Their use is essential against pluriserotipic viruses or against latent infections : anyway in acute viroses replication usually preceed symptoms and antimicrobials can't prevent infection. They can interfere with ...

• ... viral molecules (direct-acting antivirals (DAAs)) : low toxicity but virus-specific approach with high likelihood to develop drug-resistance. Targeted proteins are necessary for ...
• ... stabilization of the free virion
• 4,6'-dichloroflavan
• calchons :
• 4'-ethoxy-2'-hydroxy-4,6'-dimethoxycalchon
• WINs (effective against Picornaviridae) :
• WIN51711 / disoxaril
• WIN52035
• WIN52084
• WIN54954
• WIN56291
• R61837
• R77976
• SCH48973

• capsid inhibitors :

• pocapavir [V-073] has potent activity against poliovirus and appears to be safe and well tolerated (10). In the United States, this drug is available by special request from the Food and Drug Administration^ref
• pirodavir analog BTA-798
  
• pleconaril / VP63843 (source : ViroPharma) (effective against Picornaviridae) binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating^ref. It is used for treatment of the common cold^ref, infantile enterovirus meningitis^{ref1, ref2}



• WIN 61209
• WIN 58934
• WIN 65099
• 2,2'-dithiobisbenzamide (effective against HIV-1)
• ... receptor binding
• polyanionic compounds (gp120 inhibitors)
• polysulfates :
• heparan-SO₄^2-
• dextran-SO₄^2-
• polyvinylsulphate (PVS)
• polysulfonates :
• polyvinylsulfonates (PVAS)
• polycarboxylates :
• heparin
• polyoxomethalates
• polynucleotide :
• zintevir
• chicoric acids
• cosalane derivatives
• negatively charged albumins
• soluble receptor decoys
• sCD54 / ICAM-1 (effective against Rhinoviruses)
• CD4 antagonists^ref :
• PRO 542
• BMS-378806
• TNX-355
• PRO 2000
• cyanovirin-N (CV-N)
• CD4 downmodulators : 9-benzyl-3-methylene-1,5-di-p-toluenesulfonyl-1,5,9-triazacyclododecane (CADA) cyclotriazadisulfonamide^ref
• attachment inhibitors (AIs) : BMS-488043
• ... sialidase / neuraminidase (NA) inhibitors (NAI) (for Influenzavirus A and Influenzavirus B^{ref1, ref2}) :

	pharmacokinetics	schedule for pre- and post-exposure prophylaxis	schedule for therapy	side effects	benefits	resistance (de novo never recognized; frequency of emergence is lower than with M2 inhibitors : reduced transmissibility of NAI-resistant variants leads to negligible community spread of such variantsref. Partial cross-resistance between oseltamivir and zanamivir has been reported)	storages (% of population) [WTO regulations allow for drug patents to be violated in the event of medical emergencies, providing the patent holder is compensated at a later dateref]
zanamivir / GG167 / 4-guanidino-Neu5Ac2en (Relenza®; source : GlaxoSmithKline (complete product information, revised on June 2004)), manufactured from a readily available starting material, N-acetyl-neuraminic-acid (NANA), approved in 1999. The lactose dose (80 mg per dose) is insufficient to cause symptoms in lactase-deficient individuals.	guanidino group => a dry powder that is self-administered via oral inhalation by using a plastic device (DiskHaler®) included in the package with the medication. Investigational formulations include nebulised mist, nasal spray, intravenous. After inhalation of 10 mg :  70-87% deposits in the oropharynx 7-21% reaches the lungs 4-17% is systemically absorbed (levels not therapeutic => may not be useful if extrapulmonary dissemination has occurred) plasma half-life of 2.5-5.1 hours => excreted unchanged in the urine unabsorbed drug is excreted in the feces  oral absorption < 5%; so cannot be used in distressed airways total bioavailability : 12-17% (7.8% in ferrets, 43% in mice) Tmax : 0.75-1.5 hours Cmax : 30-50 ng/ml V of distribution : 16 l IC50 NA H1 : 0.5-2.5 nmol/l interacts with acidic residues Glu119, Glu227, and Asp151 in a previously unoccupied area within the active cleft of influenza NA	not approved by FDA, but meta-analyses of clinical trials have shown that it is highly effective for postexposure prophylaxis or early treatment in compliant patients with no underlying pulmonary diseaseref1, ref2	for persons aged > 5-7 years (depending on the country) : 2 inhalations (one 5-mg blister per inhalation for a total dose of 10 mg) twice daily (approximately 12 hours apart) for 5 days : take 2 doses on the first day of treatment whenever possible if there are at least 2 hours between doses. No dose adjustment for patients with either mild to moderate or severe impairment in renal function	while there were reports of bronchospasm post-release, trials specifically targeting patients with mild-to-moderate asthma or COPD showed that zanamivir was an effbctive treatment and had a similar safety profile to the inhaled lactose placeboref. There were no adverse eff-ect on pulmonary function, however, the pack insert reports that it is not recommended for treatment for patients with underlying airway disease unless used with caution under conditions of appropriate monitoring and supportive care (including the availability of short-acting inhaled bronchodilators) due to reports of >20% decline in FEV1. Oropharyngeal or facial edema have also been reported; no increased incidence of other side effects	2-3 days of symptom relief in high-risk patients, those presenting with severe influenza-related symptoms or those who had symptoms for <30 hours. Older patients, aged >50 years, presenting with severe symptoms derived up to 7 days of benefit, as in this group, untreated patients experienced up to 11.5 days of symptoms; reduced nasal pro-inflammatory cytokine levelsref;  no definitive evidence is available regarding the safety or efficacy of zanamivir for persons with underlying respiratory or cardiac disease or for persons with complications of acute influenza. Anyway studies with nebulized and intravenous preparations suggest that zanamivir has good safety and efficacy, even in patients with underlying respiratory diseaseref1, ref2, ref3	NA : E119G (>100 fold reduction in NA sensitivity; isolated in the strain NWS:G70C, a reassortant containing the HA of A:NWS:33 and NA of A:Tern:Australia:G70C:75 (H1N9)ref, A:Turkey:Minnesota: 833:80 (A:Turkey:Minn, H4N2)ref1, ref2 (Gubareva, L.V., Bethell, R.C., Penn, C.R., Webster, R.G., 1996. In vitro characterization of 4-guanidino-Neu5Ac2en -resistant mutants of influenza A virus. In: Brown, L.E., Hampson, A.W., Webster, R.G. (Eds.), Options for the control of Influenza III, Elsevier, Amsterdam, pp. 753–760.), the B:HK:Lee strain, a reassortant with the HA of B:HK:8:73 and NA of B:Lee:40ref, and B:Beijing:1:87 (B:Beijing)ref E119A (>100 fold reduction in NA sensitivity) E119D (>1000 fold reduction in NA sensitivity), all 3 from in vitro selection against A/H1N9, A/H4N2 and B (<= reduction in infectivity for animals)ref1, ref2, ref3, ref4, ref5 R152K from an immunocompromised child infected with influenzavirus B (> 1000 fold reduction in NA sensitivity, 60-fold reduction in infectivity for animals)ref R292K from in vitro selection against A/H4N2 (10-30 fold reduction in NA sensitivity, 400 fold reduction in infectivity for animals)ref1, ref2 no isolates with reduced susceptibility to zanamivir have been reported from clinical trials, although the number of posttreatment isolates tested is limitedref and the risk for emergence of zanamivir-resistant isolates cannot be quantified	only Germany and India are storing zanamivir up in large quantities, although other European countries and the USA have some stocks. Shorter shelf-life than oseltamivir
oseltamivir phosphate / GS4104 (Tamiflu®; developed from a rare natural compound, shikimic acid, and patented by Gilead Sciences, South San Francisco, until 2016, described in 1997ref; in 1996 Roche Holding AG acquired the worldwide rights to develop and market the drug : Tamiflu was FDA approved in 1999 and launched in North America (US and Canada) and Switzerland during 1999/2000. In all key European markets, it was launched by 2002/2003. Chugai Pharmaceutical Co., Ltd. distributes it in Japan; complete product information, revised on June 2004. Available in blister packages of 10 capsules (enough for a 5-day bid course per person) (cost US$34-60, but up to US$190 on some websitesref) or in glass bottles containing 25 ml of suspension after constitution equivalent to 300 mg oseltamivir base. Seasonal flu prevention requires at least six weeks of treatment -- that is 84 pills or about $1,600 of Tamiflu based on current pricesref Gilead Sciences, the original developer, still receives a royalty from Roche equaling about 10% of sales.  US Defense Secretary Donald Rumsfeld served as Gilead (Research)'s chairman from 1997 until he joined the Bush administration in 2001, and he still holds a Gilead stake valued at between $5 million and $25 million, according to federal financial disclosures filed by Rumsfeld. The forms don't reveal the exact number of shares Rumsfeld owns. Gilead made a loss in 2003, the year before concern about bird flu started. Then revenues from Tamiflu almost quadrupled, from $35 to $44.6m, helping put the company well into the black. Sales almost quadrupled again, to $161.6m in 2005. Mr Rumsfeld sold some of his Gilead shares in 2004 reaping - according to the financial disclosure report he is required to make each year - capital gains of > $5m (£2.9 m)ref. The report showed that he still had up to $25m-worth of shares at the end of 2004. Rumsfeld isn't the only political heavyweight benefiting from demand for Tamiflu, which is manufactured and marketed by Swiss pharma giant Roche (Gilead receives a royalty from Roche equaling about 10% of sales). Former Secretary of State George Shultz, who is on Gilead's board, has sold more than $7 million worth of Gilead since the beginning of 2005. Another board member is the wife of former California Gov. Pete Wilson. In July, the Pentagon ordered $58 million worth of the treatment for U.S. troops around the world, and Congress is considering a multi-billion dollar purchase. Rumsfeld recused himself from any decisions involving Gilead when he left Gilead and became Secretary of Defense in early 2001ref. Tamiflu is currently at the center of attention in e-mail spammingref	bulky hydrophobic group => orally administered ethyl ester prodrug (absolute bioavailability = 80%; 30-73% in rodents, ferrets, dogs, and marmosets) converted by liver and gut carboxyesterases to the active metabolite oseltamivir carboxylate / GS4071 / Ro 64-0802 / 4-guanidino-2,4- dideoxy-2,3-dehydro- N-acetylneuraminic acid (4-guanidino-Neu5Ac2en). Oseltamivir activation by HCE1 is inhibited by 90% by clopidogrelref. Oseltamivir carboxylate detectable in plasma within 30 minutes and reaches maximal concentrations after 3 to 4 hours (350 ng/ml) 3% of carboxylate binds to human plasma proteins (vs. 43% of phosphate). The bioavailability of oseltamivir phosphate and oseltamivir carboxylate were assessed in healthy volunteers when delivered as a solution of the API compared with the commercial capsule formulation. The IC90 for the ratios of the 2 treatments (capsule vs. solution) were within the reference region [0.80-1.25] for AUC0-infinity (0.94-0.99) and Cmax (0.93-1.08). Thus, the 2 formulations were bioequivalent for oseltamivir carboxylateref. Tmax : 2.5-5 hours V of distribution : 23-26 l reaches therapeutic levels in many tissues, including the upper and lower respiratory tract apparent plasma half-life of 6 to 10 hours eliminated primarily by glomerular filtration and renal tubular secretion of the active metabolite via the anionic pathway. Neither compound interacts with CYP450s or glucuronosyltransferases. The pharmacokinetic profile of the active metabolite is linear and dose-proportional, with less than 2-fold accumulation over a dosage range of 50 to 500 mg twice daily. Steady-state plasma concentrations are achieved within 3 days of twice daily administration, and at a dosage of 75 mg twice daily the steady-state plasma trough concentrations of active metabolite remain above the MIC for all influenza strains tested. The IC50 and IC90 are in the range of 0.0008 mM to > 35 mM and 0.004 mM to > 100 mM, respectively (1 mM = 0.284 mg/ml) : IC50 NA H1 : 0.3-1.0 nmol/l. It binds through an interaction between the lipophilic 3-pentyloxy side chain and a hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. Exposure to the active metabolite at steady state is approximately 25% higher in elderly compared with young individuals; however, no dosage adjustment is necessary. Exposures (AUCinf) to both the parent drug and active metabolite were increased by more than 80% in the small number of very elderly subjects presented here. However, oseltamivir was well tolerated by these subjectsref a dosage reduction to 75 mg once daily is recommended for patients with creatinine clearance <30 ml/min (1.8 L/h) because renal clearance averages 23 hours and a single dose appears adequate for haemodialysis patients. For those with clearance rates < 10 ml/min caution should be used even at the lower doseref. A 30 mg dose of oseltamivir given once weekly in CAPD or after alternate sessions in HD patients provides sufficient exposure to oseltamivir carboxylate to allow safe and effective anti-influenza treatment and prophylaxisref pharmacokinetics in patients with influenza are qualitatively similar to those in healthy young adults. In vitro and in vivo studies indicate no clinically significant drug interactions other than probenecid (proposed by some to lengthen plasma half-life in cases of drug shortageref1, ref2, a technique invented during the Second World War to extend precious penicillin supplies and that is still widely used alongside antibiotics to treat gonorrhoea and syphilis, and in emergency rooms, where doctors need their patients to have high, sustained levels of antibiotics in their blood : giving oseltamivir together with probenecid doubles the oseltamivir plasma half-life and maximum blood concentration, and multiplies 2.5-fold the patient's total exposure to the drug)ref1, ref2	initially in patients > 13 years of age, but for all ages since 21 Dec 2005ref : 75 mg capsules once dailyfor at least 7 days. Therapy should begin within 2 days of exposure. Safety and efficacy have been demonstrated for up to 6 weeks, but the duration of protection last for as long as dosing is continued. In healthy unvaccinated adults (aged 13 to 65 years), oseltamivir 75 mg once daily taken for 42 days during a community outbreak reduced the incidence of laboratory confirmed clinical influenza from 4.8% for the placebo group to 1.2% (>70% protective efficacy) and from 4.4% to 0.4% (92% protective efficacy) in vaccinated (80%) high-risk (14% had COPD, and 43% had cardiac disorders) elderly residents of skilled nursing homes. In a study of post-exposure prophylaxis in household contacts (aged 13 years) of an index case, oseltamivir 75 mg once daily administered within 2 days of onset of symptoms in the index case and continued for 7 days  reduced the incidence of laboratory confirmed clinical influenza from 12% in the placebo group to 1% for the oseltamivir group. Index cases did not receive oseltamivir in the studyref	for people aged >= 1 year recommended treatment dosages for children vary by the weight of the child:  <15 kg : 30 mg twice a day 15-23 kg : 45 mg twice a day 23-40 kg : 60 mg twice a day >40 kg : 75 mg twice a day.  The treatment dosage for persons aged >13 years is 75 mg capsules bid  for 5 days. It needs to be initiated within 2 days of symptom onset achieves and maintains plasma concentrations above the antiviral IC50 for laboratory strains of influenza A and B for 12 hours after a single 75 mg dose, but the relationship between the in vitro antiviral activity in cell culture and the inhibition of influenza virus replication in humans has not been establishedref. A liquid formulation of oseltamivir (2 mg/kg twice daily for 5 days) is effectiveref.  5 day courses of 150 mg twice daily for treatment and 6 week courses of 75 mg twice daily for prophylaxis are as well tolerated as the approved dose regimensref, giving a larger reduction in viral load in respiratory secretions, as well as a shorter duration of illness in human influenza infections. However, there is currently no evidence that this higher dose of oseltamivir equates with a significant clinical advantage over the standard dosageref1, ref2, ref3. In avian influenza a higher dose of oseltamivir should be considered in the group of patients with very severe disease and especially those having diarrhoea (which may impair drug absorption) or immunosuppressed patients (including the very young and very old) who may have a high initial viral load. Duration of treatment should be extended to 2 weeks since neutralising antibodies appear much later because most humans do not have prior exposure to H5N1	of the 75 case reports presented at the  FDA’s Pediatric Advisory Committee meeting of Nov 18, 2005, 92%, originated from Japanref  12 skin/hypersensitivity events discussed at the FDA’s Pediatric Advisory Committee meeting of Nov 18, 2005  nausea (10% vs. 6% in placebo) and vomiting (9% vs. 3%; 14.3% vs. 8.5% in children) causing discontinuation in 1%, less likely when taken with a light snack (which doesn't significantly affect peak plasma levels or bioavailability), and resolve in 1–2 days despite continued drug administration in most people slight increase in headache frequency was observed in one prophylaxis study in older people worldwide : 42 people died in Japan after they took Tamiflu since 2000, but only 2 of the deaths -- a man in his 50's and another in his 80's -- resulted from using Tamifluref. The 14 children aged 1-16 years died because of suicide (1, one 1 hour after taking Tamifluref), sudden deaths (4), cardiac arrest (4) and pneumonia, asphyxiation and acute pancreatitis (3) (1 death per million Japanese children treated) and 32 suffered "neuropsychiatric events" (hallucinations, delirium, convulsions, and encephalitis. For 8 cases causal relation ruled out : 5 died of influenza-related encephalopathy (first reported by Japanese paediatricians in the 1990s, before oseltamivir was approved). Relationship was difficult to assess because of the use of other medications and/or presence of other diseasesref. A 12- and a 13-yrs old males jumped out of the second floor window of their homes after receiving 2 doses of Tamiflu, and a third 8-year old boy ran into the street because of frightening hallucinations 3 hours after receiving his first dose of Tamiflu and  was rescued by his family from potential traffic injuryref. Tatsuo Kurokawa (2nd L), the health ministry official in charge of pharmaceutical matters, announces at a predawn news conference in Tokyo on March 21 2007 that the government has recommended against the use of Tamiflu among teenagers. After taking Tamiflu, 2 12-year-old boys apparently jumped off the 2nd floor of their houses and broke their legs in February and March. In February, a 14-year-old boy fell to his death at his condominium in Miyagi after apparently taking the drug while a 14-year-old girl died in similar circumstances in Aichiref. Separately, the EMEA’s Committee for Medicinal Products for Human Use requested at its Nov. 14-17 meeting that Roche provide a “cumulative safety review of all available data on serious psychiatric disorders, including all case reports with a fatal outcome where Tamiflu was involved,” after 2 cases of alleged suicides in adolescents reported to the EMEA. In Japan, oseltamivir is often dispensed as a powder in Japan, and mixed at the pharmacy, possibly contributing to higher dosing. The length of dosing may also be different, with Japanese patients possibly stopping administration before the recommended period, contributing to recrudescenceref. Roche commissioned Ingenix to investigate the safety of Tamiflu in 63,000 children (1-12 years old) and concluded that there “was no indication of an increased risk of neuropsychiatric events for children aged 1-12 over that already posed by influenza illness alone. A case report was published about the 22-yo Cape Coral resident Lianne Espositoref. In Dec 2005 2 new cases in Japan. A man in his 50s was hospitalized due to a rash he developed 2 days after starting Tamiflu. He died 10 days from toxic epidermal necrosis, which produced fever and MOF. He had also been taking antibiotics, cold medicine and herbal medicine. A man in his 80s was hospitalized for fatigue on the 5th day on Tamiflu and died 3 weeks later from kidney failure. He had been taking 2 types of medicines to treat high blood pressure and stomach problemsref. A single-dose of 1000 mg/kg of oseltamivir phosphate to 7-day-old rats causes 10-fold higher plasma levels and 1500-fold higher brain levels (3-fold for oseltamivir carboxylate) those found in adults because of blood-brain barrier immaturity, and results in deaths, while 2000 mg/kg do not cause effects in 14-day-old rats. No adverse effect occurred at 500 mg/kg/day administered to 7- and 21-day-old rats, at which exposure to prodrug is 800-fold that expected in a 1-year old child.  haemorrhagic colitisref1, ref2 subcutaneous hemorrhagesref :  from Jan. 1, 1999, to Oct. 31, 2005, Health Canada received 19 reports of increased INR (3.2 to 10.9) suspected of being associated with  oseltamivir, including patients aged 46 to 92 years (median age 84 years) with onset ranging from the day treatment was started to 11 days after starting oseltamivir. 11 of the reports were submitted by the same source and described a suspected interaction between oseltamivir and warfarin. Creatinine clearances were provided in these cases; dosage adjustments of oseltamivir were necessary in 3 cases, as recommended in patients with a creatinine clearance rate of 10-30 mL/min. 6 patients required treatment with vitamin K. At the time of reporting, 12 patients had recovered, 2 patients had not yet recovered, and the outcome was unknown for the remaining 5 patients. In 3 cases, the warfarin dose was increased after the introduction of oseltamivir; the increases in INR occurred following these dose changes. In 3 other cases, decreases in INR occurred during the course of oseltamivir therapy without a reported change in warfarin dose. In 2 cases, clarithromycin and levofloxacin respectively were reported as co-suspect medications; these drugs are known to interact with warfarin and may cause increases in INR. Available data indicate that the potential for drug interactions with oseltamivir is minimalref. arrhythmiasref anasarcaref single doses of up to 1000 mg associated with nausea and/or vomiting pneumoniaref anaphylaxisref	reduces the median time to alleviation of all symptoms by 30% (3 vs. 4.3 days) and the median time to become afebrile by 38% (42.3 vs. 69.3 hours) compared with placebo (earlier initiation of therapy associated with faster resolution), reduces lower respiratory tract complications  resulting in antibiotic therapy, overall antibiotic use, and hospitalization in both healthy adolescents and adults (4.6% vs 10.3% with placebo, and 14.0% vs 19.1% with placebo, respectively) and "at-risk" adults (12.2% vs. 18.5% with placebo, and 0.7% vs. 1.7% with placebo, respectively) with a proven influenza illnessref, and flu-related death rates (-60-90% in children). >103 infectious units/mL of virus were detected in some of the patients who did not shed drug-resistant viruses, even after 5 days of treatmentref. Anyways zeroing of median virus titers occurs in 60 hrs vs. 108 hrs for influenzavirus A and in 24 vs. 72 hrs or influenzavirus B (Hayden F, et al. Efficacy of oral oseltamivir in experimental human influenza B virus infection. Poster presented at: 37th Annual Meeting of the Infectious Diseases Society of America; November 18-21, 1999; Philadelphia, Pa.). The durations of virus shedding from children treated with neuraminidase inhibitors were not significantly shorter than those of untreated patientsref. For later-stage intervention in severe influenza illness, please contact WHOinfluenza @who.int for information on clinical trial protocols. In studies of naturally acquired and experimental influenza, oseltamivir did not impair normal humoral antibody response to infection. Initiation of therapy within the first 12 h after fever onset reduced the total median illness duration by 74.6 h (3.1 days; 41%) >  intervention at 48 h. Intermediate interventions reduced the illness proportionately compared with 48 h. In addition, the earlier administration of oseltamivir further reduced the duration of fever, severity of symptoms and the times to return to baseline activity and health scoresref; reduced nasal pro-inflammatory cytokine levelsref. Effective and well tolerated in high-risk patients with chronic respiratory or cardiac diseasesref	develops within day 4 in 8-12% of aged < 12 yrs, 1-4% in aged > 12 yrs, but viruses that develop resistance appear to be less virulent in laboratory animals and to replicate less efficiently than parent strainsref. Mutants detected in vitro by passage of virus in the presence of increasing concentrations (underlined mutants have been found also in clinical specimens : no drug-induced HA variants have been recognised to date) HAref1, ref2 : H3N2 : A28T  R124M reassortant human/avian virus H1N9 : H154Q NA [N2 numbering of aminoacid residues] : N1 :  H274Y (104-105-fold more resistant, but replicative ability in cell culture was reduced up to 3 logs, as was infectivity in animal models)ref1, ref2 Asn294Ser (intermediate resistance) I222T  A/H3N2 : Glu119Val (E119V) against A:Wuhan:359:95 (H3N2) (20-300-fold reduction in NA sensitivity, 100-1000 fold reduction in infectivity for animals) [Covington E, Mendel DB, Escarpe PA, Tai CY, Soderbarg K, Roberts NA. Phenotypic and genotypic assay of influenza virus neuraminidase indicates a low incidence of viral drug resistance during treatment with oseltamivir. II International Symposium on Influenza and other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr] I222T Arg292Lys (R292K) (10,000 fold reduction in NA sensitivity, 100-1000 fold reduction in infectivity for animals)ref [Ives J, Caar J, Roberts N, et al. An oseltamivir-treatment selected influenza A/N2 virus with an R292K mutation in the neuraminidase gene has reduced infectivity in vivo. II International Symposium on Influenza and Other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr; Covington E, Mendel DB, Escarpe PA, Tai CY, Soderbarg K, Roberts NA. Phenotypic and genotypic assay of influenza virus neuraminidase indicates a low incidence of viral drug resistance during treatment with oseltamivir. II International Symposium on Influenza and other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr.] N9 : Glu119Val (E119V) (300-fold more resistant)  R292K R305Q  Tamiflu can relieve symptoms of human flu within 2 days, causing people to stop taking the drug early; this could encourage the growth of resistant viruses	5-year shelf life for pills. Although buying the drug in bulk form as a powder would have been cheaper and would have extended the shelf life of the government stockpile, large containers of powder can't easily be converted into pills or capsules WHO suggests countries should stockpile enough for > 25% of their population (a global stockpile of 3 million coursesref), but although Roche quadrupled its production capacity since 2003, the current supply is thought to cover just 2% of the world population and the most optimistic estimate of production in the next 5 years (300 million treatments a year by the end of 2006) gives enough to treat just 7%. Roche's discount for governments stockpiling Tamiflu is 50%ref. The API (Active pharmaceutical ingredient) is available at a significantly reduced price (developed world: € 7.70 per 1 treatment course; developing world: € 7.00 per 1 treatment course) and Tamiflu caps purchased by governments for pandemic use are at a significant discount  (developed world: € 15.00 (US$18) per 1 treatment course; developing world: € 12.00 (US$ 15) per 1 treatment course) compared to the seasonal price (seasonal ex-factory price ranges between € 20 and € 51 in Europe)ref. 7 of the 13 sites involved in making Tamiflu are controlled by subcontractors : on Dec 9 Roche reached agreements with 2 US generic drugmakers (Teva Pharmaceuticals and Mylan Laboratories, Inc.), as well as 13 other drug producers. Roche donated the WHO's stockpile 125,000 packs in 2004 and, 3 million treatment courses in Aug 2005 (enough to delay spread in an affected nation according to models), and 2 million treatment courses in Jan 2006 worldwide : 33 million people already used it in countries including USA, Japan, Canada, Australia, the EU, Switzerland and Latin America.ref  23 developed countries stockpiled itref USA : 5.5 million people (872, 386 pediatrics)  treated since 2001, 2.3 million 10-dose courses stockpiled, enough to treat < 1% of Americans, with plans to acquire another 2 million courses by the end of  2005, and projects for 80 million courses to cover about 25% of the population, but  Roche could provide only 13 million courses in 2006, followed by 70 million in 2007. On July 19, 2006, < 2 weeks before a federal deadline for states to announce their plans, at least 16 say they're undecided how much Tamiflu and Relenza they'll buy. 13 others — including California, the biggest state — say they plan to buy their full allotments. The federal government plans to buy 44 million antiviral treatment courses for the states this fiscal year and next, enough to cover 17% of their populations. It wants states to buy 31 million more courses, to reach 25%. Even at a discount — about $15 for Tamiflu, about 20% of the Internet retail rate — cost is a concern. Arizona plans to spend $1 million for 70,000 courses. The state's full allotment of 585,780 courses would cost about $8 million, draining funds needed to prepare hospitals. Colorado plans to buy 5,400 courses — not the 477,470 allotted to it. Arizona and Nebraska are trying to get local entities to pay for more anti-virals. So is Oklahoma, which now plans to buy 9% of its allotment but may increase it. Montana and North Dakota also don't plan to buy full allotments. Other states didn't respond. The Aug. 1 deadline will help drugmakers plan, but states can change orders through Dec. 31. Businesses cannot buy the drugs at the federal discountref. Canada : 16 million pills, enough for 1.2 million people China : Shanghai Pharmaceutical Co., Ltd. reached a sublicensing deal with Roche on Dec 11 Hong Kong : 3.7 million caps (for 6.8 million people), wants to buy 16.8 million more Viet Nam : 25 million tablets,  600,000 donated by Taiwan in July 2004, 420,000 allocated to hospitals; sale to privates banned to prevent overuse; local production permitted by Roche; Romania : 20,000 (2,400 packs) doses donated by Roche in Oct 2005 Brazil : 90 million doses Georgia will reserve Tamiflu for 4000 people within end of January 2006ref Slovenia : for 25% of population Turkey : 500,000 boxes requested, 5,000 donated by Roche in Oct 2005 Greece : enough to supply 5% of the country's 11 million people Italy : 170,000 courses available; 50,000 more at March 2006. 4 million couses ordered on Feb 11, enough for 7 million people (4 million (60%) as treatment and 3 millions for PEP) Taiwan : 1 million treatments available, 1.3 millions to be delivered in 2006, covering 10% of  population Japan : 24.5 million patients (11.6 mil pediatrics) treated since 2001 with 8-10 million patients prescribed the medicine each year (about 70% of the world demand). Domestic sales totaled 35.2 billion yen in 2005. Chugai Pharmaceutical is securing enough Tamiflu for the October 2005- March 2006 flu season to treat 12 million people for 5 days. Of that amount, about one-third is in syrup form. Stockpiles to provide a 3 day course for 25 million people. Currently, Chugai imports ingredients from Germany for inspections, sends them to Switzerland for syrup production and imports them for packaging and sales. Domestic production will be less expensive. Chugai Pharmaceutical has been increasing its supply of Tamiflu since a shortage occurred in the winter season from 2002 to 2003ref India : Cipla Ltd offered to pay Roche and Gilead a 4% royalty on the sale of oseltamivir but after denial she launched a generic on Feb 2006, priced at about Rs 1,000 per strip (650/pack to the wholesaler, exclusive of taxesref) of 10 tablets, in 49 countries where the patent is not applicableref. Cipla will sell Antiflu® for about $12ref. The company has the capacity to produce 100,000 packs of the medicine. Ranbaxy Laboratories Ltd. (RLL) could provide the generic to USA, but on Dec 22 Hyderabad-based  Hetero Drugs Ltd. bag the sub-license granted by Roche to make make 100,000 caps by the end of Dec 2005, 900,000 by the end of Jan 2006, and 20 million caps within Apr 2006 for exports. About 50,000 doses of Tamiflu have already been stockpiled, another 20,000 doses will be delivered at the end of Feb 2006, another 30,000 doses at the end of Marchref Kenya : 2,000 dosesref Korea : 5 companies, including Chongkeundang Corp., Hanmi Pharm Co. and Shinpoong Pharmaceutical Co., said they have successfully produced Tamifluref. UK : 4 million courses on Jan 9 2006, growing by 800,000 doses a month. The 14.6 million target is likely to be reached in late summer 2006 at a cost of pounds 200 million France : in 2005 13.8 million doses Tamiflu and Relenza (33 million in 2007) and 200 million professional-quality face masks (1 billion by the end of 2006) and 40 million doses reserved for vaccine against the transformed H5N1 virus Algeria : 7 million dosesref. On Feb 15, 2006 Algerian state pharmaceuticals group Saidal will sign an agreement with India's Hetero Drugs to produce  6 million doses of Saiflu®ref Jordan : 60,000 dosesref Kuwait : 5 million dosesref; 10 million capsules, which are sufficient to treat about 40% of the  population of 3 millionref Australia : coverage for almost 44% Malaysia : 600,000 tablets  Roche has no patents in : Thailand : 1 million capsules for 100,000 treatments, but 120 million caps needed. Government Pharmaceutical Organization (GPO) has permission to produce 400,000 capsules a day of a generic that will cost 70 baht vs. 120 baht for a Tamiflu capref. The first 200,000 tablets were successfully manufactured on Feb 19 and will be available to the public in July 2006 at 70 baht per tablet, cheaper by 50 baht per unit, compared to the 120 baht per capsule of imported Oseltamivir. GPO is capable of producing about 1 million tablets of the anti-bird flu drug, sufficient for treating up to 100,000 patients, within 15 daysref. Philippine received a donation from the Unilab enough to treat about 50,000 people, and 75,000 capsules from Roche, with another 25,000 to 35,000 expected in Jan 2006 Indonesia bought 400,000 tablets, enough to treat 40,000 people; 800,000 tablets provided by Australia, Japan and Singapore. Kimia Farma or Indofarma will produce 20 million tablets for domestic market (to cover 10% of population) starting from shikimic acid derived via fermentation or from the pod of the star-shaped anise fruit exported from Guangzhou, Chinaref. 5 of the 12 stages in the sseltamivir manufacturing process would be carried out by the private South Korean pharmaceutical company, Daewoong Chemical and 7 in Indonesia

