BSL)-3⁺ laboratory conditions for certain procedures. However, clinical specimens can be tested by PCR assays by using standard BSL-2 work practices in a Class II biological safety cabinet. Since Jan 2005, in BSL-3-enhanced labs, researchers from the Influenza Branch of the CDC in Atlanta will hybridise avian influenza A (H₅N₁) virus and human influenza viruses in a process known as sub-unit reassortment. Viable offspring will be tested in animals thought to be good surrogates for humans, to see whether the viruses can infect, can be transmitted easily from infected animals to healthy ones, and to note the severity of disease each provokes. In other words, the CDC researchers will be deliberately engineering viruses of pandemic potential. It's high-risk but crucial work. Influenza A (H₃N₂) virus, the strain that has been responsible for most human influenza in recent years, will be the 1st priority. However, Southeast Asia has recently seen activity with influenza A (H₁N₁) virus, a mild modern descendant of the strain that caused the Spanish flu of 1918. So the CDC has been sequencing H₁N₁ viruses recently isolated in Thailand to be able to work with it as well. Reassortment studies can be performed 2 ways : scientists can use reverse genetics, a procedure that allows them to custom-tailor a virus with a predetermined constellation of genes from each parent virus. The other alternative is the classical way -- simultaneously infecting tissue culture with H₅N₁ and H₃N₂ viruses and seeing what results. That approach is more time-consuming and laborious, but more closely mimics evolution of influenza viruses in nature.
No attempt has ever been made to alter the natural course of a pandemic near its start. The WHO identified 4 trigger points for attempting rapid containment of a virus:
• moderate-to-severe respiratory illness or death in 3 or more healthcare workers with no known exposure to H₅N₁ other than contact with ill patients, and laboratory confirmation of H₅N₁ infection in at least one of those workers.
• moderate-to-severe respiratory illness or death in 5 to 10 people with evidence of human-to-human transmission to at least some of them, and lab-confirmed H₅N₁ diagnoses in more than 2 of them.
• compelling evidence that more than one generation of human-to-human transmission has occurred.
• isolation of a novel virus with avian and human genes, or a virus with an increased number of mutations not seen in avian isolates, from one or more people with moderate-to-severe respiratory illness, along with epidemiological evidence of a change in transmission pattern.
The WHO also identifies situations in which rapid containment should not be tried: when studies fail to confirm H₅N₁ or another novel influenza A virus; when the affected population is too big to provide for basic needs and emergency care during containment; and when more than 4 to 6 weeks have passed since the initial cluster was identified. Roche stands ready to ship 1.5 million doses of oseltamivir stored in the USA and another 1.5 million doses stored in Switzerland^{ref1, ref2}. However, the activity of the last 2 months in countries throughout Asia, Europe, and Africa only further supports the fact that early recognition and documentation of an emerging pandemic is going to be difficult if not impossible
Images.
2 hybrid synthetic influenza viruses made by combining a 1997 H₅N₁ avian flu virus with a human H₃N₂ flu virus turned out to be no more contagious and less pathogenetic in ferrets than the natural H₅N₁ virus. H₃N₂ reassortant virus with avian virus internal protein genes exhibited
efficient replication but inefficient transmission, whereas H₅N₁ reassortant viruses with 4 or 6 human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H₃N₂ surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H₅N₁ virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H₅N₁ viruses may require further adaptation to acquire this essential pandemic trait. Later versions of both H₅N₁ and H₃N₂ will be used to make further hybrids for testing in ferrets^ref
Web resources :
• WHO Rapid Advice Guidelines for pharmacological management of sporadic human infection with avian influenza A (H5N1) virus
• Avian influenza at WHO : fact sheet Fri 20 Jan 2006
• Avian Flu at CDC
• Nature web focus : Bird flu
• Defra's Protocol for the collection and submission of samples, including public health advice
• H₅N₂ subtype
Epidemiology :
• LPAI :
• South Africa
• South Korea : 2,000 deaths were reported on Dec 2004 at a duck farm in Gwangju (some 330 km south to Seoul), Chunchon, Kangwon Province. All 9000 ducks on the farm were slaughtered and buried Wed 22 Dec 2004
• Japan : > 40 infected farms since June 2005. There were about 10.14 million chickens at poultry farms in Ibaraki Prefecture as of February 2004, the largest population of all prefectures. Of those, about 5.78 million were raised at poultry farms where bird flu infections have since been confirmed. As of Thu 29 Dec 2005, about 2.39 million chickens had been destroyed, with about 100 000 more slated to be killed. The remaining 3.29 million will be raised in isolated poultry farms
• 800 of 25,000 chickens at a poultry farm in Mitsukaido City, northeast of Tokyo, Ibaraki Prefecture, died between April and June 2005. On 27 Jun 2005, the authorities began culling all of 25,000 chickens currently kept at the infected farm and disinfecting the site. Currently, the number of eggs laid by chickens at the farm has recovered to normal, and there have been no more mass deaths of the birds. Eggs from the infected farm have been shipped to the market through distributors in Saitama Prefecture and retailers in Tokyo. Typically, avian influenza of low pathogenicity may be, at times, clinically expressed by an ephemeral drop in egg production without further noticeable symptoms. Such cases may escape immediate diagnosis to be detected later by serological examinations. Blood tests were conducted on chickens at all 16 farms within a 5-kilometre radius of the farm with the outbreak, which are banned from shipping any birds or eggs until officials can confirm there are no further infections. On Tue 28 Jun 2005, lab tests found chickens at the 5 farms located about 0.3 miles [0.5 km] from the infected farm had developed antibodies to the virus in their blood. With no virus found at the other farms, officials believe there is no need for culling at the 5 farms where birds have avian flu antibodies. Chickens at the remaining 11 farms tested negative. Another farm tested positive on Jul 11 and 8500 chickens were culled
• on Jul 31 2005 antibodies were found in 3 outbreaks in the Ibaraki prefecture (2 in Mitsukaido city since 24 and 26 Jun, 1 in Bando city since 26 Jun), caused by an H₅N₂ LPAI virus : 115,700 birds were culled^ref
• chickens at a farm in Konosu, Tokyo's neighboring Saitama Prefecture in east Japan, tested positive on Aug 17, 2005 : the farm's 98 300 chickens will be culled. 1 of 3 Konosu's supplying farms in Ishioka, 80 km northeast of Tokyo (1.11 million chickens in 12 houses) tested positive on Aug 22 : some 100 000 chickens will be culled. The distance between the previous reported outbreak in Ishioka, Saitama province, and the location of the current outbreak, Konosu, Ibaraki province, is 44.0 miles (70.0 km)
• 5 closed-system poultry compartments of the Ogawa farm GP Center of Aikeien Co. Ltd in Nakanobu district (5 km southeast of the Minori farm, where antibodies against H₅ had been detected on 22 Aug 2005), Ogawamchi town, Higashi Ibaraki county, Ibaraki Prefecture, tested positive on Aug 27^ref. Antibody tests showed Tue 30 Aug 2005 that chickens at 7 more farms in Ibaraki Prefecture may have been infected with a bird flu virus of the H₅ strain in the past. Chickens at 2 farms near the 7 in the town of Ogawa have already tested positive for antibodies to the virus. Japan had detected bird flu cases in 28 farms in Ibaraki and one farm in Saitama since June 2005, by serological tests that indicated an H₅ past infection. In 7 farms, a virus was detected. The agriculture ministry has announced plans to cull about 1.5 million chickens following an outbreak of avian flu at poultry farms in Ibaraki and Saitama prefectures. Officials said it was a relatively weak strain of bird flu and that 504 000 chickens had already been destroyed. An additional 1.024 million birds are to be killed to prevent the disease from spreading. The area of infection has 30 farms with 4.14 million hens. 1st admin division / Lower admin division / Type epidemiological unit / Date outbreak began (2005) / species / Number of animals in outbreak: susceptible / cases / deaths / destroyed / slaughtered^ref
• 1. Ibaraki / Ishioka city / farm / 22 Aug / avi / appx 1 100 000 /--/--/ appx 100 450 /--
• 2. Ibaraki / Minori town / farm / 22 Aug / avi / appx 789 600 /--/--/--/--
• 3. Ibaraki / Namegata city / farm / 1 Sep / avi /--/--/--/ appx 40 000 / --
• 4. Ibaraki / Namegata city / farm / 1 Sep / avi /--/--/--/ appx 50 000 / --
• 5. Ibaraki / Namegata city / farm / 1 Sep / avi /--/--/--/ appx 28 601 / --
• 6. Ibaraki / Ogawa town / farm /25 Aug / avi / appx 300 000/--/--/--/--
• 7. Ibaraki / Ogawa town / farm /27 Aug / avi /--/--/--/ appx 90 000 / --
• 8. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 35 000 / --
• 9. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 30 000 / --
• 10. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 15 000 / --
• 11. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 25 000 / --
• 12. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 49 000 / --
• 13. Ibaraki / Ogawa town / farm /30 Aug / avi /--/--/--/ appx 350 000 / --
• 14. Ibaraki / Ogawa town / farm /30 Aug / avi / appx 240 000 /--/--/--/--
• 15. Ibaraki / Ogawa town / farm / 1 Sep / avi /--/--/--/ appx 80 000 / --
• 16. Ibaraki / Ogawa town / farm / 1 Sep / avi /--/--/--/ appx 30 000 / --
• 17. Ibaraki / Ogawa town / farm /1 Sep / avi / appx 90 000 /--/--/ appx 40 000 /--
• 18. Ibaraki / Ogawa town / farm / 1 Sep / avi /--/--/--/ appx 29 000 / --
• 19. Ibaraki / Ogawa town / farm / 3 Sep / avi / appx 180 000 /--/--/--/--
• 20. Ibaraki / Yasato town / farm / 8 Sep / avi /--/--/--/ appx 30 000 / --
Gene sequencing by the National Institute of Animal Health showed that the Japanese virus had 94-97% identity with virus strains from Mexico and Guatemala, too far for a migrating bird to carry into Japan. It is also different from other Central American strains of the virus previously found in other parts of Asia. If large numbers of birds, in different farms (probably administratively inter-related), demonstrate a similar level of antibodies against a common antigen, vaccination might be the etiological factor.  The use of an inactivated vaccine will cause serological reactions, but (if properly inactivated) not lead to virus isolation. The official Japanese follow-up report mentioned virus isolation among the laboratory findings. Hence, the possibility of a vaccine-related incident seems remote, unless a deficient, poorly inactivated vaccine (or a live vaccine?!) was used. On 9 Sep 2005, the Japanese government decided to ask Mexico to provide information of unauthorized vaccine. There was unconfirmed information of usage of unauthorized vaccine in Mexico. On Oct 29 2005 the Japanese Government issued an interim report on the origin of the avian influenza outbreak in the Ibaraki Prefecture in August 2005. The main findings are:
• the virus is suspected to have been spread by a vehicle from a meat processing plant which visited several chicken farms, later found infected, on the same day.
• the virus was found to have 97% identity with a Guatemalan strain from 2000-2002.
• no officially authorised and commercially available AI vaccine is known to include the said virus strain. It might be concluded that the vaccine, illegally applied in Japan, might have been an unauthorised one, produced abroad and smuggled into Japan.
The vaccine involved in the Ibaraki outbreak included a live H₅N₂ virus, which means a deficient inactivation process and lack of post-production testing. Control of production, testing, and distribution of animal vaccines is an essential part of animal disease control programmes; governments are responsible for their approval and certification. Chapter 1.5.2. of OIE's Terrestrial Animal Health Code deals with risk analysis for veterinary vaccines. The specific requirements for each vaccine are laid down in OIE's Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. A new version of chapter 2.7.12 thereof, dealing with avian influenza, was adopted by the General Session in May 2005^ref. Japan experienced a similar situation in August 2004, when the Ministry of Agriculture discovered that an outbreak of classical swine fever in Kagoshima Prefecture had been caused by an unauthorized vaccine. In view of the vital role vaccines play in disease control and the risks related to the use of unauthorised vaccines, intensification of law enforcement, coordinated internationally, deserves prioritisation. There is a much simpler explanation than that. Just look for an illegally trafficked parrot or other exotic bird -- not necessarily of a Central American variety, since the Central American traffickers also relay birds from South American, Africa, and the Caribbean. Japan is among the major destinations of illegally smuggled exotic birds.
A serosurvey showed that up to 77 out of 300 asymptomatic people (65 employees of farms and 5 prefectural government employees who were engaged in epidemic control work in Ibaraki, and 7 poultry farm employees in Saitama) are suspected of having been infected with the H₅N₂ strain of bird flu in the pas based on neutralizing antibody to the avian influenza virus strain A/Ck/Ibaraki/1/2005 (H₅N₂) (15 experienced > 4-fold increases in titers, 62 with a titer value > 40x), informed sources said on Sat 7 Jan 2006. They showed no symptoms and there is no possibility of secondary infection. Chickens at some 40 farms in central or south west part of Ibaraki Prefecture have been infected with the H₅N₂ strain of bird flu since June 2005, but the human infections probably took place before the infections were detected in chickens in the areas. The people were probably exposed to the virus while dealing with chickens and their excrement without taking proper preventive measures. This is the 1st time in the world that possible infections of humans by the H₅N₂ strain was reported^ref.
On Jan 16, 2006, a virus of the H₅ strain was detected among chickens at Moriya farm in Ibaraki prefecture, about 65 miles (104.6 km) north of Tokyo : authorities will cull another 770 000 birds
On Feb 27, 2006, 3 veterinarians at IKN Egg Farms Co, and an IKN employee were arrested with the accuse of having violated the Domestic Animal Infectious Diseases Control Law, which requires reporting any suspected contagious diseases in poultry. They also suspect an antibody test conducted at the National Institute of Animal Health in Tsukuba, Ibaraki Prefecture by a veterinarian at the request of the IKN vets was positive^ref.
• Zimbabwe : an outbreak was detected in Dec 2005 on 2 ostrich farms in Matabeleland North in the country's west. South Africa in 2004 had an outbreak of H₅N₂ among ostriches that led to around 26,000 of the birds being culled. It declared itself bird flu-free in September 2005, and the European Union in November 2005 lifted a ban on the import of South African ostriches and their meat.
• Denmark : an outbreak was reported on 1 Jun 2006 in a hatchery for farmed game birds (mallards), ducks and geese at Tommerup in Funen county : there were approximately 19 750 mallards (day-old to 5-weeks-old), 1200 [other] ducks (day-old to 2-weeks-old) and 1600 geese (day-old to 5-weeks-old). There were also 6 adult geese and 14 adult ducks^ref. In July 2006, another outbreak was reported in a hatchery for 25,000 farmed game birds (mallards and pheasants), ducks, geese and different species of ornamental birds at Loevel^ref in Viborg county^ref.
• HPAI :
• South Africa : an outbreak was reported in the Eastern and Western Cape between 2004 and 2005 was eradicated after a quarantine was imposed and > 26,400 birds were culled at 37 farms. A similar outbreak at Riversdale, the farm near the southwestern coastal town of Mossel Bay, in Western Cape province in an ostrich rearing farm (58 ostriches) aged 4 months on 1 Jul 2006^ref. A new seropositive farm has been identified in the Western Cape on a farm in Sandfontein, Riversdale (lower administrative division 34o 02' 06" S; 21o 38' 31.9" E^ref) on 19 Jun 2006. Only one additional seropositive farm (with 2 seropositive epidemiological groups) situated immediately adjacent to the index farm was detected in the 10-km-radius zone. This finding indicates that all these 3 epidemiological units (index farm and 2 epidemiological units on the adjacent farm) have been exposed to the same risk factors, probably related to clustering of wild birds at the nearby river confluence rather than being indicative of aerial spread. Sequencing of the outbreak virus at the Onderstepoort Reference Laboratory for avian influenza indicated the presence of an H₅N₂ virus not identical to the H₅N₂ ostrich isolate of 2004 from the Eastern Cape, eradicated successfully at that time. Thus, the virus described in the OIE immediate notification report dated 3 Jul 2006 is a new strain of H₅N₂ that is not identical to that found in 2004. This implies that it is a completely new infection and that no chronic carrier situation exists in ostriches for this virus in South Africa. The Eastern and Western Cape are home to some of the country's largest ostrich farms -- an industry that brings in R1.2 billion [USD 167 million] in export earnings annually. South Africa supplies about 70% of the world's ostrich meat -- about 950,000 tons a year. Ostriches are mostly used for their meat and feathers, and to make oil and leather. Ostrich leather is used in the production of clothing items, bags and luxury vehicle interiors (Sinclair, M, Bruckner, GK & Kotze, JJ. Avian influenza in ostriches: epidemiological investigation in the Western Cape Province of South Africa. Veterinaria Italiana, Vol 42 (2), April-June 2006)^{ref1, ref2}.
• Mexico : during 1994/5 and in Dec 2005 in Chiapas
• Italy (northern) : an HPAI A/H₅N₂ virus, which caused a serious outbreak in 1997/1998, was found -- in phylogenetic analysis of the HA genes -- to cluster with the A/H₅N₁, HPAI Hong Kong strains which had infected humans^ref. LPAI A/H₅N₂ was notified to the European Commission on 18 Apr 2005 concerning 10 turkey flocks in the province of Brescia. Italian authorities were now expected to carry out vaccinations in the area in question, the official said. Earlier on Wed 20 Apr 2005, Russia's Agriculture Ministry said it had suspended imports of poultry and poultry products from Italy to prevent the spread of bird flu. The Russian ban also applies to poultry meat products subjected to thermal treatment.
• Taipei China : 2 outbreaks of LPAI (based on genetic sequencing (PQREKR*GLF) and IVPI=0.0) in the central county of Changhwa, Chiayi, and Tainan Prefectures (first reports 15 Jan 2004 : smuggled bird-flu vaccines were likely to be involved) : officials ordered a pet-bird farm in Taiwan's southern Tainan County to kill about 300 birds, including Swinhoe's pheasants - a once-endangered indigenous bird with a short white crest and a blue head - after test results showed some of the birds were infected. The virus was discovered again from samples sent in by the farm in the southern Pingtung county on 1 Mar 2004, where 12 000 chickens were culled. The outbreak was promptly eradicated by the application of stamping-out policy (383,852 birds since 15 Jan 2004) without vaccination. According to their last follow-up report to the OIE^ref, an extensive targeted surveillance has not detected any evidence of the virus since 9 Mar 2004. Taiwan produced chickens and eggs worth T$37 billion (US$1.1 billion) in 2002, contributing about 0.4% to GDP. H₅N₂ LPAI virus had been detected on 2 farms during January 2004. The outbreak was promptly eradicated by the application of stamping-out policy without vaccination. According to their last follow-up report to the OIE^ref, an extensive targeted surveillance has not detected any evidence of the virus since 9 Mar 2004.
• USA : HPAI (so defined based entirely on sequencing according to OIE molecular criterion, but is not virulent for experimentally inoculated chickens throughout the 10-day observation period) was detected in a flock of 6608 broiler chickens raised for live markets in Houston, TX (where ducks commonly have AI), in Gonzales County near San Antonio, TX began on Feb 15 2004 (first detected on Feb 17, reported on Feb 23; the 1st such case in 20 years). The European Commission therefore decided to suspend the imports of live poultry, ratites, farmed and wild feathered game, hatching eggs, eggs for human consumption, and birds other than poultry (pet birds) from all of the USA. The EU is a major importer of eggs and day-old chicks from the USA until 23 Mar 2004. In 2003, approximately 9 million eggs for a value of € 20 million have been imported. This represents about 25% of the total imports of eggs in the EU. In the same year, approximately 450 000 day old chicks for a value of € 2.5 million (about 50% of the total imports in the EU) have been imported from the USA. The EU does not import poultry meat from the USA, because the current treatment of poultry carcasses in the USA is not accepted by the EU.
H₅N₃ subtype

