Table of contents :
Epidemiology :
prevalence = 3% of the world's population (180 million people
worldwide ; 3 million Americansref)
remain chronically infected. The virus claims 10,000 to 12,000
U.S. lives annually. Routine screening of blood donors for HBsAg
and the elimination of commercial blood sources in the early
1970s reduced the frequency of, but did not eliminate,
transfusion-associated hepatitis. Although the frequency of
transfusion-associated hepatitis C fell as a result of blood
donor screening, the overall frequency of hepatitis C remained
the same until the early 1990s, when the overall frequency fell
by 80%, in parallel with a reduction in the number of new cases
in injection drug users. After the exclusion of
anti-HCV-positive plasma units from the donor pool, rare,
sporadic instances have occurred of hepatitis C among recipients
of immune globulin (IG) preparations for intravenous (but not
intramuscular) use. Serologic evidence for HCV infection occurs
in 90% of patients with a history of transfusion-associated
hepatitis (almost all occurring before 1992, when
second-generation HCV-screening tests were introduced),
hemophiliacs and others treated with clotting factors, and
injection drug users; 60 to 70% of patients with sporadic
"non-A, non- B" hepatitis who lack identifiable risk factors;
0.5% of volunteer blood donors; and 1.8% of the general
population in the USA, which translates into 4 million persons.
Comparable frequencies of HCV infection occur in most countries
around the world, but extraordinarily high prevalences of HCV
infection occur in certain countries :
- Egypt : 10-15% of the population, some 8-10 million people,
are carrying anti-HCV antibodies, meaning that they either
have or at one time had the virus. 5 million of those are
actively infected. The annual infection rate is > 70 000
new cases, of which at least 35 000 would have chronic
hepatitis C. > 20% of the population in some cities is
infected. The high frequency in Egypt is attributable to
contaminated equipment used for medical procedures and unsafe
injection practices. Egypt's very high prevalence of HCV is
largely the legacy of government campaigns prior to 1980 to
treat rural populations for schistosomiasis (or bilharzia), a
water-borne disease which at one time was endemic in Egypt.
The treatment campaigns, which involved repeated injections,
did not follow rigorous hygiene standards and as such spread
blood-borne HCV throughout the population. genotype 4a is the
most prevalent HCV-genotype (85%) in Egypt (see, for example:
J Egypt Public Health Assoc. 1998;73(1-2):41-55). Only 21% of
Egyptian patients responded to interferon treatment.
- USA : African Americans and Mexican Americans have higher
frequencies of HCV infection than whites, and 30- to
49-year-old adult males have the highest frequencies of
infection. The average age at a hepatitis C diagnosis ranges
from 40 to 59 years : 4 million affected (prevalence 1%) and
10 000-20 000 deaths a year. About 9000 Montanans are known to
have hepatitis C, which the state tracks as a reportable
disease : 6% hepatitis C infection rate among women who went
to the clinics for prenatal care
- Australia : 260,000 exposed and of those about
200,000 still have chronic infection, about 40,000 of them
have significant scar tissue in their liver and all together
about 8,000 will have developed cirrhosis
- Hong Kong : the distribution of hepatitis C virus
(HCV) genotypes amongst non-drug users was 63.6% for genotype
1b, 23.6% for 6a, 4.5% for 1a, 3.9% for 3a, and 3.1% for 2a;
whereas amongst the intravenous drug users, it was 58.5% for
genotype 6a (significantly higher), 33.0% for 1b, 5.7% for 3a,
0.9% for 1a, and 0.9% for 2aref
- Pakistan : the nationwide carriage rate in 1997 was
estimated at 2.40%. 24% of patients with chronic liver disease
and hepatocellular carcinoma are viremic; 1.18% to 9% of blood
donors (1996). Seroprevalence surveys:
- 6.5% in Hafizabad, Punjab (1997)
- 5.31% of healthy persons in Islamabad (1998 to 2004)
- 15.9% in Lahore
- 23.8% in Gujranwala (Punjab)
- 3.26% in Sialkot (blood donors, 1998 to 2000)
- 4.0% in northern Pakistan (blood donors, 1996 to 2000)
- 0.44% of children by age 5 (Karachi).