Roche asked the European Medicines Agency, which regulates the use of drugs in Europe, to approve 2 smaller capsules - 30 mg and 45 mg - and planned a similar filing soon with the Food and Drug Administration in the USA. Roche has been granted an accelerated review and is optimistic that EMEA will complete their evaluation by mid 2007. The new capsules would be in addition to the 75 mg dosages already approved and will be easier to use than the liquid suspension already available for children. The capsules also will have a longer shelf life than the liquid^ref.
The anti-influenza drug oseltamivir is an ester prodrug activated by hepatic carboxylesterases. Plasma esterases also convert up to 31.8% of the parent compound to the active metabolite after 4 h ex vivo, with wide interindividual variation. This source of error is removed by adding the esterase inhibitor dichlorvos to blood collection tubes^ref.
Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir 5 mg/kg/day given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were re-challenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and that higher dose may be needed for the treatment of more virulent viruses^ref.
An influenza B virus from an infant with no history of treatment or contact with neuraminidase inhibitors demonstrated a significant reduction in sensitivity to these drugs. A mixed viral population was isolated that contained a novel D197E amino acid substitution that was responsible for this reduction^ref
The impact of various NA mutations (E119A/G/V, H274Y, R292K and N294S) on the susceptibility profiles to different NAIs (oseltamivir, zanamivir and peramivir) was evaluated using recombinant NA proteins of influenza A/WSN/33 (H₁N₁) and A/Sydney/5/97-like (H₃N₂) viruses. In the Nl subtype, the E119V mutation conferred cross-resistance to oseltamivir, zanamivir and peramivir [1,727-2,144 and 5,050-fold increase in IC₅₀ values compared with wild-type (WT)] whereas only oseltamivir-resistance (1,028-fold increase in IC₅₀) was conferred by the same mutation in the N₂ subtype. The N294S mutation conferred resistance to oseltamivir in both the NI and N2 subtypes (197- and 1,879-fold increase in IC₅₀ values, respectively) whereas the H274Y mutation conferred resistance to oseltamivir (754-fold increase) and peramivir (260-fold increase) in the N1 subtype only. The virulence of reverse genetics-rescued A/WSN/33 viruses harbouring H274Y and N294S NA mutations was investigated in Balb/c mice. The WT and H274Y recombinants had identical LD₅₀ values (103 PFUs) and generated similar viral lung titres, whereas a higher LD₅₀ (10 PFUs) and a 1-log decrease in viral lung titres were obtained with the N294S mutant. This study shows that some NA mutations at framework residues may confer resistance to 1 or 3 NAIs depending on the viral subtype. It suggests that certain drug-resistant NA mutants may still be virulent although additional studies using clinical isolates are needed to confirm these results^ref.