Epidemiology : on 19 Oct 2005, during regular monitoring activities conducted at Ribnjak 1905 fish pond, samples were taken from 26 wild ducks (Anas platyrhyncos) and sent to the Poultry Centre of the Croatian Veterinary Institute in Zagreb for analysis. Laboratory testing (after 3 passages in chicken embryos) revealed that 2 samples were positive for avian influenza virus subtype H₅, but the genetic tests revealed negative results for N1. The amino-acid chain (PQRTRGL) does not point to a virus of high pathogenicity. The nucleotide sequence (500 base pairs that encode for hemagglutinin) resembles that of influenza virus type A (Anas platyrhyncos/Chany Lake/9/03 [H₅N₃]) isolated in Western Siberia. Previously, in December 2005, investigators found an H₅N₁ in swans from the same pond. December's H₅N₁ was fingerprinted and found to be highly similar to virus subtypes previously identified in Qinghai lake (China), Turkey and Romania
• H₅N₉ subtype
Epidemiology : LPAI strains detected in Canada in 1966 and in 2 birds in the western province of British Columbia, in Nov 2005
• H₆ has healthy reservoir in water Aves spp.
• H₆N₁ subtype
• H₆N₂ subtype was found on Jan 2006 in a wild mallard duck that was shot in West Lothian, Scotland^ref
• H₆N₄ subtype
• H₆N₅ subtype
• H₆N₇ subtype
• H₆N₈ subtype
• H₆N₉ subtype
• H₇ infects seals (Phocidae) and Equus caballus and has healthy reservoir in water Avesspp.. Both low- and high-pathogenic strains exist, depending at least partly on the amino acid sequence at the cleavage site in HA1/HA2.
Epidemiology :
• Pakistan : 2 million chickens died and > 4 were slaughtered since November 2003 (first official notification 28 Jan 2004) to Mar  in 3 outbreaks at Poultry Estates 1 & 2, Korangi, Karachi, and in adjacent areas of Angara Goth, Karachi, due to H₇ and H₉ strains. Mortality ranged from 50 to 80%. All the remaining birds at affected farms have been quarantined and vaccinated using H₇ and H₉ strains. According to government estimates, chicken sale has plunged by > 50% and the prices have registered a decline > 40%
• Lebanon : on Mar 30 2006, the laboratory of the American University of Beirut found H₇ on several samples of domestic poultry from the South of Lebanon
• H₇N₁ subtype (HPAI) : a reverse genetics human vaccine was developed in 2005 by European funded project Preparing for an influenza pandemic (FLUPAN) (factsheet)^ref
• H₇N₂ subtype (LPAI), which has never been known to cause harm to humans