The major bottleneck in oseltamivir production is the availability of shikimic acid, which cannot be economically synthesized and is only effectively isolated from Chinese star anise (Illicium anisatum), an ancient oriental cooking spice; although most autotrophic organisms produce shikimic acid, the isolation yield is low. 90% of the harvest is already used by Roche in making Tamiflu. After being extracted from the seeds, a 10-stage manufacturing process procedure then converts shikimic acid into oseltamivir. The reported azide-free Roche synthesis of Tamiflu is summarized graphically below :
The synthesis commences from naturally available (-)-shikimic acid. The 3,4-pentylidene acetal mesylate is prepared in 3 steps: esterification with ethanol and thionyl chloride; ketalization with para-toluenesulfonic acid and 3-pentanone; and mesylation with triethylamine and methanesulfonyl chloride. Reductive opening of the ketal under modified Hunter conditions (JOC 1993, 58, 6756) in dichloromethane yields an inseparable mixture of isomeric mesylates. The corresponding epoxide is formed under basic conditions with potassium bicarbonate. Using the inexpensive Lewis acid magnesium bromide diethyl etherate (commonly prepared fresh by the addition of magnesium turnings to 1,2-dibromoethane in benzene:diethyl ether), the epoxide is opened with allyl amine to yield the corresponding 1,2-amino alcohol. The water-immiscible solvents methyl tert-butyl ether and acetonitrile are used to simplify the workup procedure, which involved stirring with 1 M aqueous ammonium sulfate. Reduction on palladium, promoted by ethanolamine, followed by acidic workup yielded the deprotected 1,2-aminoalcohol. The aminoalcohol was converted directly to the corresponding allyl-diamine in an interesting cascade sequence which commences with the unselective imination of benzaldehyde with azeotropic water removal in methyl tert-butyl ether. Mesylation, followed by removal of the solid byproduct triethylamine hydrochloride, results in an intermediate which was poised to undergo aziridination upon transimination with another equivalent of allylamine. With the librated methanesulfonic acid, the aziridine opens cleanly to yield a diamine which immediately undergoes a second transimination. Acidic hydrolysis then removed the imine. Selective acylation with acetic anhydride (under buffered conditions, the 5-amino group is protonated owing to a considerable difference in pK_a, 4.2 vs 7.9, preventing acetylation) yields the desired N-acetylated product in crystalline form upon extractive workup. Finally, deallylation as above yielded the freebase of oseltamivir which was converted to the desired oseltamivir phosphate by treatment with phosphoric acid. The final product is obtained in high purity (99.7%) and an overall yield of 17-22% from (-)-shikimic acid. Notably, the synthesis avoids the use of potentially explosive azide reagents and intermediates; however, the synthesis actually used by Roche uses azides. Roche has other routes to Tamiflu that do not involve the use of (-)-shikimic acid as a chiral pool starting material, such as a Diels-Alder route involving furan and ethyl acrylate or a isophthalic acid route which involves catalytic hydrogenation and enzymatic desymmetrization. Producing large amounts of Tamiflu not only takes months to complete, but is also hazardous. Some of the steps in the synthesis require careful handling and relatively mild reaction conditions, as they involve the use of potentially explosive azide chemistry. Cipla, the Indian generics company, says it could produce a version of Tamiflu within months (authorized or unauthorized), despite Roche's claims that it would take a newcomer 2–3 years to start from scratch and produce the drug. Cipla says it could create generic Tamiflu as it has experience of making the HIV drug AZT, which relies on similar chemistry. Any competent organic chemist can follow the literature and make Tamiflu in gram quantities. But making a ton of drug and manipulating tons of azide intermediate is quite a different matter. Making large-scale quantities of the drug, of course, relies on having enough starting material. But star anise takes 8 years to grow to maturity and is harvested only from March to May in only 4 provinces in China, and there aren't enough supplies to produce the number of doses needed worldwide. The northern province of Lang Son, the biggest anise-growing area in Vietnam with over 8,000 hectares of star anise, annually produces 5,000-6,000 tons of the dried fruit. Now, the country uses anise as a spice or extracts oil from it for production of traditional medicines and export. 100 grams of the fruit can turn out 5-10 g of shikimic acid (30 kg (66 pounds) of anise pod yielding only 1 kg (2.2 pounds) of acid^ref), or 6 grams of the fruit can make one Tamiflu capsule^ref. Biolyse Pharma Corp, a closely held Canadian company that extracts chemicals from plants in St. Catharines, Ontario, and now makes a generic version of the cancer drug paclitaxel from yew trees, plans to use needles from half a million Christmas trees discarded in Ontario to mine shikimic acid : it will get 500,000 trees in January from a Toronto-based firm called Gro-Bark Ltd, which recycles forest products. Biolyse has a production capacity of about 2 tons a month and plans to get more tree needles from forestry companies. 2-3% of the biomass from various pine, spruce and fir trees is extractible shikimic acid. The company is gearing up to make 50 kilograms a day of finished product or about 666,000 pills daily. It has been working on a generic version of Tamiflu for the past 2 years and has developed a process to shorten the manufacturing process to five weeks. No drug has yet been manufactured for stockpiling because of Roche's patents. Under the 2005 Jean Chrétien Pledge to Africa Act, amendments to the Patent Act give Ottawa authority to grant compulsory licenses of patented drugs for export to eligible countries in need of lower-cost medicines to address public health problems^ref. The price of the acid has risen more than 10-fold to $600 a kilogram in a year^ref. With no other agricultural sources, other than perhaps the leaves of the gingko biloba tree, the pharmaceutical industry needs to find an alternative sustainable supply. Shikimic acid, however, is also a natural intermediate in the formation of microbial amino acids. Researchers could boost production of shikimic acid in strains of the bacterium Escherichia coli by ramping up enzyme activity and feeding the microbe a diet of carbohydrates. Roche already uses engineered bacteria to boost shikimic acid production. Today, around 66% of the shikimic acid used for Tamiflu is gained from star anise and Roche also has access to the remaining shikimic acid through fermentation. This gives us more flexibility, and in particular allows us to become more independent from events such as bad harvests. Roche hopes to scale up its fermentation capacities over the next few years, but there is no firm timeline as to when this will happen. In theory, there is no limitation on how much shikimic acid can be produced by fermentation. The bioproducts industry currently produces 750,000 tons of lysine from glucose by microbial fermentation for use in products such as animal feed. If needed, the same tanks could be used to produce shikimic acid from glucose. Microbes could be engineered to make variations on the Tamiflu starting material and so allow production of Tamiflu analogues with slightly different pharmacological properties that could potentially target emerging strains other than H₅N₁. Frost, who consulted with Roche on how to grow his E. coli strain and purify the shikimic acid on a commercial scale, has started up a company called Draths Industries to help boost supplies of shikimic acid. Draths already has an agreement with one bioproducts firm to contract-manufacture 200 tons of shikimic acid by fermentation, but it could produce more. Draths Industries feels that there is enough capacity to access 1,000 tons of shikimic acid in 14 months. Roche is on record as claiming 0.13 grams of shikimic acid is needed per Tamiflu capsule. So, 1,000 tons should provide enough feedstock for > 20 million capsules a month, or enough doses for 2 million people. This would fulfill Roche's current annual production but would be inadequate for its planned production of 300 million courses of Tamiflu in 2007. But the infection rate could be as high as 50% of the global population — 3.2 billion people. Half of those people would have inapparent or asymptomatic infections — as is often the case with viral diseases — but the other 1.6 billion people could become seriously ill. It is correct that many are now saying that a severe pandemic, along the lines of 1918, would result in a 2% fatality rate among those who become ill. Even if only half of those needed treatment with Tamiflu, this would still require around 30 billion capsules. Tamiflu, however, can be synthesized from other products in a microbial fermentation tank.
• quinic acid, found in the bark of the cinchona tree of Zaire, is also in limited supply, but Frost and his team have already engineered a microbe to make quinic acid under fermentor-controlled conditions
• aminoshikimic acid is easier to isolate from fermentor broth compared with shikimic acid, which could allow Tamiflu to be made in higher yield. Introducing a nitrogen atom into the starting material would eliminate the need to deal with azide and organoazide intermediates.
• ther less well known, but more common sources of shikimic acid are grape juice and white wine. The analysis of 6 Italian and 4 Austrian white wines disclosed shikimic acid concentrations of 15-27 mg/L and quercetin concentrations up to 10.6 mg/L^ref. Unscrupulous retailers could be tempted to sell Japanese Star Anise Illicium religiosum (Shikimi) instead of the Chinese variety to meet the growing demand for the spice. Its appearance is very similar, but it is neurotoxic. Physicians should discourage the use of herbal remedies of doubtful nature and unproven efficacy in influenza. Should patients insist, physicians may recommend small quantities of grape juice, rather than letting them run useless risks^ref.
• a research team led by Masakatsu Shibasaki at the University of Tokyo can now manufacture Tamiflu using the petroleum-based chemical 1,4-cyclohexadiene via asymmetrical catalysis^ref. Tokyo University applied for a patent on 23 February 2006. Shibasaki will present his results at the Pharmaceutical Society of Japan's annual meeting on 28-30 March^ref
• a short synthetic pathway has been developed for the synthesis of oseltamivir (1) or the enantiomer (ent-1) starting with butadiene and acrylic acid, 2 of the cheapest chemicals you can buy. The intermediates and conditions for this process are summarized in Scheme 1. The synthesis provides a number of advantages: (1) use of inexpensive and abundant starting materials; (2) complete enantio-, regio-, and diastereocontrol; (3) avoidance of explosive, azide-type intermediates; (4) good overall yield (ca. 30%, still not completely optimized); and (5) scalability^ref1,ref2
The sad truth is that all of these efforts will prove to be academic if a pandemic strikes within months. Even if we wait a year before a breakout of the virus, which should give manufacturers enough time to make reasonable supplies of Tamiflu, our ability to cope with a putative bird flu pandemic shouldn't hinge on small molecules. Within the next few years, no matter what is done, there won't be enough Tamiflu to use as we like for everyone who needs it^{ref1, ref2}. Shikimic acid (1 µmol/l-10 pmol/l) has no effect on the release of 2 predictors of antiviral activity—cytokines IL-8 and IL-6—in human peripheral blood mononuclear cells or when the mononuclear cells were stimulated by LPS. Quercetin, another natural polyphenol found with shikimic acid in plants such as Hypericum laricifolium (romerillo) and Cyclopia subternata (honeybush tea)—traditional herbal remedies used in Ecuador and South Africa, was also tested. Only the concomitant presence of the 2 compounds (concentration 10 pmol/l) produced a nearly 2-fold increase in IL-8 (9440 (SD 778) pg/ml vs. 5657 (247) pg/ml) and an 18-fold increase in IL-6 (3958.63 (SD 2478.98) vs 215.83 (SD 51.56) pg/ml) versus controls^ref.
On Thu Mar 16, 2006 Roche announced it would lift production by an additional 100 million treatments to a total of 400 million treatments by the end of the year, after striking deals with > 15 external contractors located in 9 different countries around the world, including Albemarle, Ampac Fine Chemicals LLC, API Corporation, Clariant AG, DSM NV, FIS, Martek Biosciences Corporation, Novasep/Dynamit Nobel, PHT International, PPG Industries, Sanofi-Aventis, Shaanxi Jiahe Phytochem Co and Siegfried Ltd (and Hetero Drugs, Shanghai Pharmaceuticals Co Ltd, and Hangzhou Electrochemical Group (HEC) Ltd). Roche Holding AG expects 1.1 billion to 1.2 billion Swiss francs ($921 million) in sales of the drug to governments in 2006, excluding its sales as a treatment for regular influenza^ref
On Fri Mar 17, 2006, the World Health Organization (WHO) urged the medical fraternity and pharmaceutical companies to urgently conduct greater studies into the efficacy of the drug Tamiflu, manufactured by Swiss company Roche Holding AG, in preventing and treating cases of bird flu in humans^ref. In a related development, on Mar 19, the Chinese government has given the go-ahead for clinical studies of Tamiflu produced by the Shanghai Sunve Pharmaceutical Co., Ltd.^ref.
In late October 2005, Roche announced that it was suspending shipments to pharmacies in the USA and Canada until the North American seasonal flu outbreak began, to address concerns about private stockpiling and to preserve supplies for seasonal influenza. It said that when distribution resumes in Canada, the remaining available drug will be saved for use in high-risk settings like long-term care facilities and hospitals. The restrictions were lifted on Jan 24 in response to reports of increasing flu outbreaks around the country and the recent revelation of a high rate of resistance to 2 older antiviral drugs used to treat flu^ref. Sales were suspended in Hong Kong as well, and on November 8, 2005 also in China. Roche said it would instead send all supplies to China's health ministry. In January 2006 Roche is shipping Tamiflu directly to wholesalers in those markets including the parts of California, Arizona, New Mexico, Utah, Texas, Oregon, Nevada and Kansas, recently hard hit by the flu.
Both zanamivir and oseltamivir carboxylate distribute across the placenta and into breast milk in rats^ref, but no data are available for humans. Given the potential teratogenicity of the M2 inhibitors, the NA inhibitors would be preferred, especially during the second and third trimesters, when the risk of serious complications increases. The lower systemic exposure from inhaled zanamivir is a consideration in this decision.
The first package of counterfeit Tamiflu was intercepted by US customs on Nov. 26 2005 at an airmail facility near San Francisco International Airport. Since then, agents have seized 53 separate packages sent by Asian suppliers to individuals who placed orders via the Internet (so far, no shipments were bound for doctors or hospitals), each containing 10-50 fake capsules^ref. Counterfeit versions of Tamiflu have also now cropped up in Britain and the Netherlands. Initial laboratory tests have shown that the products contained vitamin C instead of the active ingredient oseltamivir. Swissmedic said that no bogus Tamiflu had made its way into the official supply chain — something Roche says would be very unlikely anyway, given the security features included in its packaging. Britain's Medicines and Healthcare Products Regulatory Agency said it had identified around 20 Internet sites that were illegally advertising Tamiflu, four of them in the UK, and was analysing test purchases from the sites for bogus ingredients^ref. Federal agents also confiscated a suspicious small package of Tamiflu in Honolulu on Dec. 22, 2005, but haven't completed a chemical analysis of it. The FDA sent warning letters to 9 Internet marketers it accused of peddling other phony remedies. Most were dietary supplements that, among other things, claimed to kill the bird-flu virus or provide ``a natural virus shield.'' Tamiflu isn't the only worrisome bird-flu remedy being promoted on the Web. The 9 Internet companies sent warning letters by the FDA offered everything from ``soil-based organisms'' to ``systemic enzymes'' for combating bird and other flu. One of the 9 is Polycil Health of Torrance, whose Web site as of Friday still touted the virus-fighting attributes of its Immunocil. The company claims the product, made from humic acid, is based on an ancient folk medicine. But the FDA says Polycil's claims that Immunocil is useful against bird-flu and other influenza ``are not supported by competent and reliable scientific evidence.'' Polycil's president, Paul Benveniste, told the Mercury News he will remove the offending references to Immunocil soon. But he added he plans to begin promoting a new substance, also containing humic acid, which he'll call his ``wellness formula.''^ref. 18 websites selling bogus Tamiflu were identified in Malta, Canada, Britain, the US, Switzerland, Bahrain, Jersey, Cyprus and Singapore^ref. On Jan 9, 2006 U.S. Customs and Border Protection officers seized 250 parcels of fake Tamiflu from Mauritius at an airmail facility in New York City. Officers also seized one package in Chicago in the biggest interception of the counterfeit flu vaccine to date^ref. A simple and rapid liquid chromatographic assay for the evaluation of potentially counterfeit oseltamivir (Tamiflu^®) has been developed and assessed. The assay uses approximately 1 mg Tamiflu^® powder when used for authentication and content estimate. The procedure was validated using 50 replicates analysed during 5 independent series with a total R.S.D. of 11.2%. The assay can also be used to monitor the exact content of oseltamivir in Tamiflu^® capsules. 1 Tamiflu^® capsule was transferred to a 250 mL volumetric flask and 150 mL water was added. The flask was placed in an ultrasonic bath at 40°C for 20min to dissolve the capsule. The solution was allowed to cool to room temperature before the flask was filled up to the mark (250 mL). A small aliquot was centrifuged and then directly injected into the LC-system for quantification. Oseltamivir was analysed by liquid chromatography with UV detection on a Hypersil Gold column (150 mm x 4.6 mm) using a mobile phase containing methanol-phosphate buffer (pH 2.5; 0.1M) (50:50, v/v) at a flow rate of 1.0 mL/min. The assay was implemented for the analysis of Tamiflu^® purchased over the Internet and at local pharmacies in Thailand and Vietnam^ref.
Officers from the government's Medicines and Healthcare Products Regulatory Agency (MHRA) carried out a raid at an undisclosed address in London on Wed Jan 11, 2006 and seized 5,000 packets, an estimated 500,000-pound (731,900 €, 885,800 US$) haul of a suspected stolen batch of genuine Tamiflu^ref.
Veterinary studies :
• pharmacokinetics of oseltamivir carboxylate (OC) in horses (n=6) after oral administration of its prodrug oseltamivir. The binding rate of OC to horse plasma proteins was negligible (<1%). Oral administration of oseltamivir of 2 mg/kg body weight of oseltamivir to horses provided a plasma concentration of OC (mean maximum concentration: 257.9 ng/ml) above the inhibitory concentrations against equine influenza A viruses determined in vitro. However, because OC is rapidly eliminated from horse plasma (mean elimination half-life: 2.5 hr), administration intervals should be less than 10 hr to retain a suitable concentration when using a single dose of 2 mg/kg oseltamivir^ref.
The safety and efficacy of repeated treatment or prophylaxis courses have not been established. The present circulating genotype Z is intrinsically resistant to amantadine and rimantadine^ref. NAIs would not be expected to affect the antibody response to injected, inactivated influenza
vaccine and this has been confirmed with zanamivir^ref. These agents could therefore be used for immediate protection in conjunction with late-season
immunisation. This means that when influenza is reported to be circulating in the community, NAIs would have the potential to provide protection to an unvaccinated target population during the 2- to 4-week period before full immunity is induced after vaccination. However, oral oseltamivir and, possibly, inhaled zanamivir might reduce the replication and immunogenicity of intranasal, live-attenuated vaccine
Prospective surveillance for influenza was performed during the 2002 Salt Lake City Winter Olympics. Oseltamivir was administered to patients with influenzalike illness and confirmed influenza, while their close contacts were given oseltamivir prophylactically. Influenza A/B was diagnosed in 36 of 188 patients, including 13 athletes. Prompt management limited the spread of this outbreak^ref.
Acyclovir in H₅N₁isolates patients :


	age, sex, location, and ID in the original paper	occupation and epidemiological information	clinical characteristics and laboratory values at admission	schedule	outcome
Hong Kong 1997 (10 out of 18 patients) : amantadine was used for most of the patients with severe infections. However, the majority of those patients were already at a critical stage of the disease when the drug was administered, and amantadine is known to be most effective when it is given at an early stage of disease (within 2 days of onset)ref	3 yrs-old male from ? (patient 1)		onset of illness on 10 May 1997; persistent MODS; no major illnesses	acyclovir started at late stage of disease	died 5 days after hospital admission

Amantadine in H₅N₁isolates patients :


	age, sex, location, and ID in the original paper	occupation and epidemiological information	clinical characteristics and laboratory values at admission	schedule	outcome
Hong Kong 1997 (10 out of 18 patients) : amantadine was used for most of the patients with severe infections. However, the majority of those patients were already at a critical stage of the disease (> 4 days after the onset of symptoms ) when the drug was administered, and amantadine is known to be most effective when it is given at an early stage of disease (within 2 days of onset)ref1 (Arabian numerals), ref2 (Roman numerals)	54 yrs-old male (patient 4)		onset of illness on 24 Nov 1997; fever (39°C) for 6 days, cough, vomiting, shortness of breath, bilateral pneumonia, persistent MODS; WBC 5.8, lymphopenia 0.3, AST 157, ALT 70, acute renal failure, ECG evidence of old myocardial infarct	amantadine started on hospital admission (6 days after the onset of illness) for 6 days	died 7 days after hospital admission with areas of hemorrhage, and fibrinous exudates in air spaces, and extensive acute tubular necrosis
	5-years old female (patient 5 or VI)		onset of illness on 7 Dec 1997; fever (37.8°C) for 3 days, rhinorrhea, vomiting and diarrhea, epistaxis, WBC 8.4, lymphocytosis 6.05, atypical lymphocyte 0.34 x 103/mL, ALT 43, AST 91, fever, URTI symptoms; no major illnesses	amantadine started on hospital admission	recovered
	24-yrs old female (patient 7)		onset of illness on 4 Dec 1997; fever (39°C) for 3 days, productive cough, rhinorrhea, sore throat, dizziness, headache, decreased appetite, right-upper abdominal pain, congested oropharynx, clinical and radiological signs of severe pneumonia, septic shock, ARDS, bilateral pleural effusion, requiring ventilatory support for 21 days, leukopenia 2.29, lymphopenia 0.28, PLT 161, aPTT 40.7, PT 12.4, AST 60	amantadine started on the third day after hospital admission	recovered
	2 yrs-old male (patient 8)		onset of illness on 12 Dec 1997; fever (39°C) for 1 day, sore throat, rhinorrhea and cough; WBC 14.66, neutrophilia 9.31 x 103/ml; glucose-6-phosphate dehydrogenase deficiency	amantadine started on hospital admission	recovered
	60-years old female (patient 12)		onset of illness on 16 Dec 1997; fever (39.7°C) for 2 days, no pulmonary symptoms or signs, progressive bilateral pneumonia, ARDS, renal failure, progressive MODS; WBC 5.4, lymphopenia 0.3, PLT 147, AST 62, ALT 52, malignant thymoma treated with radiotherapy 10 years ago	amantadine started on the 5th day after hospital admission	died 6 days after hospital admission
	25-years old Filipino female (patient 13 or V)		onset of illness on 17 Dec 1997; fever (39.6°C) for 4 days, cough, sore throat, rhinorrhea, diarrhoea, headache, dizziness, clinical and radiological signs of left-lower-lobe consolidation, persistent MODS; leukopenia 2.4, lymphopenia 0.4, PLT 106, ALT 74, ARDS, bilateral massive hemorrhagic pleural effusion, no major illnesses	amantadine started on the third day after hospital admission	died 24 days after hospital admission (1 month after the onset of illness) of acute respiratory distress syndrome and multiple organ failure showing features of reactive hemophagocytic syndrome, central lobular necrosis, fatty changes, and extramedullary hematopoiesis in the liver and prominent acute tubular necrosis, congestion, and swelling in the kidneys
	19-yrs-old female (patient 16 or IX)		onset of illness on 14 Dec 1997, fever (39°C) for 3 days, productive cough, vomiting, clinical and radiological signs or left-lower-lobe pneumonia, progressive bilateral pneumonia/ARDS requiring steroid therapy and ventilatory support for 20 days, pleural effusions requiring tube thoracotomy drainage, WBC 4.0, lymphopenia 0.6, PLT 106; no major illnesses	amantadine started on the third day after hospital admission	recovered

Ribavirin in H₅N₁isolates patients :


	age, sex, location, and ID in the original paper	occupation and epidemiological information	clinical characteristics and laboratory values at admission	schedule	outcome
Hong Kong 1997 (1 out of 18 patients)ref	13-yrs-old female from ? (patient 3 or III)		onset of illness on 20 Nov 1997; fever (39.8°C) for 4 days, rhinorrhea, cough, headache, clnical and radiological signs of patchy right-lower-lobe consolidation. Haemoptysis due to pulmonary hemorrhage; ARDS; gastrointestinal bleeding, renal failure, haemophagocytosis, leukopenia 2.54; lymphopenia 0.18, platelet 31, PT 12.8, aPTT 38.5, urea 30.6, creatinine 222; persistent MODS; no major illnesses in medical history	amantadine and intravenous ribavirin (1 g every 8 h)  started on the seventh day after hospital admission	died 25 days after hospital admission
Vietnam 2004 (2 out of 10 patients) : relatively low doses not associated with obvious effectivenessref	10 yrs-old male from Bac Nihn  (patient 3)	student; family members are farmers who kept chickens, which died unexpectedly 5 days before onset of illness; parents and older sibling healthy	cough, dyspnea, fever 39°C, ABP 105/80 mmHg, RR = 64 bpm, crackles, HGB 12.4 g/dl, WBC 2800/ml, lymphocytes 860/ml, neutrophils 1900/ml, platelets 135,000/ml, O2 saturation during receipt of 40% O2 86%, PCR for H5N1 performed on day 9 of illness	800 mg 3 times a day	died day 14
	8 yrs-old female from Ha Tay (patient 4)		cough, dyspnea, fever 38.5°C, ABP 80/40 mmHg, RR = 60 bpm, bleeding gums, HGB 12.3 g/dl, WBC 1900/ml, lymphocytes 250/ml, neutrophils 780/ml, platelets 91,000/ml, ALT 265 U/l, AST 1,217 U/l, creatinine 27 mmol/l, O2 saturation during receipt of 40% O2 50%, PCR for H5N1 performed on day 6 of illness	400 mg 3 times a day	died day 7
China 2005ref	a 9-yrs-old boy from Hunan	brother of a 12-years old girl who fell ill on Oct 8 died of ARDS, DIC, and MODS on Oct 17 but tested negative for H5-specific antibodies in both microneutralisation and haemagglutination-inhibition assays against the A/Hunan-Xiangtan-he/12/2005 virus, which was isolated from the only live chicken remaining in the household	developed fever and cough on Oct 10	amantadine, ribavirin, corticosteroids, and broad spectrum antibiotics since Oct 17	discharged on Nov 12. At his final follow-up on Dec 9, 2005, he remained asymptomatic

An H₅N₁ influenza virus, A/Hanoi/30408/2005 (the same as A/VN/HN30408 from the earlier WHO's Manila report in 6-7 May 2005^ref), was isolated on 27 Feb 2005 from a 14 year old Vietnamese girl (patient 1) who had received a prophylactic dose (75 mg once a day) of oseltamivir from 24 to 27 Feb 2005 and was given a therapeutic dose (75 mg twice daily) for 7 days starting on 28 Feb 2005. No virus was isolated from specimens after the administration of increased doses of oseltamivir. The patient recovered and was discharged from hospital on 14 Mar 2005. Direct sequencing after amplification by PCR of the virus isolated from a specimen collected on 27 Feb 2005 indicated that some of the virus population had the H274Y mutation in its neuraminidase protein^{ref1, ref2, ref3} : the IC₅₀ of oseltamivir carboxylate^ref in the isolate was 90 nM, which exceeds the IC₅₀ for oseltamivir-sensitive viruses (0.1-10 nM )^ref. 10 plaque-purified viral clones, randomly picked from the resultant plaques, were classified into 3 groups according to their response to oseltamivir: 6 were highly resistant to the drug (IC₅₀ = 763 nM), 3 were slightly resistant (IC₅₀ values between 7.1 and 12.5 nM) and one was highly sensitive (IIC₅₀ = 0.6 nM). The highly resistant viruses had Y274 in their neuraminidase, whereas those showing only slight resistance had Ser294. Patient 1 had not had any known direct contact with poultry, but had cared for her 21  year old brother (patient 2) while he had a documented H₅N₁ virus infection. The neuraminidase gene of the brother's virus was identical to clone 7 of the girl's virus. Also, the haemagglutinin gene of the brother's virus was identical to clones 2 and 9 of the girl's virus, apart from a nucleotide change at position 271. The timing of infection in these 2 patients, together with the lack of known interaction of the girl with poultry, raises the possibility that the virus could have been transmitted from brother to sister. The growth of a highly oseltamivir-resistant clone (H274Y, clone 9) and of an oseltamivir-sensitive clone (H274, clone 7) was assessed in ferrets^ref. Viral titres were higher in animals infected with the oseltamivir-sensitive virus. Oseltamivir treatment reduced viral titres in animals infected with the drug sensitive virus, but not in animals infected with the resistant virus. However, all of the viral clones, including those highly resistant to oseltamivir, were sensitive to zanamivir^{ref1, ref2} (IC₅₀ = 0.5-3.1 nM). In ferrets, zanamivir treatment reduced viral titres in animals infected with virus that was oseltamivir-sensitive or oseltamivir-resistant. The viruses bound in vitro to different configurations of sialyl glycopolymers, similar to those on the host's cell-surface receptor^ref. Binding by 2 of the viral clones (clones 7 and 9) was compared with binding by an avian flu virus (A/duck/Mongolia/ 301/2001) and another human flu virus (A/Kawasaki/1/2001) : both H₅N₁ clones bound to a-2,3-linked polymer and (less efficiently) to a-2,6-linked polymer. The A/duck/Mongolia/301/2001 virus also bound a-2,3-linked polymer but did not bind a-2,6- linked polymer at all; the A/Kawasaki/1/2001 virus bound strongly to a-2,6-linked polymer but only weakly to a-2,3-linked polymer. The broader binding properties of the H₅N₁ viral clones may reflect a degree of adaptation in human hosts. Although the findings are based on a virus from only a single patient, they raise the possibility that it might be useful to stockpile zanamivir as well as oseltamivir in the event of an H₅N₁ influenza pandemic^ref
Oseltamivir against H₅N₁ :
• Meijer, A., J.A. van der Goot, G. Koch, M. van Boven, and T.G. Kimman (2004) [Meijer, A., van der Goot, J. A., Koch, G., et al. (2004). Oseltamivir reduces transmission, morbidity and mortality of highly pathogenic avian influenza in chickens. In Options for the Control of Influenza V, (Kawaoka, Y., Ed), pp. 455–8. International Congress Series 1263, Elsevier BV, Netherlands]. Oseltamivir reduces transmission, morbidity, and mortality of highly pathogenic avian influenza in chickens. International Congress Series 1263: 495-498.  Abstract:  The effect of the neuraminidase inhibitors zanamivir and oseltamivir on the transmission of highly pathogenic avian influenza (HPAI) in chickens was studied. Per group, five chickens inoculated with HPAI A/Chicken/Pennsylvania/1370/83 H₅N₂ virus were placed 1 day post-inoculation (p.i.) in one cage with five contact chickens. Inoculated and contact chickens were treated twice daily from 1 day before inoculation up to day 7 p.i. All untreated inoculated and contact chickens became infected and 4 inoculated and 2 contact chickens died. Similarly, all of the zanamivir-treated inoculated and contact chickens became infected and all inoculated and four contact chickens died. Obviously, locally active zanamivir has no effect. In contrast, although oseltamivir could not prevent tracheal infection of the inoculated chickens, none had an infected cloaca and only one died. More important, only after stopping treatment 3 contact chickens became positive, suggesting limited transmission within or after the treatment period. In conclusion, treatment with systemically active oseltamivir limits to a large extent a severe outcome and chicken-to-chicken transmission of HPAI virus.
• Roberts, N. A., Wiltshire, H. R., Mendel, D. B. et al. Oseltamivir carboxylate is effective against all subtypes of influenza neuraminidase. Poster # 135, ASM Biodefense Research Meeting, Baltimore, March 2003.
• Balasingham S, Manvell R, Shell W, et al. Antiviral Activity of Oseltamivir Carboxylate (Tamiflu) against a potential human pandemic influenza A virus (Chicken H5N1). In Program and Abstracts of the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), American Society for Microbiology, Washington DC, USA on 31 October 2004, Abstract #3839
therapy : although there is no documented benefit from any antiviral therapy initiated later in the course of illness, oseltamivir has been used regardless of the time of the illness for very sick patients with H₅N₁, because neuraminidase inhibitors are the only potentially effective antiinfluenza drugs for H₅N₁. Since we know that in trials using Tamiflu to treat normal influenza you can increase the dosage safely, clinical trials led by the US based National Institutes of Health (NIH) and WHO with the cooperation of Roche may start as early as January 2006 in countries including Vietnam to study whether doubling the recommended dose (150 mg given twice a day for 5 days) provides more effective treatment in H₅N₁cases, Peter Horby, a Hanoi-based epidemiologist with WHO, said on Dec 9, 2005. The ministry of Health in Vietnam has already instructed doctors to lengthen the treatment time for Tamiflu to 7 days from 5 days for bird-flu patients, according to Tran Tinh Hien, deputy director of the Hospital for Tropical Diseases in Ho Chi Minh City, which has treated bird-flu patients in the past^ref. As a result, governments may be forced to stockpile far greater amounts of the drug than they had previously anticipated. Thailand joined on March 7, 2006. The 2-year Thailand-Indonesia-Vietnam joint study, lead by Associate Professor Tawee Chotpitayasunondh and called the Southeast Asia Network of Clinical Research on influenza, will focus on finding and proving the effectiveness of high doses of oseltamivir on humans who have contracted the H₅N₁cases virus, requiring about 400 volunteers in the 3 countries^ref.


	age, sex, location, and ID in the original paper	epidemiological information (occupation; source(s) of contagion)	clinical characteristics and laboratory values at admission	schedule	outcome
Hong Kong, 2003ref	33-year old man from Hong Kong	father of a family of 5 who visited Fujian province, mainland China, from Jan 26, to Feb 9, 2003. The 7-year-old daughter developed high fever and respiratory symptoms 2 days after arriving there and died of a pneumonia-like illness 7 days after the onset of symptoms. The exact cause of death could not be ascertained since the girl was buried in mainland China. The family returned to Hong Kong on Feb 9, 2003	admitted on Feb 11, 2003, with a 4-day history of fever, malaise, sore throat, cough with blood-stained sputum, and bone pain. He had a lymphocyte count of 0.6×109/L and radiological evidence of right lower-lobe consolidation.	despite treatment with intravenous cefotaxime and oral clarithromycin, clinical and radiological signs showed that the patient was deteriorating. 2 days after admission (day 6 of illness), oral oseltamivir (75 mg twice daily) was added and continued for 4 days	the patient was electively intubated because of progressive respiratory distress, but his condition worsened and he died 6 days after admission exhibiting virus in the lungs
Thailand 2004 (8 patients; 10 out of 17 patients ?) : 7 of 10 patients given oseltamivir died a mean of 11 days after the onset of symptoms (range, 5 to 22 days), as compared with 5 of 7 untreated patients. Oseltamivir was used in conventional doses (75 mg orally, twice daily for 5 to 10 days with a weight-based dose reduction in children) in the majority of recipients. Treatment tended to have been started earlier in those who survived (a median of 4.5 days from onset compared with 9 days for those who died), and both survivors who were treated received the complete 5-day course of drug, whereas 2 of 5 patients who died received the complete 5-day courseref1, ref2	2 yrs-old male from Supanburi (patient 1)	raised chickens in backyard. Chickens died unexpectedly 5 days before illness onset. Frequently played with chickens and had direct contact with carcasses.	fever, cough, sore throat, dyspnea, diarrhea, HCT 30%, WBC 4,200/ml, lymphocytes 2,646/ml, platelets 214,000/ml, peak ALT 57 U/l, peak AST 129 U/l	days 5 to 10 + corticosteroids	survived (discharged from hospital on Feb10 2004)
	31 yrs-old male from Nakornratchasima (patient 3)	raised chickens in backyard. Three days before onset, chickens started to die. The last patient died on the date he became sick. He buried all carcasses	fever, cough, sore throat, myalgia, dyspnea, diarrhea, HCT 38%, WBC 4,660/ml, lymphocytes 513/ml, platelets 171,000/ml, peak ALT 74 U/l, peak AST 41 U/l, peak BUN 10.7 mg/dl, peak creatinine 1.07 mg/dl	days 4 to 9 + corticosteroids	survived
	5 yrs-old male from Khonkaen (patient 5)	Raised fighting cocks that died 4 days before onset. Reported direct contact with carcasses. Ate chicken with suspected H5N1 influenza	fever, rhinorrhea, cough, sore throat, dyspnea, diarrhea, abdominal pain, HCT 39%, WBC 5,600/ml, lymphocytes 2,296/ml, platelets 94,000/ml, peak ALT 47 U/l, peak AST 70 U/l, peak BUN 12 mg/dl, peak creatinine 0.7 mg/dl	days 9 to 13	dead on day 13
	6 years-old male from Kanchanaburi (patient 6)	No poultry in family. Helped slaughter one ill chicken 2 days before onset	fever, rhinorrhea, cough for 5 days, dyspnea on day 6, HCT 32%, WBC 1,200/ml, lymphocytes 624/ml, platelets 89,000/ml, CXR patchy infiltrates in right lower lobe, ARDS, respiratory failure on day 8, hepatitis, peak ALT 150 U/l, peak AST 790 U/l, peak BUN 12 mg/dl, peak creatinine 0.7 mg/dl, proteinuria >= 3	days 18 to 20 + corticosteroids	dead on day 20
	6 yrs-old male from Kanchanaburi (patient 8, initials N.C.)	Chickens in backyard died unexpectedly. Grandfather slaughtered ill chickens. No direct contact with chickens but played near slaughtering area	since 24 Jan 2004 fever, cough, sore throat, myalgia, dyspnea, diarrhea, vomiting, HCT 40%, WBC 4,900 (2,900 ?ref)/ml, lymphocytes 1,763 (696 ?ref)/ml, platelets 111,000/ml, ARDS, respiratory failure on day 5, cardiac failure, renal failure, inotropic support, peak ALT 50 U/l, peak AST 280 U/l, peak BUN 22 (50 ?ref) mg/dl, peak creatinine 1.1 (4.6 ?ref) mg/dl	days 5 to 8 + corticosteroids	dead on day 8 (2 Feb 2004)
	7 yrs-old male from Supanburi (patient 9)	No poultry in family. Frequently played on ground near a chicken farm that reported unexpected poultry deaths	since 3 Jan 2004 fever, cough, sore throat, dyspnea on day 6, HCT 41%, WBC 4,100/ml, lymphocytes 1,435/ml, platelets 304,000/ml, ARDS (CXR bilateral interstitial infiltrates), respiratory failure on day 10, cardiac failure, pneumothorax, gastrointestinal bleeding, inotropic support, peak ALT 52 U/l, peak AST 120 U/l, peak BUN 10 mg/dl, peak creatinine 0.7 mg/dl	days 22 to 27ref (18 to 22 ?ref) + corticosteroids	dead on day 29 (2 Feb 2004)
	13 yrs-old male from Chaiyapoum (patient 10)	Helped raise chickens in back yard. Eight days before onset, chickens died unexpectedly and patient assisted with slaughtering	fever, cough, sore throat, dyspnea, vomiting, HCT 37%, WBC 2,000/ml, lymphocytes 580/ml, platelets 150,000/ml, ARDS, inotropic support, peak ALT 47 U/l, peak AST 34 U/l, peak BUN 132 mg/dl, peak creatinine 8.1 mg/dl	days 6 to 11 + corticosteroids	dead on day 16
	32-year-old female from Kamphaeng Phetref	buried dead chickens on Aug 30, provided bedside care in hospital for her 11-year-old niece for 12 or 13 hours on Sep 7, and attended her funeral on Sep 9 (which died as a suspected case (cremated) part of a cluster involving also her mother - embalmed)	onset of fever, myalgia, and chills on Sep 16. An upper respiratory infection was diagnosed at a clinic on Sep 19, but she had progressive difficulty breathing and was admitted to the district hospital on Sep 23 with a temperature of 39.7°C, WBC 5,400, lymphocyte 1,296, platelet 230,000, and left-lower-lobe consolidation	oseltamivir started on hospital admission	recovered (discharged on Oct 7)
Vietnam 2004 (5 out of 10 patients) : 1 of 5 recipients of oseltamivir recovered, as compared with 1 of 5 untreated patientsref : one of the 2 surviving patients did not start oseltamivir therapy until the 12th day of illness. At that point, she was still antigen-positive and PCR-positive for the virus.	8 yrs-old female from Ho Chi Minh City (patient 5)	student; patient bought duckling as pet and cared for it in her house for 5 days; duck has diarrhea and died, patient buried it, dug it up a day later and reburied it; both patient and brother handled duck; patient also ate barely cooked eggs (Vietnamese delicacy) 2 days before onset of illness; neighbors kept 40 chickens, but no illness reported in these birds; fevere developed in patient 3 days after she bought duck; no other poultry or animals at home; no other houselhold members or relatives sick	cough, dyspnea, sputum, diarrhea, fever 38.5°C, ABP 104/64 mmHg, RR = 40 bpm, crackles, HGB 11.3 g/dl, WBC 1,200/ml, lymphocytes 300/ml, neutrophils 700/ml, platelets 117,000/ml, CD4:CD8 ratio 0.71, ALT 354 U/l, AST 320 U/l, creatinine 34 mmol/l, O2 saturation during receipt of 40% O2 95%, PCR for H5N1 performed on day 12 of illness	35 mg twice daily for ? days	recovered
	16 yrs-old female from Soc Trang (patient 7)	student, extensive exposure, including handling of 10 dead or dying chickens in patient's homestead; father of patient prepared dead chickens for eating (removed feathers, washed, cut meat) 3 days before onset of illness; no other household members or relatives sick; no other poultry or animals at home	cough, dyspnea, sputum, diarrhea, fever 40°C, ABP 110/60 mmHg, RR = 60 bpm, crackles, HGB 11.9 g/dl, WBC 3,000/ml, lymphocytes 500/ml, neutrophils 2,500/ml, platelets 70,000/ml, CD4:CD8 ratio 0.62, ALT 47 U/l, AST 20 U/l, creatinine 71 mmol/l, glucose 19 mmol/l, O2 saturation during receipt of 40% O2 67%, PCR for H5N1 performed on day 5 of illness, influenza antigens negative	75 mg twice daily for up to 5 days.	died on day 14 (3 Feb 2004)
	18 yrs-old male from Lam Dong (patient 8)	farmer, direct handling of 50 chickens, including dead chickens, at home (which was also a restaurant); patient and father prepared chickens for eating; no other household members or relatives sick; no other poultry or animals at home	cough, dyspnea, sputum, diarrhea, fever 38.5°C, ABP 100/60 mmHg, RR = 60 bpm, crackles, HGB 14.7 g/dl, WBC 1700/ml, lymphocytes 500/ml, neutrophils 1100/ml, platelets 69,000/ml, CD4:CD8 ratio : 0.75, serum creatine 89 mmol/l, glucose 13.5 mmol/l, O2 saturation during receipt of 40% O2 81%, PCR for H5N1 performed on day 6 of illness, influenza antigens positive	75 mg twice daily for up to 5 days	died on day 9
	24 yrs-old male from Lam Dong (patient 9)	farmer, direct handling of chickens in patient's homestead 3 days before onset of ilness; he prepared dead chickens for eating; noone else in family sick	cough, dyspnea, sputum, diarrhea, fever 39.5°C, ABP 110/60 mmHg, RR = 50 bpm, crackles, HGB 15.8 g/dl, WBC 1900/ml, lymphocytes 800/ml, neutrophils 1100/ml, platelets 62,000/ml, CD4:CD8 ratio : 0.59, creatinine 0.43 mmol/l, glucose 11.7 mmol/l, O2 saturation during receipt of 40% O2 80%, PCR for H5N1 performed on day 5 of illness, influenza antigens negative	75 mg twice daily for up to 5 days	died day 6
	23 yrs-old male from Lam Dong (patient 10)	direct handling of sick ducks and chickens in patient's home; many sick poultry in the district; no other illness in family	cough, dyspnea, sputum, diarrhea, fever 38.7°C, ABP 120/80 mmHg, RR = 28 bpm, crackles, HGB 17.6 g/dl, WBC 2100/ml, lymphocytes 700/ml, neutrophils 1300/ml, platelets 62,000/ml,  ALT 89 U/l, AST 110 U/l, creatinine 121 mmol/l, glucose 11.7 mmol/l, O2 saturation during receipt of 40% O2 90%, PCR for H5N1 performed on day 7 of illness, influenza antigens negative	75 mg twice daily for up to 5 days	recovered
Ho Chi Minh City 2005 (10 out of 10 patients)	NA
	NA
	NA
	NA
	NA
	NA
	NA
	NA
	NA
	NA
Thailand 2005	a 6-year-old boyref1, ref2 from Bangrakam district, Phitsanulok province	patient’s family and neighbors raised chickens in the back yard and let them feed freely in the yard and surrounding areas. Chickens in the village started to die in late Dec with diarrhea and hypersecretion, more than in previous years. All of the chickens in the relative’s farm were sick and died or were culled. 4 of the 5 chickens in the patient’s house also died during that time. The patient had helped cull the sick chickens and carried one of them from the farm back home on Dec 28 : the patient’s family did not eat any sick chickens; some neighbors had symptoms of the common cold	intermittent URT symptoms since late Dec 2003, mild until Jan 9, 2004, when he developed a distinct spike of fever and shortness of breath. On Jan 12 increased tachypnea, WBC 1200 cells/mm3 (44% neutrophils, 52% lymphocytes, 2% eosinophils, 2% monocytes), PLT 89,000 cells/mm3. CXR on day 4 of illness revealed right lower lobe opacification => admission to hospital, fever 40°C, ABP 90/60 mmHg, HR = 120 bpm, RR = 48 bpm, SO2 = 85%, AST 790, ALT 150, proteinuria and hematuria	Daily ceftriaxone intramuscular injections since Jan 10 to 12 . Oseltamivir + methylprednisolone 2 mg/kg/day started on day 15 (imported from abroad) until death and with filgrastim from day 5 to day 10 of illness	died on day 17 as a result of respiratory failure (significant insults from barotraumas, including pneumothorax and pneumomediastinum after treatment with a high frequency ventilator with 100% oxygen on day 10 of illness); particles consistent with influenza virus were detected in the lungs and spleen with electron microscopy and were confirmed with reverse transcription-PCR assays, despite 3 days of oseltamivir therapy.
	a 7-year-old boy (Ronnarit Benpad) in Panuamthuan (Phanom Thuan) district of Kanchanaburi province (150 km (94 miles) west of Bangkok), which had reported fresh outbreaks of the deadly avian influenza strain in poultry around 100 km (60 miles) west of the capital, Bangkok, son of a 48-year-old male farmer named Bang-orn Benpad who developed symptoms on 13 Oct 2005, was hospitalized on 17 Oct 2005, and died on Wed 19 Oct 2005ref.	had assisted his father with defeathering of the diseased birds	developed respiratory symptoms on 16 Oct 2005 and tested positive on Oct 21	treated with oseltamivir early in his illness, recovered his appetite and his fever resolved	recovered
Vietnam 2005	a 5-year-old boy from Nakhon Nayokref	touched some chickens raised by a relative, 4 out of 10 died	fell ill with high fever, stomach pain and vomiting around Nov 25. 2 days after that he was taken to 2 private clinics, and finally moved on day 10 to HRH Princess Maha Chakri Sirindhorn Medical Centre in Nakhon Nayok's Ongkharak district	oseltamivir started on hospital admission (day 10)	died on day 12 (Dec 7, 2005)