Epidemiology : USA :
• February 2004. The "Delmarva" region, which lies between the Chesapeake Bay and the Atlantic Ocean, consists of parts of Delaware, Maryland, and Virginia and produces about 576 million birds a year for consumption. Poultry in Maryland accounted for USD 440 million or 31% of the state's $1.4 billion agriculture industry in 2002 (293 million broilers). In addition, > 60% of the state's cropland is dedicated to grains for poultry feed. There are approximately 1100 poultry farms on Maryland's Eastern Shore. Maryland's last outbreak of AI was in 1993 among game birds on a Queen Anne's county farm. US chicken producers export > 15% of their birds annually for > 1 billion dollars in revenue. In the wake of the findings in Delaware, China banned all US poultry imports, and Russia, the largest market for US poultry, banned all poultry from Delaware. Japan, Hong Kong, South Korea, China, Mexico, Poland, Singapore and Malaysia have placed various restrictions on US poultry-- a loss of USD 9 million a week. But Russia, the biggest U.S. customer, has banned only Delaware and Texas chicken thus far, so processors can substitute chickens from other states. In all, the U.S. poultry industry exports USD 1.5 billion a year worth of meat. Chicken accounts for 1/3 of US meat consumption. The government estimates each American will consume 82.5 pounds of chicken in 2004. The US broiler industry sends > 8.6 billion birds to slaughter annually. The chickens on commercial poultry farms generally are owned by large poultry companies, known as integrators. These companies, such as Perdue Farms and Tysons Foods Inc., pay farmers roughly USD 230 per 1000 birds for raising each chicken to maturity. When a flock must be destroyed because of illness, the farmer loses that payment. Farmers do not tend to be insured for such occurrences and though some integrators offer farmers a special disaster payment if a flock must be destroyed, it is "a long way" from what the farmer would otherwise have received
• 2 outbreaks in Delaware at 2 affected farms about 5 miles (8 kilometers) apart. The sequence at the cleavage site is PEKPKPR/GLF, one that is consistent with LPAI. > 226 farms have been tested, with no additional infection detected
• first detected on 5 Feb 2004 in southern Kent County (estimated date of primary infection is 2 Feb). 12,000 farm chickens were culled on Feb 6. The farm is a non-commercial operation and functioned like a backyard flock (no biosecurity, no all-in all-out system) and had a mixture of poultry breeds of different ages. The flock consisted of about 5000 chickens 5 weeks old, 4800 white roosters and 1200 red pullets, all in 1 house. Only the 5 week old chickens were clinically affected. They were separated from the rest of the birds by wire mesh, and although these older birds (the roosters and the pullets) did not appear to be clinically affected, they were directly exposed to the same agent, and therefore, presumed infected. The most likely source of the virus is live bird markets in New York state, where H₇ infections have occurred before and at which the grower had sold his chickens. The virus could have been carried back to the Delaware farm on contaminated crates). All 20 commercial farms within a 3-km radius of the outbreak tested negative for AI
• 73,800 4.5-week-old roaster-type chickens in 3 chicken houses were culled at a Perdue Farms commercial broiler operation in the eastern state's Sussex County on Feb 10
• in a commercial poultry flock in the Pocomoke City area, made up of 4 poultry houses with approximately 118,000 6-week-old broilers on Maryland's Lower Eastern Shore : there is no known connection between the Maryland and Delaware cases. The owner of this farm has an 8-house farm with 2-week-old birds about 1 mile from the first farm : because of the close relationship between these farms, and the shared personnel and equipment, as a proactive step to prevent spread of the disease, those 210 000 birds will also be depopulated. His third farm is about 2 miles from the original farm and the one-week-old birds on that farm will be observed for the time being. The new quarantine applies to the areas of Maryland south of Rt. 50 to the Virginia border : the quarantine restricts movement of poultry and poultry litter and imposes additional biosecurity precautions. There are 71 farms with 171 poultry houses within a 6-mile radius of the positive farm, all of which will be sampled for presence of AI
• 4 of the 35 live chicken markets in northern New Jersey, in cities such as West New York, Jersey City, Passaic, and Clifton. They are popular among some Hispanic immigrants who prefer freshly killed poultry.
=> in 2002, the patient was a person involved in culling activities, when an outbreak occurred among turkeys and chickens at commercial farms in Virginia. This may be the 1st recorded human case of H₇N₂ infection
=> a Caribbean immigrant living in Yonkers, Westchester County, USA, with his wife and children entered the hospital in November 2003 suffering from other serious ailments that weakened his immune system and that might have masked the symptoms of avian influenza. One official said the patient had symptoms of a respiratory illness, including coughing and an abnormal chest X-ray. Doctors at first suspected tuberculosis. The man recovered and went home after a few weeks, but it was not until 17 Mar 2004 that scientists identified the virus as H₇N₂, the same that hit chicken farms in New Jersey, Maryland and Delaware in 2004, excluding that the sample had not been contaminated in a laboratory. Doctors asked the patient for another blood sample, to compare antibody levels in it with another sample kept from the initial phase of his illness, and on April 13, the tests confirmed a recent infection with H₇N₂. None of the man's family, co-workers and close contacts had been infected. Local and federal health officials say they have found no evidence that the Westchester County patient had direct contact with contaminated birds or had traveled to any region affected by avian flu. The nature of the exposure to this low pathogenicity strain of avian influenza A virus is unknown, but the immune-deficient status of the patient may have been a relevant factor in both susceptibility to infection and the seriousness of the disease process.
In both cases, no person-to-person transmission of H₇N₂viruses was evident, and both made a full recovery from their acute respiratory illnesses.
• H₇N₃ subtype

Epidemiology : 1st detected in turkeys in Britain in 1963
• Canada : an HPAI outbreak (diseases nevere reported before in Canada), first appearing in Matsqui, about 30 km southeast of Vancouver in the Fraser Valley, on the British Columbia's Lower Mainland on Feb 17, 2004 (reported on Feb 19), in a broiler-breeding operation comprised of 2 units of approximately 9000 birds each. Tests show the virus was in the process of changing from a low to high pathogenic form in a second barn at the Loewen Acres hatchery in Abbotsford, B.C., just 2 kilometres away from the property where disease first struck. On 2 Mar 2004, the virus recovered from the flock of 52-week-old birds was confirmed as LPAI (IVPI = 0.0) and additional work on samples derived from the flock of 24-week-old birds also returned an H₇N₃ subtype with a similar nucleotide sequence at the cleavage site. However, pathogenicity determinations now completed have revealed both LPAI and HPAI strains (IVPI = 3.0) to be present within this group of birds. It was quickly quarantined; some 36 000 birds in a complex of 7 barns were slaughtered and an extensive disinfection process is underway. 5 workers fell ill with mild flu and conjunctivitis, but tests showed AI was not to blame in 3 of those cases. A third outbreak at the Fraser Valley farm, which is located within 5 km of the 2 farms where AI had already been found, caused culling of 85,000 birds on Mar 24. On May 5 there are a total of 40 infected commercial premises identified to date, some as far as Cloverdale -­ a farming region southeast of Vancouver -- in Surrey:
• 13 premises within the 5-km High-Risk Region bounded by the Fraser River, Sumas Mountain, Highway 11, and Clayburn Road, an area that is about 8 by 10 kms, where the index case was diagnosed
• 5 premises within the 10-km Surveillance Region surrounding the High-Risk Region
• 22 premises outside the Surveillance Region, but within the limits of the Control Area that was established to contain the outbreak.
These 40 farms raised a total of 1 205 133 birds, which have been eliminated. 147 birds have also been destroyed in 10 positive backyard flocks. As planned, the CFIA is now depopulating all flocks within the Control Area, targeting 1st those flocks where disease is confirmed or suspected and those premises within a 1-km radius of such premises. Authorities anticipate slaughtering 19 million birds between Vancouver and Hope by late May. Control efforts at the border to the United States have been instituted, including truck washing. Poultry from non-infected flocks will be allowed to be processed under full inspection in registered establishments and made available for sale. Some 42 countries have imposed some form of poultry ban on products from Canada or British Columbia.
Human cases :
• 2 workers who have been confirmed infected had neither taken the