• post-exposure prophylaxis :
• a 68 (61?) -year-old widow in the northern province of Thai Binh tested positive on Wed 9 Mar 2005 despite being completely healthy : she took care of her husband, who developed AI on 19 Feb after eating chicken, died on 23 Feb 2005 and tested positive on Feb 27. Other than that, she said she ate only pork, and all 4 chickens raised in her house tested negative for avian influenza. She had slaughtered a chicken for a meal on the 3rd day of the Lunar New Year Festival (early February 2005). The woman, in good health in her home town, received treatment with oseltamivir
• on 18 Oct 2004 2 small mountain hawk eagles (Spizaetus nipalensis) were discovered by customs officials at Brussels International Airport (Zaventem) in plastic tubes in the hand luggage of a Thai man carried on flights from Bangkok via Vienna. Clinical examination of the birds showed no symptoms. As import of birds and products from several Asian countries into the European Union (EU) is forbidden (DG Sanco Decision 2004/122/EC), the birds were humanely sacrificed and immediately sent to the Veterinary and Agrochemical Research Centre for routine diagnosis : as soon as the results from the tested hawk-eagles were known, 25 persons who had been in direct or indirect contact with the infected eagles in Brussels airport were rapidly traced back, examined, and given oseltamivir prophylaxis. The Thai man and a close contact went to the police immediately after hearing about the diagnosis of avian influenza in the news. They were immediately brought to Antwep University Hospital, put into an isolation unit for 4 days, monitored, and given oseltamivir prophylaxis. All exposed persons remained asymptomatic, except for the veterinarian who euthanized the birds; bilateral conjunctivitis developed 2 days after he examined the birds, so he was admitted to Erasmus Medical College, Rotterdam, but recovered. His family was given prophylaxis as well. Results from the tear swab were negative for H₅. Unless there is a previous positive test from the veterinarian, there may never be any definitive evidence for an association of his symptoms with the strain reported here^{ref1, ref2}
• on October 14, 2005, Taiwan central Taichung harbour coast guard intercepted a Panama-registered cargo ship from the southeast Chinese city of Fuzhou (capital of Fujian province, southeast China) prior to landing and found 1037 smuggled birds of 19 species (276 dead), mice and turtles : one crew member from the ship, a Chinese national, was held in quarantine in the Taichung Detention Center on the charge of smuggling and was interrogated : he had no symptoms but was treated oseltamivir. The remaining 24 crew members left Taiwan with the ship on Mon Oct 17^ref
• a 50-year-old woman from Bangkok developed symptoms on 26 Oct 2005 and was admitted to a Bangkok hospital on Sat 29 Oct 2005 after telling her doctor she had visited her husband in Nonthaburi Province, north of Bangkok (one of 6 provinces hit by a fresh rash of bird flu outbreaks), on 23 Oct, helping her husband clean up a farm where chicken were culled after laboratory tests showed they had the virus : later it emerged that she took AI from the environment and was the 8th person to contract the virus without touching a dead chicken (6 of those were children who might have caught the virus while playing near infected chicken feces or fluids). She was confirmed to have contracted avian influenza on Mon 31 Oct 2005 after all 3 lab tests showed positive results : her condition is improving. Field investigations have not found any indications of respiratory illness in close contacts of the patient. The husband raised about 30 fighting cocks and bantams in his backyard. When some of them died, he decided to cull the rest because he suspected that they might have been infected with the avian-flu virus. He said he did not report the deaths to authorities as required because he thought culling the infected poultry and burying them properly was enough. He said that was aware that he could have contracted the virus while burying the dead poultry, so he wrapped his hands in plastic bags before touching the dead animals. Although he was in good health, doctors prescribed 10 anti-viral Tamiflu tablets for him to take as a preventive measure. The husband was away when his wife went to the Nonthaburi house to visit him : after visiting the house, the woman became ill, tired and found it difficult to breath. She later went to see doctors at Siriraj Hospital because she thought she might have avian influenza. Dr. Kumnuan went to the house in Nonthaburi and said it was very likely that the woman contracted the virus either through exposure to infected feces around the house or from eating fruit that she had picked up off the ground. There were stains of chicken feces on a makeshift wooden stretcher that she sat on while she waited for her husband. Next to the stretcher was a small pond of filthy water containing chicken feces. The woman admitted to eating a piece of fruit that fell from the ground after cleaning it with the dirty water^ref.
• 17 people, including 4 family members who had associated with the 5-year-old boy from Nakhon Nayok, Thailand, died on Dec 7, were put under observation for 10 days and given a preventive dose of oseltamivir since Dec 9 2005, and 23 hospital staff involved with the care and treatment of the boy were put under a similar monitoring routine^ref.
• in animals :
• 83 tigers, aged between 8 months and 2 years, either died or were culled due to AI within 3 days following the onset of clinical signs with severe pulmonary lesions, including 30 put down on Oct 20 to prevent the virus from spreading after they had been fed raw chicken at the private Sri Racha Tigers Zoo, in eastern Thailand 80 km (50 miles) east of Bangkok, since 14 Oct 2004. The zoo had been closed since 19 Oct, and about 30 of 441 tigers in the zoo were ill. The zoo was raided and 441 tigers were confiscated, 53 of which tested positive for H₅N₁. 147 out of 441 tigers were loss due to the virus or culling. The at-risk group of tigers was re-treated with oseltamivir, administered twice daily (morning and evening) in a piece of meat for 5 consecutive days (< 60 kg body weight: 75 mg/animal/dose; 60 kg body weight and above: 150 mg/animal/dose). No clinical disease or deaths have been observed in any species in the zoo since 28 Oct 2004. 5 keepers had been put under surveillance after showing flu-like symptoms, but the other 800 workers employed by the zoo and the processing firm had shown no signs of illness. Medical check-ups on 500 zoo employees found no bird-flu infections. The crude attack rate for AI in these zoo tigers is 12.5%, but this may be a minimum estimate, as the report does not indicate whether all tigers in the zoo were fed contaminated feed. Indeed, it may be impossible to know except in the unlikely situation in which the feed source is still available and excellent records were kept. Influenza A virus was isolated from the sick tigers' nasal swabs; A/Tiger/Thailand/CU-T3/04 (HA and NA gene; AY842935-6; PB2 and NS gene; AY907672-3) was initiated from those pretreated with oseltamivir, and A/Tiger/Thailand/CU-T7/04 (hemagglutinin [HA] and neuraminidase [NA] gene; AY866475-6, PB2; AY907671 and NS; AY907674) was isolated from those posttreated. Sequencing and phylogenetic analysis of the HA and NA genes of the H₅N₁ isolates in this outbreak showed that they were similar to each other as well as to those of the virus obtained from the earlier cases identified in 2004. The HA gene contained a glutamine at position 222 (226 in H3) in HA1 and 4 polybasic amino acid insertions at the cleavage site, which had also been found in other recent H₅N₁ isolates from chicken and tigers. However, no mutation of histidine to tyrosine was seen at position 274 of the NA molecule after oseltamivir treatment^ref.
• pre-exposure prophylaxis : the only one option for extinguishing an emerging human pandemic is rapid identification of cases, and treatment of patients and all their contacts with oseltamivir within 30 days^ref. In Thailand models, 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.
• 5 people (4 workers and 1 firefighter) out of a group of about 60 made up of mostly Kyoto Prefectural Government employees involved in the clean-up at the Asano Nosan's Funai Nojo chicken farm in the town of Tamba, Kyoto Prefecture, affected by influenza A(H₅N₁) Kyoto strain (A/Chicken/Kyoto/3/2004) at the end of February 2004 all made complete recoveries after being affected by minor ailments : one farm worker, who exhibited seroconversion, had a sore throat for several days just after participating in the operation. However, this person did not develop fever or other systemic symptoms.  4 other people also failed to show systemic flu-like symptoms such as fever. Micro-neutralizing assays showed a significant increase of neutralizing antibody titers (> 1:10) against the influenza A(H₅N₁) Kyoto strain (A/Chicken/Kyoto/3/2004), suggesting their bodies had adjust to fight off the disease. All clean-up workers took oseltamivir phosphate, wore protective clothing and surgical masks.
• in animals :
• 80 BALB/c mice were given oseltamivir (0.1, 1, or 10 mg/kg/day) twice daily by oral gavage; the first dose was given 4 h before inoculation with H₅N₁ A/Vietnam/1203/04 (VN1203/04) virus. 5- and 8-day regimens were evaluated. Oseltamivir produced a dose-dependent antiviral effect against VN1203/04 in vivo. The 5-day regimen at 10 mg/kg/day protected 50% of mice; deaths in this treatment group were delayed and indicated the replication of residual virus after the completion of treatment. 8-day regimens improved oseltamivir efficacy, and dosages of 1 and 10 mg/kg/day significantly reduced virus titers in organs and provided 60% and 80% survival rates, respectively. Overall, the efficacy of the 5- and 8-day regimens differed significantly (death hazard ratio, 2.658). The new H₅N₁ antigenic variant VN1203/04 was more pathogenic in mice than was A/HK/156/97 virus, and a prolonged and higher-dose oseltamivir regimen may be required for the most beneficial antiviral effect. Oseltamivir prophylaxis is efficacious against lethal challenge with VN1203/04 virus in mice. Viral virulence may affect the antiviral treatment schedule^ref
• oseltamivir phosphate is effective against H₅N₁ and H₉N₂ viruses. GS4071 (the active metabolite of oseltamivir) inhibited viral replication in MDCK cells (EC₅₀ values, 7.5-12 mM) and neuraminidase activity (IC₅₀ values, 7.0-15 nM). When orally administered at doses of 1 and 10 mg/kg per day, GS4104 prevented death of mice infected with A/Hong Kong/156/97 (H₅N₁), mouse-adapted A/Quail/Hong Kong/G1/97 (H₉N₂), or human A/Hong Kong/1074/99 (H₉N₂) viruses and reduced virus titers in the lungs and prevented the spread of virus to the brain of mice infected with A/Hong Kong/156/97 (H₅N₁) and mouse-adapted A/Quail/Hong Kong/G1/97 (H₉N₂) viruses. When therapy was delayed until 36 h after exposure to the H₅N₁ virus, GS4104 was still effective and significantly increased the number of survivors as compared with control. Oral administration of GS4104 (0.1 mg/kg per day) in combination with rimantadine (1 mg/kg per day) reduced the number of deaths of mice infected with 100 MLD₅₀ of H₉N₂ virus and prevented the deaths of mice infected with 5 MLD₅₀ of virus. Thus, GS4104 is efficacious in treating infections caused by H₅N₁ and H₉N₂ influenza viruses in mice^ref. The sensitivity of murine influenza infections to NA inhibitors is dependent on the virus challenge dose : oral therapy with oseltamivir can be delayed until 36 h after exposure to the H₅N₁ viruses^ref and 48 to 60 h or more after infection with H₁N₁ virus^ref.
Oseltamivir is dangerously being used by veterinarians for both prevention and therapy of canine influenza (not easily diagnosed; prevalence = 15%; mortality = 5%) and parvovirus infection despite lack of published studies, but based only on case reports on the Veterinary Information Network^ref.
Patients with influenza and prolonged fever despite receiving oseltamivir should be given antibiotics since throat cultures obtained on the first or fourth day of treatment with oseltamivir were positive for pathogenic bacteria in all patients with fevers for 4 or more days^ref
Intranasal zanamivir 50 mg/kg administered twice daily 1 day before and 6 days following infection with influenza A virus effectively abolished fever and reduced nasal virus titres in ferrets. Oral oseltamivir is also effective in this animal model. When administered to ferrets twice daily for 3 days starting 2 hours after inoculation with influenza A (H₃N₂), oral oseltamivir 5 mg/kg and 25 mg/kg dose-dependently lowered the febrile response to infection by 58% and 93%, respectively. In addition, oseltamivir treatment reduced nasal signs and lethargy, lowered the numbers of inflammatory cells in nasal lavages and reduced peak viral titres. Similar effects were seen with influenza A (H₁N₁), A(H₃N₂) and influenza B induced infections (F.Hoffmann-La Roche, data on file). Intranasal zanamivir (1 mg/kg) is effective prophylactically against lethal influenza A(H₇N₇) infection in chickens, but not against infection by other virulent viruses of the N₁, N₂ or N₃ subtype. However, oseltamivir carboxylate was effective against H₅N₁ and H₉N₂ avian influenza viruses in vitro (EC₅₀ values from 7.5 to 12 mm in MDCK cells) and oral oseltamivir at 1 and 10 mg/kg/day prevented death, inhibited lung viral titres and prevented the spread of virus to the brains of mice infected with these viruses. Neither zanamivir nor oseltamivir administration has been shown to adversely affect the primary immune response to influenza infection in animals.

Consideration of use of neuraminidase inhibitors such as oseltamivir and zanamivir in IgA nephropathy^ref.

Influenza virus with resistance to antiviral drugs emerges with increased frequency in immunocompromised patients and can limit the benefit of M2 and neuraminidase (NA) inhibitors. 3 cases of influenza have been documented in severely immunocompromised patients from whom virus variants with molecular markers of resistance to anti-influenza drugs were recovered. Virus variants recovered from 2 patients had mutations in the M2, NA (with a previously recognized Glu¹¹⁹Val NA substitution), and hemagglutinin genes. The novel Asp¹⁹⁸Asn NA mutation was described in an influenza B virus and its decreased susceptibility to both oseltamivir and zanamivir^ref.

Oseltamivir 5 mg/kg oseltamivir for 5 days (equivalent to the approved human treatment dose of 75 mg twice daily) 4 hours after inoculation prevented viral replication in the upper respiratory tract and effectively treated all infected ferrets, with no deaths. All ferrets in the control group died. Resistance mutations of the H₅N₁ avian influenza virus were not detected^ref.

TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/ml and allowed the detection of the mutation in a mixture of wild type and mutant^ref

Zanamivir protects mice against lethal challenge with A/HK/156/97 (H₅N₁) influenza virus^ref and protects chickens from A/chick/Victoria/1/85 (H₇N₇), a highly pathogenic virus^ref, but fails to protect chickens from other highly virulent viruses of the NA subtypes N₁, N₂, N₃, N₇, and N₈^{ref1, ref2}. There have been recent reports of enhanced potency of multivalent zanamivir by Sankyo in Japan^{ref1, ref2} and Biota in Australia^ref, where a single dose of drug may be sufficient for protection. Zanamivir dimers 8 and 13 are highly potent neuraminidase inhibitors which, along with dimer 3, are being investigated as potential second generation inhaled therapies both for the treatment of influenza and for prophylactic use. Dimer 3 shows NA inhibitory activity against N₁ viruses including the recent highly pathogenic H₅N₁ A/Chicken/Vietnam/8/2004^ref.


Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. Reverse genetics and the recombinant baculovirus expression system were used for investigating reasons for the lack of isolation of zanamivir-resistant H₃N₂ viruses and for further exploring subtype-specific oseltamivir resistance. H₃N₂ viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivir-treated patients. Mutations conferring oseltamivir resistance in N₁ (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. These data suggest that zanamivir-resistant H₃N₂ viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance^ref.
An influenza A/H₃N₂ variant with NA mutations E59G, E119V, and I222V was first detected after 38 days of oseltamivir treatment. In an NA inhibition assay, this variant was 274 times more resistant to oseltamivir than the original isolate but was susceptible to zanamivir. The I222V substitution enhanced the level of oseltamivir resistance that was primarily conferred by the E119V mutation in recombinant NA proteins. Remarkably, the E119V mutation persisted for 8 months after cessation of oseltamivir. Amantadine therapy led to rapid emergence of the M2 mutation S31N, which is known to confer amantadine resistance. The patient shed the virus intermittently while receiving nebulized zanamivir therapy despite the absence of a resistance phenotype, which could be the result of nonoptimal drug delivery and impaired host immunity^ref.
Other treatments may have to be considered in patients with rapid disease progression. Intravenous ribavirin was tried in one case without success. The use of immune serum from convalescent patients may be another option worth consideration since neutralising monoclonal antibodies have been effective in treating established influenza A virus infection in mice with severe combined immunodeficiency^ref. The efficacy of convalescent immune serum in human beings remains speculative, but combination therapy with immuneglobulin with high neutralising antibody titre and aerosolised ribavirin has resulted in improved outcome in adult bone-marrow-transplant recipients with respiratory syncytial virus disease^ref
Recombinant viruses possessing the 1918 NA or both the 1918 HA and 1918 NA were inhibited effectively in both tissue culture and mice by zanamivir and oseltamivir. A recombinant virus possessing the 1918 M segment was inhibited effectively both in tissue culture and in vivo by amantadine and rimantadine^ref.
4 household-based, randomized clinical trials, two each of zanamivir and oseltamivir, were designed primarily to estimate the effect of postexposure prophylaxis on preventing influenza illness in household contacts. However, the effect of influenza antivirals on infectiousness as well as on the ability of the virus to cause disease--the pathogenicity--have important public health consequences. The authors show how such studies can provide estimates of pathogenicity, antiviral efficacy for pathogenicity, and the antiviral effect on infectiousness. Analysis of the four studies confirmed the high prophylactic efficacy against illness of both zanamivir (75%, 95% confidence interval (CI): 54, 86) and oseltamivir (81%, 95% CI: 35, 94). The effect on reducing infectiousness was 19% (95% CI: -160, 75) for zanamivir and 80% (95% CI: 43, 93) for oseltamivir. Pathogenicity in controls ranged from 44% (95% CI: 33, 55) to 66% (95% CI: 48, 72). Efficacy in reducing pathogenicity for zanamivir was 52% (95% CI: 19, 72) and 56% (95% CI: 14, 77) in the two studies; for oseltamivir, it was 56% (95% CI: 10, 73) and 79% (95% CI: 45, 92). Studies of influenza antivirals in transmission units would be improved if randomization schemes were used that allow estimation of the antiviral effect on infectiousness from individual studies^ref.
NAIs reduce NO production in influenza virus-infected and IFN-g-activated RAW 264.7 macrophages^ref
2 mechanisms of resistance have been recognised :

• NA-independent resistance : reduction of virus susceptibility to the NA inhibitors was observed on early passages of different viruses in the presence of several NA inhibitors. Mutations in or close to the HA receptor-binding site reduced efficiency of virus budding to cellular receptors. This causes decreased dependence on NA function, such that resistant viruses demonstrate broad cross-resistance to NAIs in cell culture. In addition, in vitro-selected mutants may demonstrate drug-dependence in cell culture, although it remains unknown if drug-dependent variants might occur in vivo.
• NA-dependent resistance : resistance also results from amino acid substitutions at the conserved residues in both structural and catalytic residues^{ref1, ref2, ref3, ref4}. Differences in the degree of cross-resistance result from differences in the geometry of drug interactions with aminoacid residues of NA^ref. For example, an amino acid substitution at position 292 leads to moderate zanamivir resistance and high level oseltamivir carboxylate resistance^{ref1, ref2}, whereas both oseltamivir carboxylate and RWJ-270201 retain activity against the zanamivir-resistant Glu119Gly variant. NA mutations compromise enzyme function and result in reduced enzyme stability^{ref1, ref2}, instability of the NA tetramer^ref, or a change in the pH optimum^ref. Resistant variants with NA mutations replicate efficiently in cell culture^{ref1, ref2} [Ives J, Caar J, Roberts N, et al. An oseltamivir-treatment selected influenza A/N2 virus with an R292K mutation in the neuraminidase gene has reduced infectivity in vivo. II International Symposium on Influenza and Other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr] but most show reduced infectivity and virulence in animal models^{ref1, ref2, ref3} [Ives J, Caar J, Roberts N, et al. An oseltamivir-treatment selected influenza A/N2 virus with an R292K mutation in the neuraminidase gene has reduced infectivity in vivo. II International Symposium on Influenza and Other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr]. Because replication of influenza virus in the respiratory tract imposes higher requirements on HA and NA functions, changes in these surface glycoproteins are likely to decrease influenza virus virulence and transmissibility in vivo, unlike the experience with M2 inhibitors [Hayden FG. Amantadine and rimantadine—clinical aspects In: , Richman DD, ed. Antiviral drug resistance. Chichester: Wiley,  1996: 59-77]
Resistance in man : in the absence of a reliable cell-culture method for resistance surveillance, screening is based on NA inhibition phenotype and NA and HA sequence analyses. During clinical trials of zanamivir, no loss of NA susceptibility and no changes in the receptor-binding site of the HA were observed [Gor D, Cadman A, Dempsey M, et al. Antiviral efficacy and sequence analysis of HA and NA genes of influenza isolates from zanamivir phase II/III clinical efficacy trials. 39th ICAAC (San Francisco, September, 1999); abstr 284; Boivin G, Goyette N, Aoki FY, Wagner A, Trottier S. Clinical and virological efficacy of zanamivir in the treatment of influenza A virus infections during the 1997–98 flu season. 39th ICAAC (San Francisco, September, 1999); abstr 289a]. Immunosuppression provides a higher risk environment for the development of drug resistance. A 2-week long treatment of a bone-marrow transplant recipient with influenza B infection led to the emergence of a zanamivir-resistant virus^ref. Oseltamivir-resistant variants have been recovered in only 1% or so of isolates from immunocompetent adult patients in the active site of the NA that are associated with greatly impaired replication in animals. No drug-induced HA variants have been recognised to date. Clinical resistance requires further study, especially in children and immunocompromised hosts, but data available so far suggest that resistance is infrequent and unlikely to limit the clinical usefulness of these drugs in most settings.
Resistance to zanamivir and oseltamivir can be induced in influenza A and B viruses in vitro^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}, but induction of resistance requires multiple passages in cell culture. By contrast, resistance to amantadine and rimantadine in vitro can be induced with fewer passages in cell culture^{ref1, ref2}. Development of viral resistance to zanamivir and oseltamivir during treatment has been identified but does not appear to be frequent^{ref1, ref2, ref3} (Jackson HC, Roberts N, Wang ZM, Belshe R. Management of influenza: use of new antivirals and resistance in perspective. Clin Drug Invest 2000;20:447--54). Available diagnostic tests are not optimal for detecting clinical resistance to the neuraminidase inhibitor antiviral drugs, and additional tests are being developed^{ref1, ref2}. Postmarketing surveillance for neuraminidase inhibitor-resistant influenza viruses is being conducted^ref.
None of the 4 antiviral agents has been demonstrated to be effective in preventing serious influenza-related complications (e.g., bacterial or viral pneumonia or exacerbation of chronic diseases). Evidence for the effectiveness of these 4 antiviral drugs is based principally on studies of patients with uncomplicated influenza (Food and Drug Administration. Subject: safe and appropriate use of influenza drugs [Public Health Advisory]. Rockville, MD: US Department of Health and Human Services, Food and Drug Administration, 2000). Data are limited and inconclusive concerning the effectiveness of amantadine, rimantadine, zanamivir, and oseltamivir for treatment of influenza among persons at high risk for serious complications of influenza^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11} (Martin C, Mahoney P, Ward P. Oral oseltamivir reduces febrile illness in patients considered at high risk of influenza complications [Abstract W22-7]. In: Options for the control of Influenza IV. New York, NY: Excerpta Medica, 2001:807--11). Fewer studies of the efficacy of influenza antivirals have been conducted among pediatric populations, compared with adults^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. One study of oseltamivir treatment documented a decreased incidence of otitis media among children^ref. Inadequate data exist regarding the safety and efficacy of any of the influenza antiviral drugs for use among children aged <1 year^ref.
No published data are available concerning the safety or efficacy of using combinations of any of these 4 influenza antiviral drugs. For more detailed information concerning potential drug interactions for any of these influenza antiviral drugs, package inserts should be consulted.
Oseltamivir is less effective for influenza B than for influenza A with regard to duration of fever and virus persistence, irrespective of patient age or the timing of administration of the first dose^ref.
Aiming to understand at the molecular level why oseltamivir (OTV) cannot be used for inhibition of human influenza neuraminidase subtype N1 as effectively as for subtypes N2 and N9, molecular dynamics simulations were carried out for the 3 complexes, OTV-N1, OTV-N2 and OTV-N9. The 3D OTV-N2 and OTV-N9 initial structures were represented by the X-ray structures while that of OTV-N1, which its X-ray structure is not yet solved, was built up using the aligned sequence of H₅N₁ isolated from humans in Thailand with the X-ray structure of the N2-substrate as the template. In comparison to the OTV-N2 and OTV-N9 complexes, dramatic changes were observed on the OTV conformation in the OTV-N1 complex in which 2 of its bulky side chains, N-acethyl (-NHAc) and 1-ethylproxy group (-OCHEt2), were rotated in order to adjust its size to fit into the N1 catalytic site. This change leads directly to the rearrangements of the OTV's environment, which are: (i) Distances to its neighbors, W178 and E227, are shorter while those to residues R224, E276 and E292 are longer. (ii) Hydrogen bonds to the two nearest neighbors, R224 and E276, are still conserved in distance and number as well as percentage occupation. (iii) The calculated ligand/enzyme binding free energies of -7.20, -13.44 and -13.29 kcal/mol agree with their inhibitory activities in terms of the experimental IC50 of 36.1 - 53.2 nM, 1.9 - 2.7 nM and 9.5 - 17.7 nM for the OTV-N1, OTV-N2 and OTV-N9 complexes, respectively. (iv) Hydrogen bond breaking and creation between the OTV and neighborhood residues are accordingly in agreement with the ligand solvation/desolvation taking place in the catalytic site^ref.
• DANA / Neu5Ac2en


• cyclopentane derivatives
• peramivir / BCX-1812 / RWJ-270201 / 1S,2S,3R,4R)-3-[(1S)-(acetylamino)-2-ethylbutyl ]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (source : Biocryst Pharmaceuticals; p.o.; entered clinical trials in 1999) : a recent product of structure-based drug design with a negatively charged carboxylate group, a positively charged guanidino group with an orientation unlike that in zanamivir, and lipophilic side chains^ref. NA IC₅₀ = 0.9-4.3 nM, superior to that by zanamivir and oseltamivir carboxylate. The different structures of the three NA inhibitors suggest that they may differ in their antiviral activity and in their susceptibility to the emergence of mutant variants. In fact, RWJ-270201 has been shown to retain its inhibitory activity against the zanamivir-resistant Glu-119 variant of influenza A virus NA (L. V. Gubareva, D. Schallon, and F. G. Hayden, 2nd Int. Symp. Influenza Other Respir. Viruses, abstr. P24, 1999). RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (EC₅₀ = 0.5-11.8 mM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H₅N₁) and A/quail/Hong Kong/G1/97 (H₉N₂) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H₅N₁ virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death^ref.
• Gubareva LV, Schallon D, Hayden FG. Activity of the neuraminidase inhibitor RWJ-270201 (BCX-1812) against influenza A and B viruses in the neuraminidase inhibition assays. II International Symposium on Influenza and other Respiratory Viruses (Dec 10–12, 1999, Grand Cayman); abstr.
• Bantia S, Ananth SL, Andries K, Horn K, Parker C, Chand P. In vitro activity of the neuraminidase inhibitor RWJ-270201 (BCX-1812) against influenza virus. 39th ICAAC (San Francisco, September, 1999); abstr 947.

Peramivir potently inhibits the neuraminidase enzyme N₉ from H₁N₉ virus in vitro with a IC₅₀ of 1.3+/-0.4nM. On-site dissociation studies indicate that peramivir remains tightly bound to N₉9 NA (t_1/2 >24 h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme (t_1/2 =1.25 h). A single intramuscular injection of peramivir (10 mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H₁N₁, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N₁ NA enzymes similar to that observed for N₉ enzyme. Additional efficacy studies indicate that a single injection of peramivir (2-20 mg/kg) was comparable to a q.d.x5 day course of orally administered oseltamivir (2-20 mg/kg/day) in preventing lethality in H₃N₂ and H₁N₁ influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak^ref. 288 susceptible, healthy volunteers (ages 18-45) were inoculated intranasally with A/Texas/36/ 91/H₁N₁ or B/Yamagata/16/88 virus in 4 randomized, double-blind, placebo-controlled trials. For treatment dosing was initiated at 24 h after inoculation with peramivir doses ranging from 100-800 mg/day p.o. for 5 days. For prophylaxis dosing was initiated 24 h before inoculation and continued for 4 days with peramivir doses ranging from 50-800 mg/day. The primary outcome measure for treatment was quantitative viral detection defined by the area under the curve (AUC) for nasal wash viral titres. For prophylaxis the primary outcome measure was the incidence of virus recovery. In influenza A treatment, peramivir 400 mg q24h and 200mg q12h, but not lower doses, resulted in significant reductions in viral titre AUC. In influenza B treatment, both 400 and 800/400 mg once daily dose groups reduced AUC values. In influenza A prophylaxis, the percentage of individuals with nasal viral shedding did not differ significantly in the placebo (58%), 50 mg (61%), 200 mg (37%) and 400 mg (31%) dose groups. In influenza B prophylaxis, shedding frequencies were similar in placebo (55%), 200 mg (41%), 400 mg (35%) and 800 mg (47%) dose groups. The drug was well tolerated in all four studies, with nausea and headache being the most common side effects. No drug-resistant variants were detected. Early treatment with peramivir was associated with significant antiviral effects in experimentally induced influenza in humans. Prophylaxis did not significantly reduce viral shedding. The relatively low blood peramivir concentrations observed may explain the lack of more robust antiviral effects, and parenteral dosing should be studied^ref. In a phase I clinical rial, > 60 healthy volunteers were given intravenous peramivir each day for up to 10 days in doses ranging from about 30 mg to 600 mg. All doses were well tolerated with no serious adverse effects, researchers independent of the company told a meeting in San Francisco of the American Society for Microbiology on Sep 30, 2006^ref
An influenza A/WSN/33 (H₁N₁) recombinant virus was passaged under peramivir or zanamivir pressure. The peramivir-selected variant had a H274Y mutation in the neuraminidase (NA) gene conferring resistance to peramivir and oseltamivir but susceptibility to zanamivir. The zanamivir-selected variant had a massive deletion in the region encoding the NA active center and an A200T hemagglutinin mutation. This variant exhibited reduced susceptibility to zanamivir with a drug-dependent phenotype^ref
• BCX-1827
• BCX-1923
• laninamivir octanoate / CS-8958 (Inavir^®), an ester prodrug of the NAI laninamivir, can be used in children with oseltamivir-resistant influenza A (H1N1) virus infection
  
• hemagglutinin-neuraminidase (HN) inhibitors for human parainfluenza viruses^{ref1,ref2, ref3} :
• BCX-2855
• BCX-2798
• pyrrolidine core^ref
• prodrug A-322278 => A-315675 / 5-[(1R,2S)-1-(acetylamino)-2-methoxy-2-methylpentyl]-4-[(1Z) -1-propenyl]-(4S,5R)-D-proline^ref (source : Abbott). Resistance is due to E119D (60-fold), R233K, S339P (310-fold), and mutation in the N₉ NA gene in vitro in the presence of increasing concentrations : these mutants show >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal^ref


Oral oseltamivir and A-322278 (both given at dosages of 10 mg/kg/day) have comparable efficacy in reducing viral replication, decreasing weight loss, and prolonging survival in an immunocompromised murine model of influenza virus infection. Once the treatment was discontinued, severe combined immunodeficient (SCID) mice had progressive viral replication and clinical decline. Drug-resistant variants were detected in 4 (29%) of 14 and 2 (13%) of 15 mice (both BALB/c and SCID) treated with oseltamivir or A-322278, respectively; no resistant variants were detected in placebo-treated mice. Amino acid substitutions in the hemagglutinin receptor-binding site at aa 137 or 225 were detected in cloned resistant isolates. A substitution in the neuraminidase (NA) active site (Arg292Lys) was detected in the cloned virus recovered from an oseltamivir-treated mouse. This model would be useful for elucidation of the molecular mechanisms of resistance to NA inhibitors and for testing of anti-influenza therapy options that might prevent the emergence of resistant variants^ref
• PM-523 : a polyoxometalate that inhibits fusion between the virus envelope and cell membrane and inhibits the penetration of the virus into cells. This compound has shown potent anti-influenza activity and synergistic inhibitory activity in combination with ribavirin or zanamivir in vitro and in vivo^ref
• 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) inhibits the mutlicycle replication of influenza viruses in tissue culture. Influenza virus grown in the presence of FANA contains neuraminic acid on its envelope which then serves as receptor for other virus particles causing extensive aggregation. Thus, FANA inhibits influenza virus replication by preventing the enzymatic removal of neuraminic acid from the virus envelope.
• T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H₅N₁ influenza virus in MDCK cells, the EC90 values ranging from 1.3 to 7.7 mM using virus yield reduction assay. The efficacy was less than exerted by oseltamivir carboxylate or zanamivir, but greater than ribavirin. Experiments run in mice lethally infected with influenza A/Duck/MN/1525/81 (H₅N₁) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h post-virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg/day were well tolerated; each prevented death, lessened decline of arterial oxygen saturation (SaO₂), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented deaths in the mice. Oseltamivir (20 mg/kg/day), administered p.o. twice daily for 5 days, was run in parallel in two experiments; it was only weakly effective against the infection. The four times daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented deaths and SaO₂ decline. Characterization of the pathogenesis of the duck influenza H₅N₁ virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H₅N₁ virus. These data indicate T-705 may be a useful means for the treatment of avian influenza virus infections^ref
• a research group at the Academy of Military Medical Sciences discovered on 26 Dec 2005 a novel NAI that will be produced by domestic labs and stockpiled for use in the event of a bird flu pandemic. Chinese scientists are currently seeking for an injectable formula to ease treatment to patients in critical conditions^ref. The cost is only a quarter to a third of Tamiflu that sells at 29.8 yuan (US$3.73) for each capsule in China, 92.6% (162 of 175) of the Vietnam isolates had mutations that conferred resistance to amantadine, as did all the isolates from Thailand (58 of 58), Cambodia (9 of 9), and Malaysia (2 of 2). In contrast, only 13% (16 of 123) of the isolates from China and 6.3% (2 of 32) of those from Indonesia had such mutations. None of the 8 isolates from Japan and Korea, collected from poultry in late 2003, showed resistance. The dual mutation motif Leu26Ile–Ser31Asn (leucineisoleucine at aa 26 and serineasparagine at aa 31) was found almost exclusively in all resistant isolates from Vietnam, Thailand, and Cambodia, suggesting the biological selection of these mutations^ref
• 4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethyl-2-pyrimidinyl)-benzenesulphonamide (SPIII-5H) and related compounds were tested for antiviral activity against influenza A (H₁N₁, H₃N₂, and H₅N₁) and B viruses in Madin Darby canine kidney (MDCK) cell culture. Among the compounds tested, SPIII-5H and four derivatives (5-chloro [SPill-5Cl], 5-bromo [SPIII-5Br], 5-methyl [SPIII-5Me] and N-acetyl [SPIII-NA]) showed similar antiviral potencies, with only the 5-fluoro (SPIII-5F) derivative being ineffective. 50% effective concentration (EC₅₀) values were determined in cytopathic effect (CPE) inhibition assays quantified by neutral red dye uptake. By this method, the active compounds were inhibitory to the H₁N₁ strain of influenza A at 2.7-5.2 mg/ml, to the H₃N₂ strain of influenza A at 13.8-26.0 mg/ml, to the H₅N₁ strain of influenza A at 3.1-6.3 mg/ml and to influenza B at 7.7-11.5 mg/ml. Confirmatory virus yield reduction studies against influenza A (H₁N₁) virus demonstrated antiviral activity (90% inhibition) at concentrations of 2-10 mg/ml. No cytotoxic effects were evident in actively growing uninfected cells or stationary monolayers at 100 mg/ml. Potencies of the compounds were similar to those of ribavirin, but much less than those of oseltamivir carboxylate against the various viruses. Time-of-addition studies indicated the compounds inhibited an early step in the virus replication cycle, probably virus adsorption/penetration, and no virucidal activity was evident. The basic molecule is amenable to diverse chemical modifications, which may improve water solubility and antiviral potency^ref.
• triazoverin^ref
• ¹H-1,2,3-triazole-4-carboxamide derivatives
• 5-substituted-2-(4-substituted phenyl)-1,3-benzoxazoles
• benzbromane and diclazuril
• aminoalkyl rupestonates
• 1,3-diarylprop-2-en-1-ones
• saponin derivatives
• DAS-181 (Fludase^®), an antiviral drug with sialidase activity, potently inhibited replication of wild-type influenza A(H7N9) and its oseltamivir-resistant R292K variants in mice^ref
  
The susceptibilities of 5 zanamivir-resistant and 6 oseltamivir-resistant influenza viruses were assessed against 4 NAIs, including peramivir and A-315675, by a fluorometric NA activity inhibition assay. The enzyme activity of a majority of the variants was effectively inhibited by either A-315675 or both peramivir and A-315675 (IC₅₀ <10 nM). A novel oseltamivir-resistant influenza virus B variant carrying substitution at residue 198 (Asp->Asn) (N2 numbering) retained susceptibility to peramivir and A-315675. In vivo, the Asn198 variant showed no apparent fitness impairment as judged by its recovery on day 5 from the nasal washes of ferrets coinfected with equal doses of the wild-type virus and the Asn¹⁹⁸ variant. Based on the sequence analysis of the virus in the nasal washes, oseltamivir treatment (5 mg/kg twice daily for 5 days) did not provide growth advantage to the Asn¹⁹⁸ variant. Nevertheless, treatment with A-315675 (prodrug A-322278) reduced the number of the animals (2 of 7) shedding the Asn¹⁹⁸ variant. These studies indicate that different patterns of susceptibility and cross-resistance between NA inhibitors may prove important if antiviral resistance to zanamivir and oseltamivir were to emerge^ref
The combined use of 2 or more drugs for which there are different mechanisms of resistance can reduce the effect of resistance to a single drug. Therapy with synergistically active antiviral drugs that target different viral proteins and have different mechanisms of action may provide several advantages over single-agent treatment, such as greater potency, superior clinical efficacy, reduction of the drug dosages needed, reduction of respiratory complications requiring antibiotic therapy, reduction of cellular toxicity and side effects, and greater cost-effectiveness.  An important initial step in evaluating combination therapy is to determine whether the combined agents reduce influenza virus replication additively or synergistically in an in vitro system. A number of reports address the anti-influenza activity of drug combinations :
• oseltamivir + zanamivir : the latter provides drugs predominantly to the upper respiratory tract. Since human infection with current strains of influenza H₅N₁ can be associated with disseminated infection and replication in the lower respiratory tract and extrapulmonary sites^{ref1, ref2, ref3}, combined treatment with nebulized zanamivir and oral oseltamivir would be likely to result in monotherapy in the lower respiratory tract and nonrespiratory sites^ref
• the NA inhibitor 4-guanidino-Neu5Ac2en was found to effectively inhibit plaque formation of influenza A clinical isolates that were resistant to amantadine and rimantadine^ref
• treatment with zanamivir reportedly ended an outbreak of influenza that amantadine had failed to control (and from which amantadine-resistant variants were isolated) in a nursing home^ref
• ribavirin + rimantadine were reported to cause additive and, in specific concentrations, synergistic reduction of influenza A/FPV^ref, influenza A/Texas/77 (H₃N₂), and influenza A/USSR/77 (H₁N₁) virus yield in MDCK cells^ref
• IFN-a + rimantadine or ribavirin additively or synergistically reduce the yield of clinical H₃N₂ or H₁N₁ influenza A isolates in primary rhesus monkey kidney cells^ref
• rimantadine + ribavirin were associated with enhanced survival in a mouse model and were significantly more effective than either drug alone^{ref1, ref2}
• rimantadine + protease inhibitor aprotinin highly protected mice against lethal influenza virus challenge^ref
• zanamivir + rimantadine, ribavirin, or 2'-deoxy-2'-fluoroguanosine showed additive effects against influenza A viruses in MDCK cells (Madren, L. K., C. Shipman, Jr., and F. G. Hayden. 1995. In vitro inhibitory effects of combinations of anti-influenza agents. Antivir. Chem. Chemother. 6:109-113)
• peramivir + ribavirin reduce influenza A virus infection in cell culture and in mice^ref.
• NAIs + rimantadine : synergism between was observed at a wide range of concentrations, against both the H₁N₁ and H₃N₂ influenza virus subtypes, at reducing the extracellular H₁N₁ and H₃N₂ influenza virus yield in MDCK cells in comparison to the yield obtained after treatment with either single drug. The exact mechanism by which these 2 drugs synergistically reduce influenza virus yield in MDCK cells is not clear. The inhibition of the M2 protein function by the amantadine and its derivative rimantadine causes a decrease in pH within intracellular compartments and thus may play a role in the maturation of HA glycoprotein in trans-Golgi regions of the exocytic pathway^ref. Treatment with amantadine and rimantadine could cause specific conversion of the native HA conformation to the low-pH HA conformation^ref; HA in this conformation has a tendency to aggregate and therefore may interfere with the pinching off of transport vesicles, causing enlargement of trans-Golgi structures in a manner analogous to the inhibition of virus release from the cells. This effect could not only decrease the numbers of virus particles released from the cells but also alter the balance between HA and NA so that less NAI is required to achieve the same biological effect
• rimantadine hydrochloride and oseltamivir phosphate on mice infected with influenza A/Aichi/2/68 (H₃N₂) virus. Compounds were simultaneously administered in a 5-day-treatment course, starting 4 h before intranasal infection with 10 or 20 viral 50% mouse lethal doses. Initially, we tested combinations of oseltamivir (0.05, 0.1 and 0.2 mg/kg/day) and rimantadine (2.5, 5.0 and 7.5 mg/kg/day). Significant differences were recorded between combination-treated groups, and groups with separately applied compounds and the placebo group, such as: protection index of oseltamivir with 5.0 or 7.5 mg/kg rimantadine varied between 34-41% and 43-87%, respectively, whereas the individual effects of oseltamivir, 5 mg/kg of rimantadine and 7.5 mg/kg of rimantadine were 0-10%, 0% and 18.7-29.6%, respectively; mean survival time in combination-treated groups was lengthened by 3.1-6.9 days, in oseltamivir groups by 0-1.9 days, and in rimantadine groups by 0.8-1.3 days at 5 mg/kg and 2.6-3.2 days at 7.5 mg/kg. The three-dimensional method of Prichard and Shipman characterized the combination effect as synergistic. Further, the activity of 0.05 mg/kg/day of oseltamivir combined with 5 mg/kg of rimantadine was studied. Lung virus titre in Madin Darby canine kidney cells, lung index and consolidation score proved the high effectiveness of the combination. When compared with the placebo group, a 2.8 log₁₀ lower titre of 50% cell culture infectious dose (CCID₅₀) was recorded in the combination-treated group at 48-60 h post-infection (the peak of lung virus growth). This is in contrast to the 0.1-1.0 log10 and 1.1-1.4 log10 reduction in CCID₅₀ titre observed in the oseltamivir and rimantadine groups, respectively. These data emphasize the high anti-influenza A potential of the combination^ref.
• amantadine + low doses of oseltamivir carboxylate (</=1 mM) significantly reduced (P<0.005) virus yields of human A/Nanchang/1/99 (H₁N₁), A/Panama/2007/99 (H₃N₂), and A/Hong Kong/156/97 (H₅N₁) viruses in MDCK cells. After 5 sequential passages in MDCK cells, the M2 protein of viruses cultivated with amantadine alone mutated at positions V27A and S31N/I. Viruses cultivated with oseltamivir carboxylate (>/=0.001 mM) possessed mutations in the hemagglutinin (HA) protein. These variants showed reduced efficiency of binding to sialic acid receptors and decreased sensitivity to NA inhibitor in plaque reduction assay. Importantly, no mutations in the HA, NA, and M2 proteins were detected when the drugs were used in combination. Combination chemotherapy with M2 blocker and NA inhibitor reduced the emergence of drug-resistant influenza variants in vitro. This strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored^ref. In mice^ref
• oseltamivir + ribavirin : mouse models have been widely used for evaluating potential influenza virus inhibitors. However, the viral strains traditionally used in these models are fairly old and do not represent currently circulating viruses in nature. 2 new lethal infection models were developed in mice using mouse-adapted influenza A/New Caledonia/20/99 (H₁N₁) and influenza B/Sichuan/379/99 viruses. Both virus infections were used to study oral treatment with oseltamivir and ribavirin, both alone and in combination. Oral treatments were given twice daily for 5 days starting 4 h before infection in initial studies. Against influenza A, oseltamivir was active at 10, 20, and 40 mg/kg/day, protected 80-100% of mice from death and reduced lung consolidation - ribavirin was similarly effective at 20, 40, and 80 mg/kg/day. When treatments were initiated after virus challenge, delaying treatment with oseltamivir even 1 day caused it to be ineffective. Ribavirin prevented mortality by 50-80% when treatments were delayed 1-4 days after infection. The combination of the two drugs (oseltamivir at 20 mg/kg/day and ribavirin at 40 mg/kg/day) was no better than ribavirin alone. In contrast to what we observed with influenza A virus infections, oseltamivir and ribavirin showed similar dose-related antiviral activities against influenza B virus infections. The compounds both significantly increased survival when treatments started up to 4 days after infection, but ribavirin was more active than oseltamivir (50-80% survival compared to 30-40% survival, respectively, when starting treatments on days 2-4 after infection). By varying the doses of each drug that were used in combination (oseltamivir at 1.25, 2.5 and 5 mg/kg/day; ribavirin at 5, 10 and 20 mg/kg/day) certain dosage combinations were superior to either compound used alone as assessed by decreased mortality, lung virus titre, lung score and lung weight parameters. These activities differed from published results with older, more established virus strains as oseltamivir was less effective and ribavirin was more active than previously reported^ref
Combination therapy against influenza virus infection has been investigated^{ref1, ref2, ref3}. However, most studies have tested experimental compounds, such as polyoxometalate PM-523^ref, nucleoside analogue 2'-deoxy-2'-fluoroguanosine (Madren, L. K., C. Shipman, Jr., and F. G. Hayden. 1995. In vitro inhibitory effects of combinations of anti-influenza agents. Antivir. Chem. Chemother. 6:109-113), or infusions made from the natural antiviral agent Flos verbasci^ref. Additional information is needed about combination therapy with antiviral agents approved for use against influenza virus infection^ref. On Jan 26, 2006, WHO doctors who set up a virtual network to compare notes on treating bird flu agreed to try hitting the next serious cases with oseltamivir combined with amantadine^ref.
During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, 8 (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, 1 (0.22%) in 1999 to 2000, 3 (0.36%) in 2000 to 2001, and 4 (0.41%) in 2001 to 2002. 6 had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use^ref
The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H₃N₂) recombinant virus background, we characterized 7 charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N₂ subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure^ref. Biologists have uncovered subtle variations in the structure of a key flu virus enzyme, a finding that might lead to better anti-flu drugs. As well as offering a new target for drugs, the work helps our understanding of how drug resistance develops. All flu viruses have the proteins haemagglutinin and neuraminidase on their coats. Haemagglutinin (H) helps the virus attach to cells and has 16 known forms. Neuraminidase (N) is an enzyme that comes in nine forms. It allows the virus to be released from infected cells and infect new ones. Different flu virus strains are named after the different forms of 2 proteins that they carry. The chemists who developed NAIs based their designs on the structures of the N₂ and N₉ forms of the enzyme, because these were the only ones available at the time. But both drugs are also effective against flu viruses with other types of neuraminidase. Scientists assumed this was because the 'active site' — the part of the enzyme that cuts viruses free from cells and also where the drugs bind — must be identical in all of the types. This made sense because the sequences of amino acids in all neuraminidase active sites are virtually identical. But John Skehel from the National Institute for Medical Research in London, UK, had a hunch this might not be the case. With his colleagues he used X-ray crystallography to determine the structures of 3 other types of neuraminidase: N₁, N₄ and N₈. the 3d structures of the active site were indeed different to those in N₂ and N₉. When N₁, N₄ and N₈ bind their normal targets, a small cavity close to the active site opens up. It seems that amino acids well outside the active sites influence whether or not such a cavity opens. So drug developers were lucky that oseltamivir and zanamivir were able to block all neuraminidase subtypes. They could now exploit information from his studies to find new drugs — ones that may even be subtype-specific, or perhaps less likely to allow resistance to develop. We have to remember, though, that there is more to making a drug than just getting a small molecule to fit into an active site. It has to be well tolerated by patients and have the right chemistry to enable it to get to the virus in the body. At the moment, researchers may have a bit of time to develop new leads, since resistance to oseltamivir seems to be developing relatively slowly. And pharmacologists still have a few tricks up their sleeves. Other neuraminidase inhibitors are starting to enter clinical trials, including an intravenous versions of zanamivir and another promising drug called peramivir. Zanamivir is already known to be effective — it's just that the inhaled version is impractical. I'm very excited about an intravenous neuraminidase inhibitor. It answers a real medical need because there is currently no injectable drug that can be given to very sick people^ref.
NAIs have a reasonable side effect and adverse effect profile if they are to be used to treat or protect patients against a life-threatening disease. However, if these compounds are to be prescribed in situations in which avoidance of inconvenience or minor discomfort is hoped for, then the balance of harms to benefits will be more difficult to judge^ref.
NAIs are effective in shortening illness duration in healthy children with influenza, but efficacy in 'at risk' children remains to be proven. Oseltamivir is also effective in reducing the incidence of secondary complications, and may be effective for influenza prophylaxis^ref.
Even if antiviral treatment or prophylaxis leads to the emergence of a transmissible resistant strain in as few as 1 in 50,000 treated persons and 1 in 500,000 prophylaxed persons, widespread use of antivirals may strongly promote the spread of resistant strains at the population level, leading to a prevalence of tens of percent by the end of a pandemic. On the other hand, even in circumstances in which a resistant strain spreads widely, the use of antivirals may significantly delay and/or reduce the total size of the pandemic. If resistant strains carry some fitness cost, then, despite widespread emergence of resistance, antivirals could slow pandemic spread by months or more, and buy time for vaccine development; this delay would be prolonged by nondrug control measures (e.g., social distancing) that reduce transmission, or use of a stockpiled suboptimal vaccine. Surprisingly, the model suggests that such nondrug control measures would increase the proportion of the epidemic caused by resistant strains. The benefits of antiviral drug use to control an influenza pandemic may be reduced, although not completely offset, by drug resistance in the virus. Therefore, the risk of resistance should be considered in pandemic planning and monitored closely during a pandemic^ref.
11 functional residues were highly conserved except for AAF02313 with the H₅N₁-NA (BAE46950) strain, which is the first reported oseltamivir-resistance virus strain. However, the framework residues have remarkable mutations from N9-NA to H₅N₁-NA, and a few mutated residues were observed in different H₅N₁-NAs. A partially hydrophobic site S5 (formed by Ala²⁴⁶ and Thr²⁴⁷) in N9-NA is changed to a hydrophilic site (formed by Ala²²⁷ and Asn²²⁸) in H₅N₁-NA, while a hydrophilic site S6 (formed by Asn³⁴⁶ and Asn³⁴⁷) in N9-NA was replaced by a hydrophobic site (formed by Ala³²³ and Tyr³²⁴). All these mutations might be the reason for the oseltamivir-resistance by some H₅N₁ viruses. In order to find the possible drug-resistant H₅N₁ virus, similarity analysis was performed using the BAE46950 sequence as the benchmark template, and 21 sequences were found from the database of the 108 H₅N₁ NAs that had over 95% sequence similarity with BAE46950^ref.
Web resources : Neuraminidase Inhibitor Susceptibility Network (NISN)^ref