anti-viral drug nor received a vaccination. They have fully reeovered and are not being monitored further
• a Canadian Food Inspection Agency contract worker, who was involved in the culling of infected birds on 13-14 Mar 2004 in Matsqui. On 13 Mar 2004, he may have been accidentally exposed in the eye. On 16 Mar, he reported conjunctivitis and nasal discharge. Treatment with oseltamivir began on 18 Mar. On 30 Mar, Health Canada concluded that this case was caused by avian influenza A (H₇) virus and informed WHO on 31 Mar: he was held in isolation at a health unit set up by the health authority
• on 2 Apr 2004, WHO was informed by Health Canada of a second poultry worker in British Columbia infected with avian influenza A (H₇) virus infection. This worker developed conjunctivitis on 25 Mar after close contact with infected birds. He was treated with oseltamivir on 25 Mar
• 12 other workers  who were outfitted with gloves, goggles, and protective gear for their feet and hands, and who have been working in the area in the past few weeks have also developed conjunctivitis and upper respiratory ailments such as coughing and sore throats
There are 294 licensed chicken producers in B.C. and 50 broiler breeders, which provide eggs for hatching to other farms but do not produce chickens for food. Canada has had 3 cases of low pathogenic H₅ and H₇ viruses since 1975, the latest of which was reported in 2000 in Ontario. Canada only exports about 7% of the chicken it produces. The value of all exports involving B.C. poultry, eggs and egg products is about $22.5 million [almost USD 16.9 million], with over half the product shipped to the United States. Canadian poultry exports are worth $125 million [USD 93.7 million]. The poultry industry is worth nearly US$1 billion to British Columbia each year, of which 80% comes from the Fraser Valley
A total of 167 people (20% to 25% of all workers) participated between May 7 and July 26, 2004. Of these, 19 had experienced ILI and 21 had experienced red or watery eyes. There was no significant association between illness reports and exposure to infected birds. Among 65 people who entered barns with infected birds, 55 (85%) had received influenza vaccine, 48 (74%) had received oseltamivir, and 55 (85%), 54 (83%) and 36 (55%) reported always wearing gloves, mask or goggles, respectively. Antibody to the H₇ subtype was not detected in any sera. During the BC outbreak, compliance with recommended protective measures, especially goggles, was incomplete. Multiple back-up precautions, including oseltamivir prophylaxis, may prevent human infections and should be readily accessible and consistently used by those involved in the control of future outbreaks of avian influenza in poultry. Localized human avian influenza infections may not result in serologic response despite confirmed viral detection and culture^ref.
• USA, Texas :
• mid-May 2004 : 48 000 chickens owned by Pilgrim's Pride at a farm about 5 miles from another farm where bird flu was first detected
• June 2004 : a small flock of 14 birds in Hopkins County in northeast Texas, located about 4 miles from the depopulated commercial operations, was immediately euthanized and incinerated
• Taiwan : detected in 2005 on April, Nov 14 (HPAI H₇N₃ was found in droppings left by a migratory bird in marsh land in the southern city of Tainan. It was the 2nd time the strain has been detected in Taiwan. The 1st case of H₇N₃ was discovered in the outskirts of Taipei in April 2005. COA experts also ruled out infection by lethal bird flu after examining the > 200 dead birds at the Metropolitan Park in Taichung City, central Taiwan. They rushed to the park after receiving reports about the > 300 pet birds forsaken by their unknown owners. A total of 212 birds already died, probably from the sudden drop in temperature and hunger. Officials have sent the 114 live ones to a zoo nearby for shelter) and December
• UK (last seen in the UK in 1979)^ref : after H₇N₃ bird flu was found on Apr 26, 2006^ref among dead birds on Witford Lodge farm in Hockering, 13 miles west of Norwich^ref, (at Mattishall^ref, near the market town of Dereham, in the eastern county of Norfolk), 35,000 chickens were culled^{ref1, ref2, ref3}. A poultry worker suffered from conjunctivitis after contracting H₇N₃. The worker has not required hospital treatment. He or she was given Tamiflu as a precautionary measure on Thu 27 Apr 2006 as soon as the HPA was told about the outbreak. An unconfirmed number of other workers are already taking the same drug to prevent the illness. They are also being offered the seasonal flu jab to prevent the H₇ virus from mixing with any human flu viruses that may be circulating. Nose and throat swabs and blood tests are also being taken from the other workers, and their health is being closely monitored. Close contacts of the infected worker are also being given advice and preventative medication as appropriate^ref. The1st birds are believed to have died on Sat 22 Apr 2006, with the number totalling about 400 birds, about 0.02% of the total population. Owners Banham Poultry submitted blood from the birds for testing after workers noticed some were showing reduced appetite and poor egg-production, with test results confirming the virus late on Tue 25 Apr 2006. Kimblewick Equestrian Centre, run by Mrs Moore, is the farm's closest neighbour and the owner, fears her family may have already been unwittingly exposed to the virus. On Mo 24 Apr 2006, their Rottweiler-cross Ziggy dumped a dead chicken from the farm in their garden; the stinking carcass lay in a pile of manure at the stables until Defra vets took it away late yesterdayDefra experts recommended that both Mrs Moore and Nikkita take Tamiflu and are given a flu vaccination^ref. On Sun Apr 30, 2 farms (Norfolk Road Farm and Mowles Manor Farm, both in North Tuddenham) registered new outbreaks, while 3 other poultry workers with conjunctivitis at Witford Lodge Farm tested negative for H₇N₃^ref
• Italy^ref
• H₇N₇ subtype / A/equine 1 : it affects Equus caballus (equine influenza) and birds (Gallus gallus, Meleagris gallopavo, Anas spp., and Sus scrofa), but cannot be transmitted through eggs and is harmless to humans. A report was published in 1942 in the Japanese Journal of Veterinary Science, Vol. 4, on page 511-523 about natural and experimental infection in cats with H₇N₇ : a cat, which had been kept in the laboratory for the purpose of controlling wild rats in the food store-cottage, developed severe nervous symptoms one day and died after an acute course on the 3rd day. In the morbid anatomy of the cat, no clear lesions were found. In the histological study, severe nonsuppurative meningo-encephalomyelitis was confirmed. Bacteriological examinations of the organs were negative. Fresh emulsions of the cat's liver and spleen were inoculated into mice (i.c.), fowls (i.m.), and developing eggs (on chorio-allantoic membrane). The results were completely negative. The brain emulsion was inoculated into 2 rabbits (i.c.), 2 guinea pigs (i.c.), 5 mice (i.c.) 4 fowl (i.c. and i.m.), and 5 developing eggs ( on c.a.m.), of which 1 mouse, 1 fowl (i.m.), and 2 eggs died. The virus transfer experiments from these carcasses were further carried out on the same animal species as the first generation. The results were remarkable: all the inoculated animals, i.e., 10 mice, 4 fowl, and 6 eggs died in 3-7 days, 40-65 hours, and 40 hours, respectively, and revealed lesions indistinguishable from those of animals that had died of fowl-pest. Bacteriological examinations were again negative. Further transfers of the causative virus were easily continued in fowls and eggs. On the spleen virus obtained from the 3rd-generation fowl, neutralizing effects of hyperimmune fowl-pest fowl serum, hyperimmune Chosen disease (Newcastle disease) fowl serum, and normal fowl serum were compared. The results showed that the fowl-pest serum was strongly active on this isolated virus, while the other sera were completely inactive. In the control experiment, in which Chosen disease virus was used for the substitution of the cat virus, the chosen disease serum alone proved strongly effective. Thus the cat virus was specifically neutralized by the anti-fowl-pest serum. Judging from the above results, the authors believe that the cat died of a natural infection with fowl-pest virus. The per os infection of healthy cats with fowl-pest virus was attempted. All of the 4 cats fed with Chiba strains, i.e. 2 with infected mice and the other 2 with infected fowl organs, developed distinct nervous symptoms and died in 7~13 days after feeding, except one, which recovered after several days. Histological examinations proved encephalomyelitis in 2 cases and myelitis in the other. In each of the dead cats, virus was recovered from brain, but not from the spleen and liver. The lung was virulent in only one case. Each of the 2 cats fed with Doerr strain, i.e., infected fowl organs, died 7 days after feeding. They did not reveal distinct nervous symptoms nor inflammatory changes in the brain and spinal cord. In one of the cats, however, fresh emulsions of spleen plus liver as well as the lung were found to contain enough virus to infect test fowls and mice. The virus search in the other cat resulted negatively. Organs of fowls infected with the Fukuda strain, were given to 2 cats. One of them developed general symptoms after a long incubation period (24 days) and died suddenly in 24 hours. Weak virus was recovered from the brain, but not from the other organs. The other cat remained healthy. The per os infection of the cat with fowl-pest virus thus proved to be positive and the animal appears to have a comparatively strong susceptibility to the virus.
Epidemiology :
• influenza A/H₇N₇ in humans was 1st reported in 1959^ref
• LPAI was detected in Netherlands at the end of 2002 : presumably, the virus mutated to become a HPAI variant and caused an outbreak in chickens on 28 Feb 2003. The Dutch Ministry of Agriculture announced a ban on the export of all poultryand poultry-related products on 1 Mar 2003. In a week Dutch authorities slaughtered around 26 million of the 100 million poultry at some 225 farms in the country to fight the outbreak. The Netherlands is the biggest poultry exporter in the European Union : in 2003 Singapore imported from the Netherlands 447 tonnes of chicken meat, which was valued at SGD 850,550 [USD 502,986] and constituted 0.26% of total poultry consumption and imported 2,066 live ornamental birds valued at SGD 103,390 [USD 61,141]. Worries exist that migrating wild birds could spread the disease : the chief form of transmission, however, is consumption of infected materials or feces. The disease also spread to Belgium and Germany, although on a smaller scale than in the Netherlands. All 3 countries later resumed poultry exports. Many Sus scrofa developed antibodies against H₇N₇. An estimated 1,000 people, possibly more, have been infected. 1/3 of the poultry farmers whose holdings were cleared reported stress reactions, fatigue and depressive symptoms. 453 individuals with health complaints were identified from an at-risk population of individuals with direct contact with poultry or poultry products with possible H₇ infection or individuals with close contact with an H₇-infected person. Of those with health complaints 349 reported conjunctivitis, 90 had influenza-like illness (ILI), and 67 had other complaints. The researchers detected influenza A/H₇ in conjunctival samples of 78 (26.4%) of people with conjunctivitis only, in 5 (9.4%) of those with ILI and conjunctivitis, in 2 (5.4%) of those with ILI only, and in 4 (6%) of individuals with other symptoms. Of the 453 individuals who reported health complaints, H₇ was detected in 19.6% overall. Of those with laboratory confirmed H₇, 41.2% were chicken cullers, 26.3% were veterinarians, and 14.7% were poultry farmers or family. Of those exposed to 83 cases of confirmed A/H₇ infection, 70 individuals reported conjunctivitis, 13 reported ILI, and 14 reported other illnesses. The investigators confirmed A/H₇ in 3 individuals exposed to other humans with A/H₇ infection. Antibodies were found in 59% of infected poultry workers' family members. Of the 500 tested persons who had handled infected poultry, about 50% showed an antibody response^ref. Following the March 3 confirmation that A/H₇N₇ was the cause of the avian influenza outbreak, poultry workers were advised to use of personal protection gear. By the March 14 virology update, which included data on 19 confirmed cases of A/H₇ and the first confirmed contact transmission, all workers received prophylactic oseltamivir and influenza virus vaccination. At this point 56% of the A/H₇ infections had already occurred^{ref1, ref2} :
• 3 family members of 2 poultry workers have also fallen ill with a minor respiratory disease and conjunctivitis (a 12-year-old child, had significant ILI as well and was repeatedly positive for A/H₇), suggesting a possible chain of human-to-human transmission
• a 57-year-old veterinarian from Rosmalen died from acute respiratory distress syndrome (ARDS) on 17/4/03 in Hertogenbosch [Den Bosch], The Netherlands : he became ill 2 days after working on a farm in in Teefelen that had been infected with avian virus (in throat swabs collected at hospital admission on 11/4 and on 13/4 and sent to another laboratory, the presence of HHV-1 / HSV-1 was indicated - for which he was treated with aciclovir)^{ref1, ref2}
It would be interesting to know the relationship of antibody response in this group to age and previous history of exposure to influenza. On the other hand, any direct disease response in humans was mild, and oseltamivir appears to have provided adequate protection to those handling infected birds. Nor has there been evidence of interaction between avian and human influenza viruses in the aftermath of the outbreak, which might have provoked a new outbreak in either the human or avian population. Virological sequence data published elsewhere indicate that the virus strains were of entirely avian origin, with no human genes. The virus isolated from the fatal case had, however, accumulated significant mutations, which may have been associated with enhanced virulence in this case. 25% of all Dutch poultry (30.7 million birds at 1300 farms) were slaughtered to contain the outbreak. The measures to prevent simultaneous co-infection of humans with HPAI and human influenza viruses included mass anti-viral treatment and vaccinations of all poultry workers and their families in a 3-km radius of infected poultry farms and those suspected of having the infection. Treatment with oseltamivir was recommended for all conjunctivitis cases, and a prophylactic regimen of oseltamivir was given to all people handling potentially infected poultry for 2 days after their last exposure. Vaccination of poultry was considered as a substitute for a stamping-out policy, but remained theoretical.
A questionnaire survey was carried out as a follow-up to the outbreak^ref. Approximately 400 poultry farmers and their families and almost 900 people who were involved in controlling the epidemic participated in this investigation. Blood samples were taken from 500 of these participants to determine possible infection with the avian influenza virus. Additional studies were performed for 62 household contacts of 25 persons with avian influenza virus infection. Routine serological tests failed to detect any antibodies, even in the group of persons with confirmed avian influenza virus conjunctivitis. A modification of the haemagglutination assay was developed, based on observations that avian influenza viruses favour binding to red blood cells from horses rather than turkeys^ref. As > 50% of the people exposed to infected poultry had H₇ antibodies detectable with the modified assay, it was estimated that avian influenza A/H₇N₇ virus infection occurred in at least 1000, and perhaps as many as 2000 people. The seroprevalence of H₇ antibodies in people without contact with infected poultry, but with close household contact to an infected poultry worker, was 59%. This suggests that the population at risk for avian influenza was not limited to those with direct contact to infected poultry, and that person-to-person transmission may have occurred on a large scale. Specificity of the unconventional assay was confirmed by the absence of reactivity in sera from 100 controls recently vaccinated with influenza vaccine (2002/2003) (specificity 100%). Assay specificity was further supported by the results of the cohort study: having measurable antibodies was associated with having conjunctivitis (RR 1.72; 95% CI 0.99-2.99), and a lower proportion of the exposed persons who took prophylactic antiviral medication developed antibodies (corrected OR 0.48; 95% CI 0.25-0.89). Neither poultry farmers nor those engaged in controlling the epidemic complied satisfactorily with preventive measures. Only 6% of farmers reported consistent use of facial masks and 1% reported consistent use of goggles while working with infected poultry. In cullers, compliance was only slightly better: 25% consistently used facial masks and 13% used goggles. The results of the epidemiological study suggest that oseltamivir protected against conjunctivitis (corrected OR=0.14; 95% CI=0.08-0.27) as well as against infection without specific symptoms. No protective effect was demonstrable for safety goggles or mouth-nose masks^ref. A review of the outbreak and control efforts in the Netherlands highlights important lessons for preparedness: while separate systems are in place to signal and control animal diseases and human diseases, an outbreak of a zoonotic disease illustrates the importance of coordination between the 2. In the Netherlands, the people infected came from a wide geographic region and included foreign poultry workers. While the movement of animals was restricted, these people were out of the reach of the public health authorities while infectious and shedding the virus. Although the disease in humans is more severe for A/H₅N₁, both avian influenza outbreaks illustrate that crossing the species barrier is less rare than previously recognised, that avian influenza virus adaptation occurs rapidly, and that if such jumps between species occur, human behaviour in the broad sense may accelerate dissemination^ref.