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• ... coreceptor binding inhibitors (CRI) and fusion inhibitors (FI)
  
• for Influenzavirus A :
• M2 ion channel inhibitors (exceptedA(H₅N₁)) : tricyclic antiviral (TAV) drugs / adamantane or aminoadamantane derivatives (the less expensive class of anti-influenza drugs)
• amantadine (1-adamantan amine) (Amantan^®, Antadine^®, Contenton^®, Mantadine^®, Mantadan^®, Mantadix^®, Mantaviral^®, PK-Merz^®, Protexin^®, Symadine^®, Symmetrel^®, Virofral^®) was approved in 1966 for chemoprophylaxis of influenza A (H₂N₂) infection and was later approved in 1976 for the treatment and chemoprophylaxis of influenza type A virus infections among adults and children aged >1 years (also approved for the treatment of Alzheimer's disease and Parkinson's disease). Careful observation is advised when amantadine is administered concurrently with drugs that affect CNS, including CNS stimulants. Concomitant administration of antihistamines or anticholinergic drugs can increase the incidence of adverse CNS reactions. No clinically substantial interactions between rimantadine and other drugs have been identified. Schedule : 200 mg p.o. q day (peds 2.2-4.4 mg/kg bid, max, 75 mg bid) for at least 10 days (prophylaxis) or for 1-2 days after symptoms resolve (treatment)
• 2-amantadine (2-adamantanamine) isomers
• rimantadine (a-methyl-1-adamantane methylamine) (Flumadine^®) was approved in 1993 for treatment and chemoprophylaxis of influenza A infection among adults and prophylaxis among children. Although rimantadine is approved only for chemoprophylaxis of influenza A infection among children, certain specialists in the management of influenza consider it appropriate for treatment of influenza A among children (American Academy of Pediatrics. Influenza. In: Pickering LK, editor. 2000 red book: report of the Committee on Infectious Diseases. Elk Grove Village, IL: American Academy of Pediatrics, 2000:351--9). Schedule : 100 mg p.o. bid (peds < 10 y.o. 5 mg/kg Q24h, max 150 mg/day) (elderly and adults with renal or hepatic impairment 100 mg p.o. Q 24 hr) (prophylaxis) or for 7 days (treatment)
• tromantadine (Tromantadine hydrochloride^®)
• somantadine
• memantine (1-amino-3,5-dimethyl adamantane) (Ebixa^®, Namenda^®)
• carbobenzoxy-D-Phe-L-Phe-nitro-L-Arg

Resistance : amantadine-resistant viruses are cross-resistant to rimantadine and vice versa^ref. Drug-resistant viruses can appear in approximately 33% of patients when either amantadine or rimantadine is used for therapy^{ref1, ref2, ref3}. During the course of amantadine or rimantadine therapy, resistant influenza strains (a single point mutation at amino acid positions 26, 27, 30, 31 (expecially Ser31Asn), or 34 of the M2 protein can confer cross-resistance to both amantadine and rimantadine) can replace sensitive strains within 2-3 days of starting therapy^{ref1, ref2}. Resistant viruses have been isolated from persons who live at home or in an institution where other residents are taking or have recently taken amantadine or rimantadine as therapy^{ref1, ref2}; however, the frequency with which resistant viruses are transmitted and their effect on efforts to control influenza are unknown. Amantadine- and rimantadine-resistant viruses are not more virulent or transmissible than sensitive viruses^ref. The screening of epidemic strains of influenza A has rarely detected amantadine- and rimantadine-resistant viruses^{ref1, ref2, ref3}. Persons who have influenza A infection and who are treated with either amantadine or rimantadine can shed sensitive viruses early in the course of treatment and later shed drug-resistant viruses, including after 5-7 days of therapy^ref. Such persons can benefit from therapy even when resistant viruses emerge. The possibility exists for primary M2 inhibitor resistance in a pandemic strain : complete cross-resistance occurs with these compounds and is associated with a single nucleotide change in the M2 protein. Resistant variants are transmissible and fully pathogenic.
Side effects : both amantadine and rimantadine can cause CNS and gastrointestinal side effects when administered to young, healthy adults at equivalent dosages of 200 mg/day. However, incidence of CNS side effects (e.g., nervousness, anxiety, insomnia, difficulty concentrating, and lightheadedness) is higher among persons taking amantadine than among those taking rimantadine. In a 6-week study of prophylaxis among healthy adults, approximately 6% of participants taking rimantadine at a dosage of 200 mg/day experienced >1 CNS symptom, compared with approximately 13% of those taking the same dosage of amantadine and 4% of those taking placebo. A study of older persons also demonstrated fewer CNS side effects associated with rimantadine compared with amantadine. Gastrointestinal side effects (e.g., nausea and anorexia) occur in approximately 1-3% of persons taking either drug, compared with 1% of persons receiving the placebo. Side effects associated with amantadine and rimantadine are usually mild and cease soon after discontinuing the drug. Side effects can diminish or disappear after the first week, despite continued drug ingestion. However, serious side effects have been observed (e.g., marked behavioral changes, delirium, hallucinations, agitation, and seizures). These more severe side effects have been associated with high plasma drug concentrations and have been observed most often among persons who have renal insufficiency, seizure disorders, or certain psychiatric disorders and among older persons who have been taking amantadine as prophylaxis at a dosage of 200 mg/day. Clinical observations and studies have indicated that lowering the dosage of amantadine among these persons reduces the incidence and severity of such side effects. In acute overdosage of amantadine, CNS, renal, respiratory, and cardiac toxicity, including arrhythmias, have been reported. Because rimantadine has been marketed for a shorter period than amantadine, its safety among certain patient populations (e.g., chronically ill and older persons) has been evaluated less frequently. Because amantadine has anticholinergic effects and might cause mydriasis, it should not be used in patients with untreated angle closure glaucoma. Only two cases of amantadine use for severe influenza illness during the third trimester have been reported. However, both amantadine and rimantadine have been demonstrated in animal studies to be teratogenic and embryotoxic when administered at substantially high doses
• stachyflin, acetylstachyflin, and deacetyl SQ-02-S-V2 produced by Stachybotrys sp. RF-7260, were found to have potent anti-Influenzavirus A activity. Stachyflin is a new class of hemagglutinin fusion inhibitors of influenza A virus. Several derivatives were synthesized from acetylstachyflin and subjected to preliminary examination of their structure-activity relationships. Among them, the 3-oxo and 3,8'-dioxo derivatives showed potent antiviral activity similar to stachyflin. The 3-epi derivative was 4 times less active than stachyflin. Modification of the 6'-hydroxy group and the C-5' position markedly diminished the antiviral activity^ref.
• cleavage and activation, attachment, and fusion steps are unique to HA
• transcription initiation step is unique to the viral RNA polymerase
• the capped short RNA inhibited the viral RNA polymerase
• the peptide derived from matrix protein inhibited the RNA polymerase activity
• antisense oligonucleotide
• DNA enzyme
• RNAi are also available to inhibit gene expression of influenza virus.
• recombinant collectins inhibit replication of influenza by binding to the viral haemagglutinin as well as altering phagocyte responses to the virus
• inhibitors of binding to CXCR4 (effective against HIV-1 T-tropic or X4 strains) :
  
• bicyclam AMD3100
• T22
• T134
• ALX40-4C
• CGP64222
• AMD-070
• CS-3955
• inhibitors of binding to CCR5 (effective against HIV-1 M-tropic or R5 strains) :
  
• TAK779
• SCH-C / SCH351125
• SCH-D
• TAK-220
• AK-602
• maraviroc / UK-427857 (Celsentri^®)
• inhibitors of gp41 :
  
• enfuvirtide / T20 / T-20 / DP178 / pentafuside (Fuzeon^®; source : Roche and Trimeris) :
  
• betulinic acid derivatives
• T1249 / T-1249
• RSV and paramyxovirus fusion inhibitor
• VP14637 (source : ViroPharma) : a triphenol-based molecule that interacts with a transient conformation of the RSV F protein
• JNJ2408068
• R 170591
• NMS03
• GS-5806^ref
• 5-helices
• peptide n inhibitor
• CTC96
• effective against enveloped viruses
• LJ-001
• dUY11
• epigallocatechin gallate
• FGI-103
• FGI-104
• FGI-106
  
• n-docosanol / behenyl alcohol (10% cream : Abreva^®) (a 22-carbon-long saturated alcohol that inhibits fusion of plasma membrane with a broad spectrum of lipid-enveloped viruses in vitro including HHV-1 / HSV-1, HHV-2 / HSV-2, HHV-3 / VZV, HHV-5 / CMV, HHV-6, HHV-8, RSV and HIV-1)
• ... viral assembly and disassembly
• NCp7 zinc finger-targeted agents
• 2,2'-dithiobisbenzamides (DIBAs)
• azodicarbonamide (ADA)
• capsid assembly inhibitor (CAI) : formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. A 12-mer peptide binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1^ref. Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. The 1.7-Å-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature- and mature-like particle assembly in vitro was reported. The peptide inserts as an amphipathic a-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact 5-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy^ref.
• ... transcription
• microRNA inhibitors :
• miravirsen acts by inhibiting microRNA-122, important for HCV replication
  
• fluoroquinolines
• K-37 / 7-(3,4-dehydro-4-phenyl-1-piperidinyl)-1,4-dihydro-6-fluoro -1-methyl-8-trifluoromethyl-4-oxoquinoline-3-carboxylic acid
• bistriazoleacridone derivatives
• temacrazine
• CGP64222
• IFNs
• ... translation
• IFNs
• isatin-b-thiosemicarbazone
• N-methylisatin-b-thiosemicarbazone / methisazone (Marboran^®)
• ... v-polyprotein cleavage
• inhibitors of serine proteases
• inhibitors of serine protease of HRV
• 3C inhibitors :
• rupintrivir / AG7088
  
• inhibitors of serine proteases of HHVs
• thieno[2,3-D]oxazinone
• aryl-hydroxylamine derivatives
• monobactams
• pyrrolidin-5,5'-lactams
• 1,4-dihydroxynaphtalen
• naphtoquinones
• inhibitors of serine proteases of HCV
  
• valopicitabine / NM283 : the 3'-O-l-valinyl ester prodrug of the pyrimidine nucleoside analogue 2'-C-methylcytidine, suffers from a low oral bioavailability^ref, whose activity is antagonized in vitro by ribavirin^ref
• NS3.4A protease inhibitors^ref
• ABT-450
• asunaprevir / BMS-650032
• boceprevir (Victrelis^®)
  
• ciluprevir / BILN-2061
• danoprevir
• narlaprevir
  
• SCH-503034 (Schering-Plough Research Institute)
• simeprevir (Olysio^®)
• telaprevir / VX-950 (Incivek^® and Incivo^®; Vertex Pharmaceuticals Inc./Janssen Pharnmaceutica NV/Mitsubishi Pharma Corp.)^ref rapidly reduce the plasma viral load of patients chronically infected with genotype 1 HCV by a mean approximately 3 log₁₀ in 2 days^ref
• TMC435
  
• vaniprevir
  
• NS5A inhibitors
  
• daclatasvir / BMS-790052
• ACH-3102
• GS-5885
  
• ledipasvir (LDV) (; in combination with sofosbuvir, Gilead)
• ombitasvir
• nonnucleoside NS5B inhibitor
• dasabuvir
  
• aspartate proteases inhibitors (PI) (in HIV-1) ("-navir") (also directly inhibit the growth of Plasmodium falciparum in vitro at clinically relevant concentrations^ref)
• peptides containing double bonds
• amprenavir (APV) / VX-478 / 141W94 (Agenerase^®). Side effects : Steven-Johnsons's syndrome
• BMS-232632
• darunavir (Prezista^®)
• fosamprenavir / GW433908, 908 (Lexiva™)
• indinavir (IDV) / L-735,524 (Crixivan^®)
• lopinavir / ABT-378/r (as fixed dose coformulation with ritonavir in Kaletra^®)
• nelfinavir (NFV) (Viracept^®)
• ritonavir (RTV) / ABT-538 (Norvir^®; as fixed dose coformulation with lopinavir in Kaletra^®)
• saquinavir mesylate (SQV) (Invirase^®; previously known as Fortovase^®, which was discontinued by Roche in 2006)
In a new study of 100 patients who had not taken an AIDS drug before Kaletra^®, 64% of patients have < 50 copies of the HIV/mL of blood.
They are pumped out by ABCB1 / P-glycoprotein (P-GP) / MDR1.
PIs cannot be suspended
Side effects :
• they inhibit the peptidase (mitochondrial processing) a (PMPCA) and the subsequent mitochondrial failure leads to hypertriglyceridemia, fat redistribution (fat decrease in hips and retroorbital regions, fat increase in breast, back and interscapular region), a condition termed lipodystrophy or Launois-Bensaude's syndrome. The severity relates also to administration of NRTI, which inihibits mitochondrial DNA polymerase g, leading to mutations in mtDNA and hyperlactacidemia. They also cause coronary calcification.
• premature carotid atherosclerosis (CA)
• non peptidic compounds
• azapeptidic scaffold
• atazanavir / BMS 232632 (Zrivada^®, Reyataz^®); resistance is associated with an I50L substitution in HIV protease. Side effects : urolithiasis composed of a mixture of 60% atazanavir metabolite and 40% calcium phosphate (carbonate apatite). The stone contained atazanavir crystals and not metabolites adsorbed into the apatite^ref. Atazanavir is metabolized and excreted mainly through the liver, and only 13% of a single radiolabeled 400-mg dose was recovered in the urine, of which 7% constituted unchanged drug
• brecanavir (discontinued by GSK on 2006)
• cyclic urea
• mozenavir dimesylate / DMP-450
• 4-hydroxy-2-pyrone
• tipranavir (Aptivus^®)
• L-mannaric acid
• GW640385
• TMC114 / darunavir (Prezista^®; source : Tibotec Pharmaceuticals, Ltd)
• 4-hydroxy-5,6-dihydro-2-pyrrone
• VX-175/GW433908
• cysteine proteases (in Rhinoviruses)
• AG7088
• ... genome replication
• inhibitors of v-DNA polymerase
• nucleoside analogues (chain destabilizers or chain terminators)
• heteroyclic or carbocyclic alogenated or alkylated thymine derivatives
• 5-iodo-2'-deoxyuridine (idoxuridine, IDV, IDUR / NSC-39661) (Herplex^®, Dendrid^®)
• 5-trifluoromethyl-2'-deoxyuridine (trifluridine / TFT / trifluorothymidine) (Viroptic^®)
• (E)-5-(2-Br-vinyl)-deoxyuridine (BVDU)
• (E)-5-(2-I-vinyl)-deoxyuridine (IVDU)
• (E)-5-ethyl-2'-deoxyuridine (EDU)
• (E)-5-(2-chloroethyl)-2'-deoxyuridine (CEDU)
• (E)-5'-F-uracil (5-FU)
• arabinofuranosylnucleosides
• 9-b-D-ribofuranosyladenine / vidarabine / adenine arabinoside / araA / ara-A (Vira-A^®)
• 1-b-D-ribofuranosylcytosine / citarabine / citosine arabinoside / araC
• 1-b-D-ribofuranosylthymidine / araT
• alogenated derivatives
• 5 alogenated derivatives
• BVaraU
• 5 and 2' alogenated derivatives
• FBVAU
• FMAU
• FEAU
• FIAC
• guanine analogues
• cyclic guanine analogues
• A5021
• D- or L-cycloesenylguanine
• acyclic guanine analogues : they can be transformed into acyclic GMP exclusively by HHV-1 / HSV-1-tk or HHV-3 / VZV-tk or by HHV-5 / CMV-PK. Then they are transformed into acyclic GTP.
• 9-(2-hydroxyethoxymethyl)-guanine (acyclovir / acycloguanosine / ACV (Aciclin^®, Zovirax^®; ME609 / Zovirax Duo^® in combination with hydrocortisone) ; oral prodrug : valacyclovir (Talavir^®, Valtrex^®, Zelitrex^®)

Desensitization therapy^{ref1, ref2, ref3}.

• 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir / GCV / DHPG (Cytovene^®, Cymevene^®, Vitrasert^® (sterile intravitreal implant) ; prodrug : valganciclovir (Valcyte^®))

During the lytic form of infection, 2 virally encoded kinases, the HHV-4 / EBVthymidine kinase (EBV-TK) and the BGLF4 gene product, which phosphorylate the prodrug GCV and convert it into its active cytotoxic form, are expressed. Phosphorylated GCV inhibits not only the virally encoded DNA polymerase but also the cellular DNA polymerase, leading to premature termination of the nascent DNA and cell death^{ref1, ref2, ref3}. In addition, phosphorylated GCV can be transferred to adjacent cells, thus inducing "bystander" killing^ref. Lytic EBV infection also confers sensitivity to the cytotoxic effects of zidovudine (AZT), possibly by inducing AZT phosphorylation^{ref1, ref2, ref3, ref4}.
Side effects : myelosuppression, urticaria
Desensitization therapy^ref

• penciclovir / PCV (Denavir^®, Vectavir^®) ; oral prodrug : famciclovir / FCV (Famvir^®; source : Novartis)
• maribavir / 1263W94
• BDCRB
• FV-100, the 5'-valine ester of Cf1743, the most potent congener of the bicyclic nucleoside analogues (BCNAs)
  
• oxetanocins, which are partially dependent on HHV-3 / VZV-tk
• lobucavir
• acyclic thymine analogues : they can be transformed into acyclic TMP exclusively by HHV-1 / HSV-1-tk or HHV-3 / VZV-tkor by HHV-5 / CMV-PK. Then they are transformed into acyclic TTPs.
• 5-(2-bromovinyl)-2'-deoxyuridine (BVDU) / brivudin (Helpin^®, Zostex^®)


• sorivudine / (1-b-D-arabinofuranosyl E-5 [2-bromovinyluracil] (BV-araU) (Bravavir^®, Bristol-Myers Squibb)
• fialuridine / 2'-fluoro-2'-deoxy-1-b-D-arabinofuranosyl-5-iodo-uracil (FIAU)
• fiacitabine
• netivudine
• non-obligate RNA chain terminator :
• BCX4430^ref effective against Ebolavirus
• inhibitors of RNA replicase
• pesti- (hepaci-, flavi-) virus : VP-32947
• inhibitors of HCV NS5B RNA-dependent RNA polymerase
• NS5B ‘nucleoside’ inhibitors :
  
• INX-189
• GS-7977
• mericitabine
• NS5B ‘non-nucleoside’ inhibitors :
• tegobuvir
• setrobuvir
• filibuvir
• HCV-796 (source : Viropharma, Inc.)
• sofosbuvir (SOF) (Sovaldi^®; in combination with ledipasvir, Gilead)
• inhibitors of the HSV helicase-primase / UL5 (HPI)
  
• pritelivir / AIC316^ref
  
• AIC316 / BAY 57-1293
  
• aminothiazolylphenyl-containing compounds
• BILS 179 BS
• BILS 45 BS
• BILS 22 BS, a potent analog of BILS 45 BS.
• inhibitors of the HSV terminase complex
• inhibitors of HHV-5 / CMV terminase complex (interfering with the viral pUL56 gene product and in the process disrupting the viral terminase complex)
  
• letermovir / AIC246
  
• inhibitors of the HSV portal protein
• inhibitors of the HSV UL97 protein kinase
• inhibitors of integrase (INI) :
  
• against HIV-1 IN :
• L-879812 / L-870,812
• PDPV-165
• raltegravir / MK-0518 (Isentress^®)
• elvitegravir (Vitekta^®; Stribild^® in combination with cobicistat, emtricitabine, and tenofovir); Quad pill = elvitegravir + cobicistat + tenofovir + FTC, cobicistat being a
  pharmacoenhancer, intended to boost the anti-HIV activity of the INI elvitegravir)
  
• dolutegravir (Tivicay^®)
• zintevir / AR177 / T30177
  
• L-chicoric acid
• diketoacids
• 4-aryl-2,4-dioxobutanoic acid derivatives
• L731,988
• L708,906
• inhibitors of capsid proteins :
• PA-457
• a-HCG
• inhibitors of v-reverse transcriptase (RT) (antiretroviral (ARV) drugs)
• nucleoside RT inhibitors (NRTI) / nukes : 2',3'-dideoxynucleoside analogues (or their 5'-monophosphate prodrugs stabilized by phosphoramidate or cyclosaligenyl to by-pass the first phosphorylation step)
• against HIV-1 RT
• (-)-2',3'-dideoxy-3'-thiacytidine / lamivudine (LAM) / 3TC (Epivir^®, Zefix^®, Zeffix^®; Combivir^® in combination with zidovudine; Epzicom^® in combination with abacavir; Trizivir^® in combination with abacavir sulfate and zidovudine; Emtri Suspension^® in combination with nevirapine and stavudine)) : in 20% of patients HBV develops at least partial resistance within a year, a figure that raises to 53% after 3 years.