The occurrence of infection with AI A virus subtype H₇N₇ in household contacts of human A/H₇N₇ confirmed index cases has been described in the absence of contact with infected poultry. 33 of 56 household members (58.9%) had an A/H₇N₇ infection confirmed by RT-PCR or serology, 4 of 62 household members (6.5%) met the possible case definition of conjunctivitis, and all 4 cases (100%) had positive H₇ serology. The authors assume that the presence of H₇ antibodies is indicative of a past AI A/H₇N₇ infection. This is supported by the results of another study in which the prophylactic use of oseltamivir was found to significantly reduce the seroprevalence of H₇ antibodies in professionals exposed to infected poultry using the same serological test (Bosman A, Mulder YM, de Leeuw JRJ, Koopmans MPG, Meijer A, Du Ry van Beest Holle M, et al. Vogelpest Epidemie 2003: gevolgen voor de volksgezondheid. RIVM Rapport in het kader van project GezondheidsOnderzoek Vogelpest Epidemie (GOVE, V/630940/05), 2004). In that study, a significant association was found between the presence of H₇ antibodies and the occurrence of eye symptoms, after correcting for prophylactic use of oseltamivir. When using the adjusted HI assay, but not when using the microneutralization assay, we detected a measurable antibody response in a high proportion of sera from persons exposed to laboratory-confirmed A/H₇N₇ infected persons. Evidence that these antibodies are real comes from 3 observations. 1st, any cross reaction of the A/H₇ specific HI-assay with antibodies against A/H1 or A/H₃ viruses would have been detected in the sera from persons recently vaccinated with the seasonal human influenza vaccine, but no reaction (0%) in the A/H₇ HI assay was found. 2nd, as the sera of the recently vaccinated persons were collected in autumn 2002, just before the H₇ epizootic started, the anti-H₇ antibodies in the household contacts can not be explained as being the result of previous circulation of A/H₇ virus. 3rd, none of the samples collected in autumn 2002 from 100 recently vaccinated persons had reactivity with the adjusted H₇ assay^ref. This suggests that our results cannot be explained by a specific reactivity of the adjusted HI-assay. During the outbreak of avian influenza A virus, subtype H₇N₇, household members of poultry workers were at increased risk of avian influenza either by direct (person to person) or by indirect (fomite) transmission. Previous observations of influenza transmission within households had shown secondary attack rates among household members of influenza cases in the same high range as observed in our study^ref. These high secondary attack rates are in contrast with findings for subtype A/H₉N₂ and A/H₅N₁, where no to limited secondary transmission was observed among healthcare workers and household contacts of cases^{ref1, ref2, ref3, ref4, ref5}. However, we used a method for the detection of antibodies against the H₇ virus which has a high analytical sensitivity. Detailed studies to analyze person to person transmission of H₅ and H₉ with the same methodology are sparse. Interestingly, for H₉, a recent publication showed that in 44.6% of suspected cases of H₉N₂ infection and in 33.5% of the general population in Shantou city in China, antibody titres against H₉ could be detected (Lin JM, Cai FC, Zhong XZ, Lin WS. Serological investigation on the population infection of avian influenza H₉N₂ virus in Shantou city. Strait J Prev Med. 2004;10:16-7). This observation suggests that secondary transmission of H₉ viruses may be more common than has previously been assumed. In addition, the primary site of infection, the conjunctiva for H₇ virus and the airway epithelium for H₅ and H₉ virus, and the possible difference in virus receptor expression on the conjunctiva and the airway epithelium together with the difference in affinity of the respective viruses for these receptors, may also account for the observed differences. Although sharing bath towels and washcloths, and using cloth handkerchiefs seemed to increase the risk of clinical conjunctivitis, none of these observations was statistically significant, presumably due to lack of study power. However, it seems plausible to assume that patients with a viral conjunctivitis are more likely to expose household members to virus when sharing towels and washcloths or using cloth handkerchiefs. This is supported by our observation of higher seroprevalence among people using cloth handkerchiefs and of lower seroprevalence among those using paper (disposable) handkerchiefs, all of which were statistically significant. Studies on transmission of other viral conjunctivitis within households identified crowding and high numbers of persons per bathroom as risk factors^{ref1, ref2, ref3, ref4, ref5}. Seroprevalence was significantly higher among those who had at least 2 toilets in their homes than among those who had only one toilet. We have no explanation for this result. Hygienic measures, such as using soap for hand washing and good hygiene by the index case, associated with seropositivity, were of borderline significance. Although we observed higher seroprevalence in those household members who had pet birds kept indoors at home, this cannot account for all seropositive secondary cases, as only 7 of all 33 cases had indoor birds at home. However, this finding raises the question of whether indoor pet birds could play a role in the household transmission of avian influenza virus, especially since 6 of 7 cases with pet birds in the home were part of the same household. It is conceivable that these animals could serve as an amplifier for multiplication and shedding of the virus in the home environment. This deserves further attention in future outbreaks, for example, by monitoring and screening pet birds in the homes of poultry workers. It was not possible to perform binomial regression for the outcome of A/H₇N₇ infection, presumably due to low numbers in the cohort. If the detection of H₇ antibodies is indicative for human (subclinical) influenza A/H₇N₇ infection, then the secondary spread of A/H₇N₇ to household contacts is on an unexpectedly large scale. Although the pathogenity of the A/H₇N₇ virus seemed to be low, the high transmissibility is directly related to an increased risk for double infection and reassortment. Current outbreak control measures did not take transmission to household contacts into account. This also raises the question of whether or not subclinical A/H₇N₇ cases can transmit the virus efficiently to other close contacts, which would imply that outbreak control strategy for A/H₇N₇ should be thoroughly revised. Consideration may be given to early isolation of cases and quarantine of contacts. Prophylactic treatment with oseltamivir should be considered for all household contacts of poultry workers during outbreaks of avian influenza, although its role must be further assessed in order to determine the risk of developing antiviral resistance. Moreover, in order to assess the role of fomites in secondary transmission of the A/H₇N₇ virus, further studies of contacts outside the household should be performed, as well as investigations to obtain background information on the spread of A/H₇N₇ in the general population of the Netherlands. The study had the following limitations. Non-response was high and may be associated with rates of illness (selection bias), but we see no reason why it would have differed between exposed and non-exposed members of the cohort, therefore not biasing the estimate of the risk ratios. However, selection bias is not likely to play a major role with respect to seroprevalence, since most household members with detectable antibodies were asymptomatic. In conclusion, our study suggests that human-to-human transmission of HPAI A/H₇N₇ can occur within household contacts in the absence of contact with infected poultry. Monitoring of clinical symptoms alone in household contacts of confirmed A/H₇N₇ cases underestimates the extent of human-to-human spread. In addition, our results suggest that cloth handkerchiefs, having indoor pet birds at home or having at least 2 toilets at home could be risk factors for household transmission A/H₇N₇. Taking all the results together, we recommend that during an outbreak of AI:
• household members should be encouraged to use paper handkerchiefs instead of cloth handkerchiefs;
• household members of poultry workers exposed to A/H₇N₇ should be advised on enhanced general hygiene measures;
• in the case that oseltamivir prophylaxis is offered to exposed poultry workers in future A/H₇N₇ epizootics, this should also be considered for household members of A/H₇N₇ cases
• indoor pet birds of poultry workers should be screened and monitored during future outbreaks of avian influenza in order to determine the role of indoor birds in household transmission of the virus
• further seroprevalence studies among contacts of asymptomatic persons with positive H₇ serology should be conducted in order to assess the risk of person to person transmission, and consequently the potential for a new pandemic strain, in the absence of symptoms.
The significant conclusion extracted from the above article applicable to other situations, such as the A/H₅N₁ outbreaks in East Asia is that: Current outbreak control measures did not take transmission to household contacts into account. This also raises the question of whether or not subclinical A/H₇N₇ cases can transmit the virus efficiently to other close contacts, which would imply that outbreak control strategy for A/H₇N₇ should be thoroughly revised. Consideration may be given to early isolation of cases and quarantine of contacts. Prophylactic treatment with oseltamivir should be considered for all household contacts of poultry workers during outbreaks of avian influenza, although its role must be further assessed in order to determine the risk of developing antiviral resistance. Some other factors, such as the higher vulnerability of households with 2 toilets over those with one, are not easily interpretable^ref.
On Sat 13 Mar 2004, the government decided to cull all 22 000 asymptomatic free-range chickens at a farm in Uithuizermeeden (Eemsmond municipality, Groningen province) in the northeast of the country as a precautionary step, after blood samples taken there indicated birds had developed antibodies to H₇ LPAI.
Anouther outbreak of LPAI in a poultry farm in the central region of Gelderse Vallei on 1 Aug 2006^ref : 25,000 chickens were destroyed by authorities at a poultry plant at Voorthuizen^ref in the eastern province of Gelderland on Wed 2 Aug 2006^ref
On 27 Mar 2005 bird flu outbreaks was confirmed at 3 chicken farms owned by the same company within a 4-km radius in Pyongyang, including 1 in Hadang (one of 5, constructed in December 2002, that breed and process chickens in Pyongyang) (160 000 - 200 000 layers), and 2 in farms approximately 4 km from the Hadang farm with 560 000 and 300 000 layers, respectively : the farms had slaughtered and buried hundreds of thousands of chickens infected by the disease, and no people working at the farms were found to be infected. This is in line with article 1.1.2.3. of the Terrestrial Animal Health Code (2004) which says: "Veterinary Administrators shall send to the Central Bureau a notification from the Delegate of the country by telegram, fax or e-mail, within 24 hours, of any of the following events:
• a. 1st occurrence of a listed disease and/or infection in a country or zone/compartment;
• b. re-occurrence of a listed disease and/or infection in a country or zone/compartment following a report declared the outbreak ended;
• c. 1st occurrence of a new strain of a pathogen of an OIE listed disease in a country or zone/compartment;
• d. a sudden and unexpected increase in the distribution, incidence, morbidity or mortality of a listed disease prevalent within a country or zone/compartment;
• e. an emerging disease with significant morbidity or mortality, or zoonotic potential;
• f. evidence of change in the epidemiology of a listed disease (including host range, pathogenicity, strain), in particular if there is a zoonotic impact."
Seemingly, the 1st (a) of the said notifiable events is the one applicable to the North Korean situation, the relevant listed disease being HPAI. Data on the subtyping of the HPAI virus involved, with special attention to subtype H₅N₁, are of particular importance. The 2 Koreas are technically at war, because the 1950-53 Korean War ended in a truce and not a full peace treaty. According to some rumors North Korea may have culled as many as 10 million chickens in the Pyongyang area alone : FAO and OIE report that North Korea has culled 219,000 birds at 3 chicken farms. On 4 Apr FAO tests confirmed those done by the North that the birds were infected with the H₇ strain -- not the H₅ strain ! However, H₇ is not innocent regarding its potential as a human pathogen. One of the (at least, theoretical) sources of a HPNAI H₇ virus is a live AI virus vaccine. Most of North Korea's annual 25 million birds are produced on larger farms, one of the few growing sectors in a country battling severe food shortages. The number of the country's poultry was estimated at 25.5 million in 2004, about 2 times the level of 1997. North Korean authorities had buried the dead chickens but some residents had dug up the poultry to sell or consume. North Korea has for more than a decade relied on food aid from donor countries to meet the needs of its 23 million people. A confirmed outbreak of bird flu would necessarily trigger a vast cull of poultry, a main sources of animal protein for North Koreans. The current import prohibition by South Korea and Japan may be explained by the unclear epidemiological situation in N.Korea. After receiving news of the unconfirmed outbreak, Porky Trading Korea, a firm in Seoul, asked its North Korean counterparts to delay chicken shipments : the trader was ready to send a ship to the North Korean port of Nampo to ship 40 tons of North Korean chicken. The company had planned to buy 2000 tons of North Korean chicken in 2005. The culling of 218 000 chickens and vaccination of 1.1 million poultry in affected areas had ended the outbreak.Korea (Dem. People's Rep.) is a member-country of the World Organisation of Animal Health (OIE) since March 2001, obliged to report suspected outbreaks of infectious animal diseases such as avian influenza even prior to their final confirmation. No disease notification reports -- be they weekly, monthly or annual -- from N.Korea are available on OIE's web-sited database, dating back to 1996. An avian-influenza outbreak was initially suspected in N. Korea on 15 Mar 2005, and, according to the authorities, could eventually be officially announced in the country on 27 Mar 2005. On 7-8 Apr 2005, the national authorities officially informed the OIE about the diagnosed H₇ outbreak. The notification added detailed information, including the following statement: "Tests are currently under way to determine the N subtype of the virus". The results of the said tests, as well as the source/origin of the outbreak/infection, are still pending.
Prophylaxis : vaccination with the current influenza vaccineand treatment with neuraminidase inhibitors for agricultural workers in contact with H₇N₇-affected poultry. The vaccine applied in Northern Italy during the 2000-2002 epizootic caused by an H₇N₁ HPAI virus was an inactivated, heterologous subtype HPAI vaccine (strain A/Chicken/Pakistan/1995-H₇N₃). The reason for the selection  of a heterologous vaccine was the possibility of using it as a natural "marker" or a differentiating infected from vaccinated animals (DIVA). This enabled the EU to approve another AI vaccination scheme in Northern Italy that has been operational since 31 Dec 2002, using again a heterologous vaccine, this time an HPAI H₇N₁ subtype, to protect against the current H₇N₃ LPAI field strain. The H₇N₃ vaccine used in Italy in 2000-2002 is one of the 2 vaccines approved by the Dutch Ministry of Agriculture for the vaccination of susceptible animals in zoos : Nobilis
Influenza H7^® manufactured by Intervet (Boxmeer) and Fluvac H7^®, manufactured by  Merial B.V. (Amstelveen). Both include a heterologous neuraminidase (not N₇). The dead vaccine for s.c. injection can be administered in 2 shots, about 14 to 30 days apart (other methods include the use of aerosol mists that can be taken into the birds' nasal passages or oral vaccines that can be delivered with a dropper).
• H₇N₈subtype
• H₇N₉subtype

Epidemiology : during February–May 2013, the initial outbreak of human infection in China resulted in 133 cases^ref. Influenza A(H₇N₉) virus reemerged in southern China in October 2013 and had caused 85 laboratory-confirmed cases of infection in Guangdong Province as of March 7, 2014. Targeted surveillance for influenza A(H₇N₉) identified 24 cases of infection with this virus in Guangzhou, China, during April 1, 2013–March 7, 2014. The spectrum of illness ranged from severe pneumonia to asymptomatic infection^ref. It is unclear how the H₇N₉ virus re-emerged and how it will develop further; potentially it may become a long-term threat to public health. H₇N₉ viruses have spread from eastern to southern China and become persistent in chickens, which has led to the establishment of multiple regionally distinct lineages with different reassortant genotypes. Repeated introductions of viruses from Zhejiang to other provinces and the presence of H₇N₉ viruses at live poultry markets have fuelled the recurrence of human infections. This rapid expansion of the geographical distribution and genetic diversity of the H₇N₉ viruses poses a direct challenge to current disease control systems. H₇N₉ viruses have become enzootic in China and may spread beyond the region, following the pattern previously observed with H₅N₁ and H₉N₂ influenza viruses^ref

• H₈ has healthy reservoir in water Aves spp.
• H₈N₄ subtype
• H₉ infects humans and Sus scrofa has healthy reservoir in water Aves spp. and poultry : in Colombia it was discovered in October 2005 in chickens at 3 farms in Tolima state in western Colombia, and the affected flocks were immediately quarantined to prevent the spread of the disease
• H₉N₁ subtype
• H₉N₂ subtype
Epidemiology : the main worry regarding this strain is its documented potential to infect humans, as reported in Hong Kong in March 1999 (isolated by the Virus Unit of the Hong Kong Department of Health from 2 girls (aged 1 and 4 years old) exposed via unreported circumstances. Influenza A (H₉N₂) viruses had been isolated from ducks and chickens for several years previously^ref) and December 2003 (a 5-years old boy). All H₉N₂ infections were associated with uncomplicated ILI, and no evidence of person-to-person transmission of H₉N₂ viruses has been reported^ref. Infection was identified on nasopharyngeal aspirates from 2 children; H₉N₂ virus was confirmed by the WHO influenza reference laboratories in both London, UK and Atlanta GA, USA. The illness in both children was mild [fever, URI] and self-limited. A history of probable contact with poultry was reported for one of the children. It has been postulated that shared gene constellations in avian influenza viruses H₉N₂ and H₅N₁ may confer the ability to cause infection and disease in humans. Besides the legal aspects, the epidemiological hazards of unauthorised introduction of the virus into a country seem to be mainly related to the risk of virus escape; the vaccines are inactivated. 2% of chickens were infected between 2001 and 2003. In 1999, detection of antibody to H₉N₂ among persons in northern and southern China and poultry workers in Hong Kong suggests additional unrecognized infection^ref. It is perhaps worth adding that as part of a study of avian influenza viruses isolated in Hong Kong markets, cross-reactive cellular immunity induced by H₉N₂ influenza virus infection protected chickens from lethal infection with H₅N₁ influenza viruses but did not abort virus shedding in the feces. They concluded that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds housed in live markets and create a situation for the perpetuation of H₅N₁ influenza viruses^ref. Subsequently, Xu et al, in a study of influenza viruses recovered from ducks in southern China, found that some H₉N₂ viruses were double or triple reassortants that had amino acid signatures in their hemagglutinin, indicating a potential to directly infect humans. Some of these H₉N₂ reassortants contained genome segments related to those of H₅N₁ and H₅N₂ avian viruses. They concluded that the 2-way transmission of influenza viruses between terrestrial and aquatic birds had generated novel reassortant H₉N₂ influenza viruses that might directly or indirectly play a role in the emergence of the next pandemic influenza virus^ref
• H₉N₃ subtype
• H₉N₅ subtype
• H₉N₇ subtype
• H₉N₉ subtype
• H₁₀ has healthy reservoir in water Aves spp.
• H₁₀N₇ subtype

Epidemiology : isolated on May 2004 from 2 infants, both aged 1 year, residents of Ismaillia, Egypt, who recovered after presenting a fever and cough. The father of one of them is a poultry merchant who frequently traveled between Isamillia and Damietta. In this last town, avian influenza virus A (H₁₀N₇) was isolated from 5 wild ducks between 18 and 22 Apr 2004, from samples taken from a market of hunted migratory birds. Egypt is predominantly a Muslim country, where pig breeding is not expected; however, a substantial number of pig holdings are maintained by the Coptic population. The influenza A (H₁₀N₇) virus has reportedly been occasionally isolated from wild and domestic avians in various parts of the world, including ratites (emu), as well as from mammals (minks). Its pathogenicity is regarded as being of low magnitude. During 3 described LPAI outbreaks in turkeys (Minnesota 1979-1980), the clinical signs ranged from severe, with a mortality rate as high as 31%, to sub-clinical^ref. The replication of A(H₁₀N₇) virus in human volunteers seemed to be limited and without a detectable antibody response^ref.

• H₁₀N₈ subtype

Epidemiology : In China, H₁₀N₈ virus was isolated from the environment of Dongting Lake in Hunan Province in 2007^ref and from a duck in a live poultry market (LPM) in Guangdong Province in 2012^ref. This AIV was not then known to directly infect humans or other mammals. In December 2013, H₁₀N₈virus infection in a person was reported in Nanchang, Jiangxi Province, China^ref; 2 more human cases followed^{ref1, ref2}. The initial reported case was in a 73-year-old woman who visited a local LPM 4 days before the onset of her illness^ref. The emergence of the novel influenza A(H₁₀N₈) virus has become an urgent public health concern^{ref1, ref2}. A preliminary genomic analysis showed that the emerging influenza virus was genetically distinct from the avian influenza A(H₁₀N₈) viruses previously identified in China, and scientists have postulated that the virus resulted from multiple reassortments of subtype H₉N₂ strains that circulated widely in poultry in China^ref. The virus was conserved in chicken eggs but presented substantial adaptive mutations in MDCK cells^ref. 

• H₁₀N₉ subtype
• H₁₁ has healthy reservoir in water Aves spp.
• H₁₁N₁ subtype
• H₁₁N₈ subtype
• H₁₁N₉ subtype
• H₁₂ has healthy reservoir in water Aves spp.
• H₁₂N₁ subtype
• H₁₂N₄ subtype
• H₁₂N₅ subtype
• H₁₃ has healthy reservoir in water Aves spp.
• H₁₃N₆ subtype
• H₁₄ has healthy reservoir in water Aves spp.
• H₁₅ has healthy reservoir in water Aves spp.
• H₁₅N₈ subtype
• H₁₅N₉ subtype
• H₁₆
• H₁₆N₃ subtype was isolated in 2005 from black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H₁₆ and the previously described subtypes H₁ to H₁₅. Genetically, H₁₆ HA was found to be distantly related to H₁₃ HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H₁₃ and H₁₆ HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H₁₆ viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N₃ genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. Sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes^ref
• H₁₇
• H_17^N10 from bats^ref
• H₁₈
• H₁₈N₁₁ from bats^ref
• hybrid subtypes

Susceptibility : humans who had previously encountered an influenza virus with the N2 neuraminidase may have been partially protected in the 1968 H₃N₂ pandemic that followed the global circulation of H₂N₂ viruses^ref. There is also indirect evidence of short-term immunity between subtypes of influenza viruses^ref (Y. Xia, J. R. Gog, B. T. Grenfell, Appl. Statist. 54, 659 (2005)), which could play a role in the early spread of pandemics^ref. In addition, cross-reactive T cells also may contribute to heterosubtypic immunity to influenza and reduce viral shedding^ref.
Pathogenesis : during the course of a single-cycle infection, human viruses preferentially infected non-ciliated cells, whereas avian viruses, as well as the egg-adapted human virus variant with an avian virus-like receptor specificity, mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (a2-6-linked sialic acids) on non-ciliated cells and of receptors for avian viruses (a2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a non-optimal cellular tropism^ref. The influenza virus hijacks receptor-based endocytosis to invade a cell, moving from early endosomes through to late endosomes before fusing with the endosomal membrane to release its genetic material. This complex process is thought to involve 2 pH changes, one from extracellular pH to early endosome pH (~pH 6) and a second change to late endosomal pH (~pH 5), with the late pH change an absolute requirement for influenza fusion. The trajectory from the cell periphery, moving to the perinuclear region and finally fusing with a late endosome follows a previously unknown 3-step pattern: the virus tracks slowly in a actin dependent manner through the cell periphery (stage I), increases in speed in a microtubule dependent manner (stage II), and then slowed again before fusion with an endosome (stage III). The initial acidification step to ~pH 6 occurred after the rapid stage II movement in the perinuclear region. Previous studies have indicated that early endosomes are the site of initial acidification, but these results suggest that virus-containing endocytic compartments leave the early endosomes before this initial acidification.