Side effects : inhibition of TERT
• 3'-azido-2',3'-dideoxythymidine / zidovudine / AZT (Retrovir^®, Videx EC^®, Combivir^® in combination with lamivudine; Trizivir^® in combination with lamivudine and abacavir sulfate)

Side effects :
• inhibition of TERT^{ref1, ref2, ref3, ref4, ref5}
• inhibition of DNA polymerase g^{ref1, ref2, ref3, ref4, ref5} => ragged-red fibers => toxic myopathy : fatigue, myalgia, mild hyposthenia, mild CK increase, myelotoxic and neurotoxic (it crosses both the blood-brain and the blood-placenta barriers)
• 2',3'-dideoxycitosine / zalcitabine / ddC (Epivir^®, Hivid^®)
• 2',3'-dideoxyadenosine
• 2',3'-dideoxyinosine / didanosine / ddI (Videx^®)
• 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC)
• abacavir / ABC / 1592U89 (Ziagen^®; Epzicom^® in combination with lamivudine; Trizivir^® in combination with lamivudine and zidovudine) => carbovir triphosphate. Side effects : Steven-Johnsons's syndrome and abacavir hypersensitivity syndrome (AHS) (a potentially life-threatening illness occurring in 4-8% of those initiating the drug,  specifically and exquisitely restricted by HLA-B*5701 and mediated by CD8⁺ lymphocytes; anyway 45% of those carrying HLA-B*5701 can tolerate abacavir^ref)
• amdoxovir
• (-)-dOTC / BCH-10652
• 3'-deoxy-2:3'-didehydrothymidine / stavudine / d4T (Zerit^®; Emtri Suspension^® in combination with lamivudine and nevirapine)
• emivirine / MKC-442
• PSI-5004
• DPC-817
• elvucitabine
• alovudine
• MIV-210
• DOT
• L-697639 binds at a site near, but not in, the active site
• against HBV RT
• 9-[2-phosphonylmethoxyethyl]-adenine / PMEA / adefovir
• adefovir dipivoxil (ADV) (Hepsera^®) can be used as a licensed drug for the treatment of chronic HBV infection^{ref1, ref2, ref3}. Virtually 100% efficacy has been suggested in lamivudine-resistant HBV infections^ref, but in 2 reports^{ref1, ref2} only 39 of 46 patients and 13 of 18 patients, respectively, had a marked treatment response. Resistance to lamivudine developed in 3 patients with chronic hepatitis B, and all 3 had a response to tenofovir but not to adefovir. HBV isolated from all 3 patients before adefovir therapy had the same rtI233Vmutation in the RT domain^ref, which is very rare (3 of 152 strains) among patients with the genotype D worldwide^ref. The 3 patients had genotype D (subgroup D3)^ref and had closely related but not identical RT and S gene sequences. A GenBank search revealed that only 3 of about 500 previously reported HBV strains had the rtI233V mutation. 2 of these strains were from Southeast Asia and were genotype C^ref, and the third was a HBV strain found in gibbons^ref. A lamivudine-resistant genotype C strain found in Chinese patients had a threonine at that position, but this variant may not have been viable because it had a truncated S gene^ref. Thus, there is very little variation at rt233 and no obvious genotypic association. There is, however, at least one other example of primary adefovir resistance. 2 of 3 cases of in vivo adefovir resistance that we discussed in a previous report^ref involved HBV strains that had an rtL217R mutation before adefovir therapy. This mutation is present in most HBV strains with genotype A2, which are prevalent in Europe and the USA, but not in strains with other genotypes, including A1, which are found in Africa and Asia^{ref1, ref2}. Some naturally occurring HBV strains are primarily resistant to adefovir. The rapid and strong effect of tenofovir in all three patients along with the in vitro data suggest that the patients' nonadherence to the medication was not the reason for the failure of adefovir therapy. Reactivation of HBV replication, as occurred in Patient 1, has also been reported in three patients by Van Bömmel and Berg^ref, but sequencing data for HBV DNA have not been reported by these authors, and therefore it is not known whether these patients carried relevant HBV variants. In agreement with a previous report^ref, the in vitro efficacy of tenofovir against wild-type HBV was similar to that of adefovir. Van Bömmel et al. reported that tenofovir was generally superior to adefovir in vivo^ref, a finding that was most likely not caused by the presence of reverse-transcriptase variants. They ascribed this general superiority of tenofovir in vivo to the fact that the dose of the drug can be increased by a factor of 10, whereas the dose of adefovir is fixed^ref. Since the dose of adefovir cannot be increased, the presence of naturally occurring HBV variants, as described here and in our earlier report^ref, may decrease its efficacy
• pradefovir mesylate (PDV) is a cytochrome P450 (CYP) 3A4-activated, liver-targeted prodrug of PMEA with potentially improved efficacy and less nephrotoxicity than ADV
• clevudine (CLV) / L-FMAN
• b-L-deoxynucleosides^ref :
• telbivudine (Tyzeka^®)
• valtorcitabine => torcitabine
• entecavir (Baraclude^®; source : Bristol-Meyers Squibb). Side effects : headache, abdominal pain, diarrhea, fatigue and dizziness
• emtricitabine / FTC (Coviracil^®, Emtriva^®; source : Gilead; Truvada^® in combination with tenofovir disoproxil ; Atripla^® in combination with tenofovir disoproxil and efavirenz; Complera^®/Eviplera^® in combination with tenofovir disoproxil + rilpivirine)
  
• b-L-deoxythymidine (L-dT)
• nonnucleoside RT inhibitors (NNRTI) / non-nukes (allosteric inhibitors) :
• against HIV-1 RT :
  
• first generation :
• capravirine / AG-1549
• delavirdine (DLV) (Rescriptor^®)
• efavirenz (EFV) / DMP266 (Sustiva 600^® (source : BMS), Stocrin^® (source : Merck) outside the USA, Canada and certain European countries; Atripla^® in combination with tenofovir disoproxil and emtricitabine)
• GW-420867X
• nevirapine (NVP) / BI-RG-587 (Viramune^®; Emtri Suspension^® in combination with lamivudine and stavudine)
• thiocarboxyanilide / thiocarboxanilide / UC781
• second generation :
• etravirine / TMC125 / R165335 (Intelence^®)
  
• third generation :
• DPC083 / DPC-083
• +/- calanolide A
• GW5634
• MIV-150
• SJ-3366 (also interferes with HIV-1 entry via an intermediate target formed after virus-cell attachment)
• pyridine oxide derivatives (also against HIV-2 RT : they also interfere with postintegration transcription of the proviral DNA to RNA and may have the potential to prevent reactivation of the virus, which is an interesting added value^ref)
• dapivirine
• rilpivirine (Edurant^®; Complera^®/Eviplera^® in combination with tenofovir disoproxil + emtricitabine)
Side effects : Steven-Johnsons's syndrome
• highly active antiretroviral therapy (HAART) / triple-therapy cocktails = IDV + NRTI (AZT + 3TC) + NNRTI + drugs effective against opportunistic infections.
• stavudine 30 mg + lamivudine 150 mg + nevirapine 200 mg (Imunaid-30^®)
• stavudine 40 mg + lamivudine 150 mg + nevirapine 200 mg (Imunaid-40^®)
Liver-related mortality is an increasing problem in HIV/HBV-coinfected patients receiving HAART. In HIV^- patients, HBV chronicity is associated with a reduction in specific T cell responses that can be partially restored by treatment with lamivudine, and reconstitution of some HBV-specific T cell responses can also occur in HIV⁺ patients after a reduction in HBV load with HAART, even if HAART regimen doesn’t include drugs with a specific anti-HBV effect. Since restoration of HBV-specific T cell reactivity is only transient, therapeutic stimulation of HBV-specific T cell responses to complement lamivudine treatment should be done early after the initiation of lamivudine. Moreover, the transient nature of the immune reconstitution may represent a favorable condition for virus reactivation once lamivudine therapy is withdrawn
Complications : immune reconstitution inflammatory syndrome (IRIS) / immune restoration syndrome or disease (IRD) is the name given to a diverse group of symptoms identified in 1999 that can occur soon after the initiation of HAART, despite satisfactory control of viral replication and improvements in CD4 lymphocyte counts : this can include the first appearance or the recurrence of previously-treated opportunistic infections, like pulmonary and CNS Mycobacterium tuberculosis^ref, HHV-5 / CMV uveitis, Pneumocystis carinii, Cryptococcus neoformans meningitis or Mycobacterium avium-intracellulare lymphadenitis, or the flare-up of viral infections that include hepatitis and herpes. Low CD4 counts at HAART initiation are the only independent predictor of IRIS as it is caused by a deferred or partial CD4 response after HAART is initiated. 22% has IRIS events, which occurr at a median of 12 weeks (range 3 - 24 weeks) following the initiation of HAART, and the majority (78%) are due to the occurrence - or the more frequent and/or more severe recurrence - of dermatological problems: HHV-2 / HSV-2(50%); HPVs (24%); molluscum contagiosum virus (9.5%); and HHV-3 / VZV (9.5%). Non-dermatological manifestations of IRIS are much less common: Mycobacterium avium-intracellulare (2%), HBV (2%), and there are rare cases of HHV-8 or Pneumocystis carinii symptoms. In MTB, the major risk factor for IRD is beginning HAART within 2 months of antituberculosis treatment. This fact has led many experts to recommend delaying HAART for 2 months, a strategy that may also reduce the incidence of adverse events. However, a recent retrospective review suggested that delaying HAART would, particularly in those with CD4 cell counts <100 cells × 10⁶/L, lead to significant increase in risk for death and new opportunistic infections^ref. IRD should always be considered along with treatment failure if a patient's clinical condition deteriorates after HAART is introduced.
Fixed-dose combinations are cheaper and easier to take than other antiretroviral drugs and are a major part of drug access programs in the developing world.
3 combination antiretrovirals (triple fixed-dose combination pills containing lamivudine, stavudine, and nevirapine in 2 different strengths, and a lamivudine plus zidovudine tablet), manufactured by Ranbaxy Laboratories Ltd. (RLL), were taken off the WHO prequalification list because a contract research organization (CRO) that had done bioequivalence studies on behalf of Ranbaxy was found to be noncompliant with good clinical and laboratory practices. In May 2004, 2 combination antiretrovirals from Cipla were withdrawn after another CRO performing bioequivalence studies failed good clinical and laboratory practice.

• nucleotide RT inhibitors (NtRTI)
• tenofovir disoproxil fumarate (PMPA) (Viread^®; source : Gilead; Truvada^® in combination with emtricitabine; Atripla^® in combination with emtricitabine and efavirenz; Complera^®/Eviplera^® in combination with rilpivirine + emtricitabine)

Side effects : Fanconi syndrome^ref

• inibitors of every polymerase
• acyclic nucleotide phosphonates (phosphonylmethoxyalkylpurines and -pyrimidines) do not depend on the virus-encoded TK for their phosphorylation but depend on cellular enzymes for conversion to their diphosphoryl derivatives which then inhibit viral DNA synthesis
• (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ((S)-HPMPA)
• (S)-cHPMPA
• (S)-HPMPDAP
• PMEADP
• cidofovir / [s]-1-[3-hydroxy-2-phosphonylmethoxypropyl]cytosine / (S)-HPMPC  (Vistide^®)
• hexadecyloxypropyl (HDP)-cidofovir / CMX001 (source : Chimerix, Inc) : cidofovir prodrug for treating resistant HSV infection in immunocompromised patients
  
• pyrophosphate analogues (allosteric inhibitors)
• foscarnet / phosphonoformate (PFA) (Foscavir^®). Side effects : nephrotoxicity, hypocalcemia, seizures.
• ... replisome destabilization
• guanidinium chloride (effective against Polioviruses)
• benzimidazoles
• 2-(a-hydroxybenzyl)-benzimidazole (HBB)
• 1263W94 (effective against HHV-5 / CMV)
• ... viral egress :
  
• ST-246 / tecovirimat inhibits egress of orthopoxviruses by targeting p37 envelope glycoprotein.
• ... cellular molecules : virus-aspecific approach, high toxicity, low likelihood to develop drug-resistance. Targeted proteins are necessay for ...
• ... transcription and translation
• alisporivir / Debio 025 / DEB025 / UNIL-025 : cyclophilin inhibitor active against HCV
• microRNA-141 targets PPARa is effective against HBV
• HBSC-11, an inhibitor of the human La protein is effective against HBV
• Matijing-Su derivatives, isolated from the Chinese ethnic drug Matijing, used as folk medicine in China is effective against HBV
• recombinant IFNs
• IFN-a₂(effective for short-lasting prophylaxis against HBVand HCV ; flu-like side effects, not recommended in pregnants and individuals affected by autoimmune diseases ; most effective in males with age > 50)
• ... nucleotide availability
• IMPDH1 and IMPDH2 inhibitors
• ribavirin / 1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxyamide (Copegus^®, Roche; Rebetol^®, Schering-Plough; Rebetron^®, Ribasphere^®, Three Rivers Pharmaceuticals; aerosol Virazole^®) is an oral nucleoside analogue with broad activity against viral pathogens^ref approved by FDA for treatment of HCV and RSV, but also somewhat effective against influenzaviruses^ref. 5 distinct mechanisms have been proposed to explain the antiviral properties of ribavirin, including both indirect mechanisms (IMP dehydrogenase (IMPDH) inhibition, immunomodulatory effects) and direct mechanisms (interference with RNA capping, polymerase inhibition, lethal mutagenesis)^ref. Ribavirin appears to have minimal direct activity against HCV replication^ref, but it may lead to rapid and lethal mutation of virions or depletion of intracellular guanosine triphosphate, which is necessary for viral RNA synthesis^{ref1, ref2}
• viramidine, the 3-carboxamidine derivative of ribavirin, was effective against a spectrum of influenza A (H₁N₁, H₃N₂ and H₅N₁) and B viruses in vitro, with the 50% effective concentration (EC₅₀) ranging from 2 to 32 mg/ml. The mean 50% cytotoxic concentration (CC₅₀) in the MDCK cells used in these experiments was 760 mg/ml. Ribavirin, run in parallel, had a similar antiviral spectrum, with EC₅₀ values ranging from 0.6 to 5.5 mg/ml; the mean CC₅₀ for ribavirin was 560 mg/ml. Oral gavage administrations of viramidine or ribavirin to mice infected with influenza A/NWS/33 (H₁N₁), A/Victoria/3/75 (H₃N₂), B/Hong Kong/5/72 or B/Sichuan/379/99 viruses were highly effective in preventing death, lessening decline in arterial oxygen saturation, inhibition of lung consolidation and reducing lung virus titers. The minimum effective dose of viramidine in these studies ranged from 15 to 31 mg/kg/day, depending upon the virus infection, when administered twice daily for 5 days beginning 4 h pre-virus exposure. The LD₅₀ of the compound was 610 mg/kg/day. Ribavirin's minimum effective dose varied between 18 and 37.5 mg/kg/day with the LD₅₀ determined to be 220 mg/kg/day. Viramidine's efficacy was also seen against an influenza A/NWS/33 (H₁N₁) virus infection in mice, when the compound was administered in the drinking water, the minimum effective dose being 100 mg/kg/day. Delay of the initiation of either viramidine or ribavirin therapy, using the approximate 1/3 LD₅₀ dose of each, was protective as late as 48 h after exposure to the A/NWS/33 virus. While both compounds appear to have similar efficacy against influenza virus infections, when one considers the lesser toxicity, viramidine may warrant further evaluation as a possible therapy for influenza^ref
• EICAR
• mycophenolic acid (MPA) (prodrug : morpholinoethyl ester)
• VX497
• ... viral genome methylation
• S-adenosylhomocysteine hydrolase (SAH) inhibitors
• carbocyclic adenosine
• carbocyclic 3-deaza-A
• neplarocin A
• 3'-deazaneplarocin A
• ... cell cycling
• CDK9 inhibitors
• fluoroquinolones
• ... viral protein glycosylation (effective against enveloped viruses)
• a-glycosidases inhibitors :
  
• castanospermine
• celgosivir, in development by MIGENIX Inc for the treatment of HCV infection, is an oral prodrug of the natural product castanospermine that inhibits alpha-glucosidase I, an enzyme that plays a critical role in viral maturation by initiating the processing of the N-linked oligosaccharides of viral envelope glycoproteins. Failed against dengue virus^ref
  
• 2-deoxy-D-Glc
• deoxynojirimycin (DNJ) and derivatives
• N-nonyl deoxynojirimycin (NN-DNJ) : inhibition of as little as 6% of cellular glycoprocessing results in a greater than 99% reduction in the secretion of HBV, while there seems to be no effect on the host cells. That's why only a small number of misfolded M proteins disrupt the symmetry characteristic of the HBV coat and prevent it from escaping the ER
• N-nonyl deoxygalactojirimycin has the same effect on HCV
• tunicamicin
• ... nuclear export
• exportin 1 (XPO1) inhibitors
• leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo
• KPT-335 / verdinexor^ref
• infection of cells with Influenzavirus A leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf/MEK/ERK signalling cascaderesults in nuclear retention of viral ribonucleoprotein (RNP) complexes, impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle. Unfortunately, the kinases also perform several important jobs in healthy cells, such as controlling how cells divide or when they die. So before inhibitors of Raf kinase can be used as anti-flu drugs, researchers will have to devise a way of making sure that they only get into cells infected with flu^ref

• rifampicin inhibits Retroviridae RT, Poxviridae, and Parvoviridae
• B-904 virucide (source : Venkateshwara Biosentry India Ltd, originally a product of the US-based Biosentry Inc) : currently being test-marketed in Maharashtra, Karnataka, Andhra Pradesh and West Bengal, it is effective in 400 ppm hardness of water and 5% organic matters, but also has antifungal properties and finds multiple applications in hospitals, healthcare facilities, labs, IVF centres and pharma and vaccine manufacturing units. It contains 3 active ingredients known for its germicidal properties — DDAC (didecyl dimethyl ammonium chloride), ADBAC (alkyl dimethyl benzyl ammonium chloride) and Tri butylin oxide^ref
• prostratin is a unique phorbol ester that stimulates membrane translocation of classical, atypical, and expecially novel protein kinase C (PKC) isoforms but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo HIV-1 infection relating to down-regulation of CD4, CXCR4 and CCR5expression yet up-regulate viral expression from latent proviruses in from CD8⁺ T lymphocyte-depleted peripheral blood mononuclear cells. Prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Prostratin forces the HIV-1 out of reservoirs in the body, thus allowing anti-retroviral drugs to attack it : it could upregulate expression from latent viruses and concluded that it may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1^ref. It works, at least in part, by stimulating IKK-dependent phosphorylation and degradation of IkBa, leading to the rapid nuclear translocation of NF-kBand activation of the HIV-1 LTR in a kB enhancer-dependent manner^ref. Under a separate agreement, AIDS Research Alliance (ARA), will return 20% of any profits from the plant-derived form of the drug to Samoa
• brincidofovir (Chimerix; NC, USA) : an antiviral developed for use against cytomegalovirus or adenovirus infections
• favipiravir / T-705 / 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (Toyama Chemical, Japan) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus and Ebolavirus^{ref1, ref2, ref3}
  
• glycyrrhizin / glycyrrhizic acid inhibits HCV, SARS coronavirus, and HIV-1
• a sulphated polysaccharide (SP-2a) from the brown alga Sargassum patens (Kutz.) Agardh (Sargassaceae) was found to significantly inhibit the in vitro replication of both the acyclovir (ACV)-sensitive and -resistant strains of HSV-1^ref

• International Antiviral Therapy Evaluation Center (IATEC)
• HIVandHepatitis.com

• Antiviral Agents: Advances and Problems by Ernst Jucker, Birkhäuser Ed., May 3, 2002, 258 pages, ISBN 3764365471

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