The Fujian strain of influenza A (H₃N₂) virus appears to be the currently predominant strain in the USA and Europe. The Panama strain, which is a constituent of the current vaccine, is responsible for the current outbreak in western Canada. Although there is believed to be sufficient antigenic cross-reactivity between the Fujian and Panama strains for the existing vaccine to be protective, it has been judged prudent to replace the Panama strain with the Fujian strain in the new Southern Hemisphere vaccine, although the Panama strain is still in circulation in some places.
The mouse has been a valuable and extensively used model to study the mechanisms that protect against or promote recovery from this infection. Evidence indicates that components of both innate^{ref1, ref2, ref3} and adaptive^{ref1, ref2} immune systems contribute to the control of the infection and that activities provided by CD4⁺ helper (T_h) and B cells^{ref1, ref2} or CD8⁺ cytotoxic T (T_c) cells^{ref1, ref2, ref3} can independently resolve it, although the latter are generally believed to be more effective. The recovery process mediated by T_h and B cells appears to depend largely on the generation of a T_h-dependent antiviral Ab response, as neither T_h^{ref1, ref2, ref3} nor B cells^ref are capable of resolving the infection on their own, while infection in SCID mice can be cured by treatment with Abs specific for the viral hemagglutinin (HA) molecule^{ref1, ref2}. The high therapeutic efficacy of these Abs appears to be due to their ability to concomitantly suppress yield of progeny virus from infected cells and prevent released progeny virus to spread the infection to new host cells^{ref1, ref2}. The T_c cell-mediated recovery process has been shown to rely mainly on the perforin/granzyme- and Fas-mediated killing of infected host cells^{ref1, ref2}, while secretion of cytokines such as IFN-g, which may inhibit virus spread by inducing cellular resistance to infection, does not appear to play a significant role^{ref1, ref2}, at least in the intact mouse^ref, but may become important if the Tc activity is being tested at its therapeutic threshold^ref. The above implies that effector T_c (eT_c) are capable of killing infected epithelial cells before the release of progeny virus. This is surprising in the case of an acute infection in which the eTc has available only a short window of time (between expression of viral peptides by MHC class I and release of virions) to perform this task^{ref1, ref2}. The massive recruitment of virus-specific eT_c into the cellular exudate of the infected airways at the time of virus clearance would be consistent with such a scenario^ref. However, since evidence for the autonomy of T_c-mediated clearance was obtained in the study of influenza viruses of low pathogenicity, such as X31^{ref1, ref2} and B/AA^ref, we wondered whether eT_c-mediated control mechanisms would be similarly effective also against a more pathogenic, and perhaps more rapidly replicating, influenza virus strain like PR8. There is evidence from other virus systems that rapidly replicating viruses such as vesicular stomatitis virus and Semliki Forest virus or more virulent variants of lymphocytic choriomeningitis virusare not effectively controlled by T_c^{ref1, ref2, ref3, ref4, ref5}. In addition, 2 of the influenza virus studies^{ref1, ref2} had been done in mice that contained B cells, although no T_h cells. Therefore, the conclusion that T_c resolved the infection autonomously in these mice assumed that the B cells made no significant contribution to recovery without help from T_h cells. B cell-deficient mice (µMT) of BALB/c background additionally depleted of T_h cells by chronic treatment with anti-CD4 Ab GK1.5 were used to test the ability of the T_c response to autonomously resolve the highly pathogenic PR8 and the less pathogenic X31 virus infections : the study confirmed that the T_c response has the basic capability to autonomously (in conjunction with innate defense) resolve these infections, but with substantial delay compared with immunologically intact mice, which resulted in high mortality in infection with the pathogenic PR8 strain. The study further showed that B cells contributed to the recovery process by a T_h-independent mechanism of still undefined nature^ref.
However, in contrast to findings in mice, the protective value of memory Tc cells in humans remains controversial. The classic study by McMichael et al.^ref indicated that presence of memory Tc cells in blood, which could give rise to Tc cells on stimulation in vitro, correlated with reduced virus shedding 3–4 days after volunteers were challenged with a wild-type virus, but had no significant effect on illness. Subsequent studies performed in children found no significant difference in shedding of attenuated vaccine strains in patients who had recovered from previous infection with a vaccine or natural strain of a different subtype than did study participants who had no evidence of previous virus exposure^ref (Wright PF, Johnson PR, Karzon DT. Clinical experience with live, attenuated vaccine in children. In: Options for the control of influenza;1986. New York: Alan R Liss, Inc. p. 243–53). Similarly, children vaccinated with an H₁N₁ strain showed no difference in attack rate and febrile respiratory illness during exposure to natural epidemic H₃N₂ virus from controls who received a placebo^ref

The pathogenesis of influenza infections has been associated with alteration in the lymphohemopoietic system^{ref1, ref2, ref3, ref4, ref5,ref6, ref7} (20, 21, 29, 31, 50-52). Experimental infection of chickens with the avian influenza virus A/Turkey/Ontario/7732/66 (H₅N₉) (Ty/Ont) resulted in the destruction of lymphocytes and histopathological necrosis of lymphoid tissues. It was further demonstrated that the lymphocyte destruction in birds was associated with virus-induced apoptosis, as Ty/Ont, but not a human strain, A/Puerto Rico/8/34 (H₁N₁), induced apoptosis of an avian lymphocyte cell line^ref. Whether an avian virus has an affinity for cells of the mammalian immune system, resulting in leukocyte death, remains an unanswered question. Infection of mice with a highly virulent H₅N₁ resulted in a decrease in peripheral blood and tissue lymphocytes and aberrant cytokine and chemokine production. An increase in apoptotic cells in the spleen and lung tissue is identified as a possible cause of lymphocyte death.

A variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses^ref

Influenza virus has been shown to induce apoptosis in tissue culture cells^{ref1, ref2} and in peripheral blood monocytes^{ref1, ref2}. A depletion of lymphocytes due to apoptosis has also been described in mice infected with a highly virulent influenza A virus (IAV) (H₅N₁) isolated from humans^ref. The immunopathological mechanisms and the role played by the virus infection of leukocytes with respect to disease pathology in general and leukocyte death in particular have not been elucidated. An early lymphopenia has been described in IAV-infected patients^{ref1, ref2, ref3}, and inoculation of humans with IAV has been shown to cause a decrease in both T- and B-cell numbers during illness^{ref1, ref2}. In the experimental infections, volunteers developed a severe T-cell lymphopenia and a moderate B-cell lymphopenia even though seroconversion occurred in 90% of the volunteers, suggesting that T- and B-cell functions were preserved^{ref1, ref2}. This observed lymphopenia could be the result of cell migration from the circulation and/or cell death caused by necrosis or by apoptosis or through suppression of hematopoeisis. Lymphocyte depletion via apoptosis after exposure to IAV could be the result of virus-induced cytokine stimulation, viral induction of Fas, or other cell-virus interactions^ref.

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4-6 h. Most macrophages died by apoptosis within 25-30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of TNF-a, IL-1, IL-6, IFN-a/b, and CC-chemokines. The basic mechanisms of virus-induced cytokine expression are still unknown and appear to involve transcription factors such as NF-kB and AP-1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC-chemokines MIP-1a, MIP-1b, and RANTES were produced while the prototype neutrophil CXC-chemoattractants IL-8 and GRO-a were entirely suppressed. This selective induction of CC-chemokines may explain the preferential influx of mononuclear leukocytes into virus-infected tissue. Monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen-specific defense^ref

During acute illness^ref or induced infection^ref, lymphopenia is evident as reduced numbers of B and T cells. This may reflect migration of lymphocytes to the site of infection, and it would therefore be reasonable to expect that lymphopenia would correlate with recovery from infection. However, in the recent influenza A virus H₅N₁ outbreak, low leukocyte counts correlated with severity of disease^ref. In addition, the T cells present during acute infection are functionally impaired, with reduced lectin-induced stimulation^{ref1, ref2}, suggesting that these quantitative and qualitative changes may not simply be due to migration of cells. A number of factors probably contribute to these observations. For example, virus load, as well as viral components that confer pathogenicity, may influence the milieu of cytokines and the composition of responding cells. These factors may act on both naïve and effector B and T cells to result in lymphopenia. The cell type that may mediate this lack of response is the dendritic cell (DC), since it transports virus to the draining lymph node^ref and has direct contact with T cells. The interactions between DC and naïve and memory T cells determine both the magnitude and quality of the immune response. Influenza virus alters this interaction in vitro^ref. In this in vitro system, we examined the effects of influenza virus infection on DC function. DC were cultured from H-2^b bone marrow and then used to stimulate H-2^d allogeneic T cells. Since this response is not virus specific, the ramifications of influenza virus infection were determined by comparing T-cell proliferation stimulated by uninfected and virus-infected DC. When DC were infected with a low dose of A/PR/8/34 (PR8), there was increased T-cell proliferation in response to influenza virus-infected DC^ref. This altered response was dependent on viral neuraminidase (NA) and did not require infection of the DC with influenza virus. One or more mechanisms may mediate this effect when sialic acid is removed from glycoconjugates at the DC surface. This may include changes that facilitate interactions between the major histocompatibility complex (MHC) class I-peptide complex with the TcR, B7-1 with CD28, and adhesins with their ligands or changes in charge at the cell's surface that result in a general increase in contact. However, our current results show that this enhanced proliferative response is not observed when DC are infected with high doses of PR8. There may be multiple reasons for the lack of an enhanced response at high PR8 multiplicity of infection (MOI). For example, since influenza virus induces apoptosis of infected cells^ref, greater numbers of virus particles may induce greater DC apoptosis, thereby reducing the number of effective stimulators in the culture. Alternatively, at high doses of virus, virions released from the DC may interact with T cells, resulting in their reduced proliferation. Viral NA could contribute to this reduced response by desialylation of T-cell surface glycoproteins. This would result in DC and T cells having equal charges, so that opposite attractive charges would no longer facilitate the interaction between them. Other reasons for the dose-dependent proliferative response may be that properties of DC that contribute to successful T-cell activation are altered at high virus doses, or that, under these conditions, cytokines that inhibit proliferation are secreted. At a high MOI, a number of changes occur in DC. The most notable physical change that provides a reasonable mechanism to explain the reduced response to DC infected with high doses of PR8 is the formation of DC clusters that exclude T cells. However, neutralization of TGF-b₁ restored the enhanced alloreactive T-cell response, suggesting that this cytokine plays a primary role in reducing proliferation^ref.

• human SAP binds much more avidly than mouse SAP to the virus, but has no effect  in human influenza infection.
• clonal characteristics of effector CD8⁺ T cells specific for :
• in HLA-A2⁺ adults is almost exclusively directed at residues 58–66 of the virus matrix protein (MP(58–66)). Vb17Va10.2 TCRs containing a conserved arginine-serine-serine sequence in CDR3 of the V segment dominate this response. Whereas the TcR typically fits over an exposed side chain of the peptide, in this structure MP(58–66) exposes only main chain atoms. This distinctive orientation of Vb17Va10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved Arg in Vb CDR3 into a notch in the surface of MP(58–66)–HLA-A2.
• nucleoprotein (NP) and acid polymerase (PA) peptides presented by H2D^b. Using a single-cell approach and determination of CDR3b profiles, a limited, predominantly "public" repertoire was found for CD8⁺D^bNP₃₆₆⁺Vb8.3⁺cells, whereas diverse and "private" TcRb clonotypes were typical of the CD8⁺D^bPA₂₂₄⁺Vb7⁺ response. This single-cell approach has now been used to relate the contributions of particular clonotypes (or affinities) to high-avidity TCRs, as defined by binding under conditions of limiting tetramer availability. At least by the measure of CDR3b usage, no difference could be found between total and high-avidity populations in the spectrum of TCR-pMHC affinities throughout the limited, and relatively public, CD8⁺D^bNP₃₆₆⁺Vb8.3⁺ populations. Conversely, the more even (by clone size), diverse, and private CD8⁺D^bPA₂₂₄⁺Vb7⁺ response was characterized by the clear partitioning of the largest T cell clones in the high-avidity compartment. These results suggest that the relatively constrained CD8⁺D^bNP₃₆₆⁺Vb8.3⁺ set utilizes a relatively narrow range of affinities, whereas the broader CD8⁺D^bPA₂₂₄⁺Vb7⁺ response is induced at a range of TCR-pMHC affinities. Thus, whereas TCR sequence (or affinity) appears to contribute substantially to the avidity profile of diverse virus-specific CD8⁺ populations, other mechanisms may be prominent where the TCR spectrum is more limited^ref
• high levels of serum IFN-a are correlated with febrile seizures in pediatric influenza
• MIF and and CXCL8 are released from lung epithelial cells rendered necrotic by influenza A virus infection.

myxovirus resistance-1 (MX1) is a member of the dynamin family of large GTPases induced by type I interferon in neutrophils that self-assembles into long filamentous homo-oligomers then rearranged into rings and more compact helical arrays that tubulate lipids in vitro, associates with the smooth endoplasmic reticulum, and promotes apoptosis of infected cells (on the contrary of MX2)

Laboratory examinations :

• observing live influenza virus in human serum or plasma is unusual. In 1963, low quantities of virus were isolated from blood of a patient on day 4 of illness^ref, and in 1970, the virus was cultivated from blood specimens from 2 patients^ref. Recently, a fatal case of avian influenza A (H₅N₁) in a Vietnamese child was reported. The diagnosis was determined by isolating the virus from cerebrospinal fluid, fecal, throat, and serum specimens^ref; viral RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness^ref. In this case, the H₅N₁ virus could be isolated from plasma on day 10 after symptoms developed. A titer of 3.08 × 10³ copies/mL was obtained from the plasma of a 5-year-old Thai boy with H₅N₁^ref. This case showed the virus in the patient's blood, which raises concern about transmission among humans. Because probable H₅N₁ avian influenza transmission among humans has been reported^ref, this case should be a reminder of the necessity to carefully handle and transport serum or plasma samples suspected to be infected with H₅N₁ avian influenza. Because viable virus has been detected in blood samples, handling, transportation, and testing of blood samples should be performed in a biosafety (category III) containment laboratory to prevent the spread of the virus to healthcare and laboratory workers.
• predictors of influenza virus infection during the influenza season have previously been evaluated in adult studies of the antiviral agent zanamivir; cough and temperature >/=37.8°C predicted influenza virus positivity in 79% of those evaluated. During the influenza season, symptomatic predictors of influenza virus infection are applicable to identification of cases in children, although confirmation of predictive values in subjects 1-4 years of age may require further study of additional signs/symptoms : a temperature >/=38.2°C plus cough predicted 83% (95% CI, 79%-88%) of illnesses that were determined to be influenza virus positive. Cough (positive predictive value, 70%; 95% CI, 64%-75%), but not fever, was the best predictor of influenza virus-positive status in children aged 5-12 years in the oseltamivir trials, but neither cough nor fever were successful predictors in young children 1-4 years of age^ref
• erythrophagocytosis and phagocytosis of inflammatory cells in the liver, spleen, and bone marrow have been reported in lethal cases of infection with the avian influenza A (H₅N₁)^ref and swine influenza A (H₁N₁)^ref viruses

=> epidemic flu or influenza / grip [Fr. grippe] after 1-3 days incubation : fever, dry cough, profound sinus tachycardia, myoarthralgia, anorexia, photophobia, acute rhinitis. Self-limiting within 3-5 days.
Therapy :

• M2 inhibitors : the largest proportion of Asian amantadine-resistant H₅ and H₉ AIV during 1991-2004 occurred in China : resistance is spreading more rapidly among avian influenza viruses of H₅N₁ subtype in Southeast Asia than in North America (Virology). Adamantanes have been used to treat influenza A virus infections for many years. Studies have shown a low incidence of resistance to these drugs among circulating influenza viruses; however, their use is rising worldwide and drug resistance has been reported among influenza A (H₅N₁) viruses isolated from poultry and human beings in Asia. We sought to assess adamantane resistance among influenza A viruses isolated during the past decade from countries participating in WHO's global influenza surveillance network. We analysed data for influenza field isolates that were obtained worldwide and submitted to the WHO Collaborating Center for Influenza at the US Centers for Disease Control and Prevention between 1 Oct 1994, and 31 Mar 2005. Pyrosequencing, confirmatory sequence analysis, and phenotypic testing were used to detect drug resistance among circulating influenza A H₃N₂ (n=6524), H₁N₁ (n=589), and H₁N₂ (n=83) viruses. > 7000 influenza A field isolates were screened for specific amino acid substitutions in the M2 gene known to confer drug resistance. During the decade of surveillance, a significant increase in drug resistance was noted, from 0.4 % in 1994-1995 to 12.3% in 2003-2004. This increase in the proportion of resistant viruses was weighted heavily by those obtained from Asia, with 61% of resistant viruses isolated since 2003 being from people in Asia. These data raise concerns about the appropriate use of adamantanes and draw attention to the importance of tracking the emergence and spread of drug-resistant influenza A viruses^{ref1, ref2}. Adamantane resistance in US H₃N₂ isolates^{ref1, ref2, ref3, ref4} : 7 of 8 resistant variants (accounting for 98.2% of the total) contained Ser31Asn mutation in M2 protein
• 1992-1995 : 0.8%
• 1996-1997 : 0.4%
• 1998-1999 : 2.2%
• 2000-2001 : 1.4%
• 2002 : 1.4%
• 2003 : 1.7%
• 2004 : 1.9%
• October 2004-March 2005^ref : 14.5%
• October-December 2005 : 92.3%. 3 influenza A(H₁N₁) viruses have been tested and have demonstrated susceptibility to adamantanes; CDC recommended not to prescribe them and to use NAIs^ref
From 1998 to 2004, resistance was observed in 2 of 589 H₁N₁ viruses collected worldwide (0.3%, Val27Ala and Glu34Lys) and in 1 of 82 H₁N₂ viruses isolated in the USA (Ala30Thr). 6 H₃N₂ viruses with Ser31Asn also contained Val27Iso in M2; 2 of 8 H₁N₁ isolates had Ser31Asn.
• neuraminidase inhibitors (NAIs) (when administered within 48 hours of symptom onset, antiviral treatment of influenza can reduce the duration of illness by approximately 1 day in healthy adults. Data on the use of any of the 4 antiviral agents during pregnancy are not available). In prophylaxis compared to placebo, NIs have no effect against influenza-like illnesses (ILI) (relative risk (RR) 1.28, 95% confidence interval (CI) 0.45 to 3.66 for oral oseltamivir 75 mg daily; RR 1.51, 95% CI 0.77 to 2.95 for inhaled zanamivir 10 mg daily). The efficacy of oral oseltamivir 75 mg daily against symptomatic influenza is 61% (RR 0.39, 95% CI 0.18 to 0.85), or 73% (RR 0.27, 95% CI 0.11 to 0.67) at 150 mg daily. Inhaled zanamivir 10 mg daily is 62% efficacious (RR 0.38, 95% CI 0.17 to 0.85). Neither NI has a significant effect on asymptomatic influenza. Oseltamivir induces nausea (odds ratio (OR) 1.79, 95% CI 1.10 to 2.93). Oseltamivir for PEP has an efficacy of 58.5% (15.6% to 79.6) for households and of 68% (34.9 to 84.2%) to 89% in contacts of index cases. Zanamivir has similar performance. The hazard ratios for time to alleviation of influenza symptoms were in favour of the treated group 1.33 (1.29 to 1.37) for zanamivir and 1.30 (1.13 to 1.50) for oseltamivir. Viral nasal titres were significantly diminished by both NIs. Oseltamivir 150 mg daily prevented lower respiratory tract complications (OR 0.32, 95% CI 0.18 to 0.57)^ref.

Prevention :

• killed, subunit and attenuated vaccines. The composition of the vaccine is changed each year in response to antigenic shifts and changes in prevalence of influenza virus strains; the vaccine is usually bivalent or trivalent, containing 1-2 influenza virus A strains and 1 influenza virus B strain. Annual immunization before November is recommended for high-risk individuals (persons over 65 years of age and persons with chronic disease). The last time the vaccine missed the mark was in 1997, when the strain A-Sydney appeared that was significantly different from the strain included in the flu shot, which remained just 30-50% protective. In the 2007–2008 season, the absolute efficacy against the influenza A virus was 72% (95% CI, 49 to 84) for the inactivated vaccine and 29% (95% CI, ?14 to 55) for the live attenuated vaccine, with a relative efficacy of 60% (95% CI, 33 to 77) for the inactivated vaccine^ref
• chemoprophylaxis : inhibitors of coreceptor binding and fusion(amantadine, rimantadine), neuraminidase inhibitors (oseltamivir) can be used for control of institutional influenza outbreaks and are approximately 70-90% effective in preventing illness in healthy adults
• post-exposure prophylaxis : PEP with oseltamivir is likely to be a cost-effective strategy for family contacts in the UK from a healthcare payer perspective when influenza-like illness contact attack rates are 8% or higher and the only treatment given is 'usual care'.^ref
• Development of strategies for mitigating the severity of a new influenza pandemic is now a top global public health priority. Influenza prevention and containment strategies can be considered under the broad categories of antiviral, vaccine and non-pharmaceutical (case isolation, household quarantine, school or workplace closure, restrictions on travel) measures. Mathematical models are powerful tools for exploring this complex landscape of intervention strategies and quantifying the potential costs and benefits of different options. A large-scale epidemic simulation was used to examine intervention options should initial containment of a novel influenza outbreak fail, using UK and the USA as examples. Border restrictions and/or internal travel restrictions are unlikely to delay spread by > 2–3 weeks unless > 99% effective. School closure during the peak of a pandemic can reduce peak attack rates by up to 40%, but has little impact on overall attack rates, whereas case isolation or household quarantine could have a significant impact, if feasible. Treatment of clinical cases can reduce transmission, but only if antivirals are given within a day of symptoms starting. Given enough drugs for 50% of the population, household-based prophylaxis coupled with reactive school closure could reduce clinical attack rates by 40–50%. More widespread prophylaxis would be even more logistically challenging but might reduce attack rates by over 75%. Vaccine stockpiled in advance of a pandemic could significantly reduce attack rates even if of low efficacy. Estimates of policy effectiveness will change if the characteristics of a future pandemic strain differ substantially from those seen in past pandemics^ref.

Complications : pregnant women are at higher risk than nonpregnant women for having complications secondary to influenza. Pregnant women who will be in their 2nd or 3rd trimester during influenza season should be vaccinated against influenza

• respiratory tropism
acute laryngitis and epiglottitis
acute bronchitis
bronchiolitis
primary viral interstitial pneumonia
secondary bacterial community acquired bronchopneumonia (e.g. CA-MRSA) after 4-5 days (leading cause of flu death) : ARDS, hemoptysis
• myocarditis :
• influenza A virus-induced rhabodmyolysis and acute congestive heart failure after high-dose therapy and hematopoietic stem cell transplantation for malignant lymphoma. Early treatment with neuraminidase inhibitor (oseltamivir 150 mg/day for 5 days) may improve the clinical outcome^ref
• neurotropism (diagnosis : PCR on CSF)
• postinfectious encephalomyelitis (PIE) (influenza-associated encephalopathy with with bilateral thalamic necrosis^{ref1, ref2} has mostly been reported in Japan) among young children and school-aged children, including adolescents.
• Reye's syndrome (aspirin and other salicylate-containing medications should be replaced by paracetamole for children with fever and respiratory illness)
• radiculitis
• neuritis
• pharyngotonsillitis
• acute liver failure
• sudden deaths associated with influenza in children occurs with atypical symptoms (e.g., abdominal pain, absence of fever, and mild respiratory symptoms)

• Influenza at WHO : the Global Influenza Programme began in 1947
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• Avian Influenza at CDC
• Avian flu at Connotea
• Immune Epitope Database and Analysis Resource (IEDB) : 412 T cell epitopes and 190 antibody epitopes from 13 viral subtypes and 58 different strains. The relatively low number of antibody epitopes was surprising, given that antibody titers are the only accepted measure of protection from flu. Only 2 H5N1 avian flu epitopes were found, both from a 2004 Vietnam strain of the virus. The lack of H₅N₁ epitope data is not surprising, given the recent emergence of the virus and the special biosecurity measures required for studying it. Antibody epitopes were identified from only 5 of the 10 viral proteins -- most of them from hemagglutinin, neuraminidase, and M2 -- whereas T cell epitopes from all 10 proteins were identified. Only one antibody epitope -- versus 160 T cell epitopes -- was identified by studying human samples (rather than animal samples). The authors comment that interpreting epitope data from human samples is more complex because people, unlike lab animals, typically have been exposed to many flu strains over many years. Using an analysis tool developed as part of the IEDB, the authors found that a higher percentage of T cell epitopes than of antibody epitopes were shared by multiple viral strains. About 11% of T cell epitopes were 100% identical in human and avian strains, while 30% of them were 90% identical, and 50% were 80% identical. In contrast, only 2.7% of antibody epitopes were 100% identical, and < 11% were found to be 80% identical. However, one protective Ab epitope from the M2 protein shows appreciable conservation among the selected human influenza strains and H₅N₁^ref
• The Desktop Flu Tracker by Roche is updated twice weekly with information from FluSTAR^TM (System for Tracking And Reporting Flu), a comprehensive surveillance system that provides reliable reporting of flu activity nationwide. FluSTAR data is gathered from four different surveillance methods, including rapid assay data, laboratory confirmation and clinical diagnosis data, which is compiled and analyzed throughout the flu season. Data for each geographical area is reported on a scale that indicates whether current flu and flu-like illness is low, moderate or high
• European Influenza Surveillance Scheme (EISS)
• Offlu : OIE/FAO network of expertise on avian influenza

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