- 1.6% of children ages 1 to 15 years in Karachi
- 68% of hemodialysis patients and 10 per cent of controls
in Lahore (1999);
- 3.7% in Islamabad.
- 4.8% of antenatal women in Islamabad (2001 to 2002)
Hepatitis C accounts for 40% of chronic liver disease, is the most
frequent indication for
liver transplantation
, and is estimated to
account for 8,000 to 10,000 deaths per year in the USA. Most
asymptomatic blood donors found to have anti-HCV and approximately
20 to 30% of persons with reported cases of acute hepatitis C do
not fall into a recognized risk group; however, many such blood
donors do recall risk-associated behaviors when questioned
carefully.
Genomics : the
complete HCV sequence has been available since 1989
ref;
genes coding for structural proteins (C, E1, E2/NS1) undergo
antigenic drift (=>
quasi-species). It replicates from
a ribonucleoprotein (RNP) complex that is associated with the ER
membrane : in response to this stress, the
unfolded
protein
response (UPR)
is
initiated by the proteolytic cleavage of a transmembrane protein,
activating
transcription factor 6 (ATF6), leading to increased
transcriptional levels of
heat
shock 70kDa protein 5 (HSPA5) / GRP78
, an ER
luminal chaperone protein. However, the overall level of GRP78
protein is decreased. While ER stress is also known to affect
translational attenuation, cells expressing HCV replicons have
lower levels of phosphorylation of eIF2
a.
Interestingly, cap-independent internal ribosome entry
site-mediated translation directed by the 5' noncoding region of
HCV and GRP78 is activated in cells expressing HCV replicons.
There are 4 hierarchical strata in the genetic heterogeneity of
HCV: group above subgroup above isolates above quasispecies. The
entire genome sequence has so far been reported for 16 isolates
which are classifiable into 3 groups and 6 subgroups.
Provisional classification of HCV is also possible using a partial
sequence of the HCV genome :
- C gene : Okamoto's
genotypes, 1992ref
: at least 28 HCV genotypes have been reported, which differ
by > 20% in the nucleotide sequence of the entire genome
(approximately 9500 nt) or the sequence of the E1 gene (576
nt). Different HCV genotypes have distinct geographical
distributions, and may be associated with variations in viral
replication and disease-inducing activity, as well as poor
response to interferons in patients with chronic hepatitis Cref
- I/1a
- II/1b
- genotype 1c appears to have evolved and remained
in Indonesiaref
- III/2a
- IV/2b
- V/3aref
- provisional genotypes 3c, 3d, 3e
and 3f have evolved and remained in Nepal, and have
not been observed in the other areas of the world
Types 1, 2 and 3 were found in patients from the UK, southern
Europe, Asia, Africa and South America. Infection with HCV
genotypes 4 through 9 is prevalent in some geographic areas
where the disease burden of chronic hepatitis C approaches
endemic levels (eg, HCV genotype 4 in Egyptref
where there is an HCV infection prevalence of approximately 18%)ref.
On the basis of sequence variation in both the coding and
noncoding regions, several classification systems have been
proposed. Enomoto et al.ref
classified HCV into two major types and noted that each genotype
could be further classified into 2 subtypes. Later, Mori et al.ref,
Simmonds et al.ref,
Stuyver et al.ref,
and Bukh et al.ref1,
ref2reported
several
additional genotypes and proposed their own classification and
nomenclature schemes. A number of HCV genotyping systems were
also developed. Nakao et al.ref
and McOmish et al.ref
genotyped HCV by restriction fragment length polymorphism.
Okamoto et al.ref
and Chayama et al.ref
genotyped HCV by PCR with genotype-specific primers. However,
current classification of HCV involves > 6 major types and a
series of subtypesref1,
ref2.
None of the reported systems was able to determine the HCV
genotype in all 6 major types and the common subtypes on the
basis of the core region of HCV. A convenient genotyping system,
based on PCR of the core region with genotype-specific primers,
which allows for the determination of HCV genotypes 1a, 1b, 2a,
2b, 3a, 3b, 4, 5a, and 6a. HCV is known to have marked genetic
heterogeneity, and it was estimated to have a nucleotide
substitution rate of between 1.44 3 1023 and 1.92 3 1023
substitution per site per yearref1,
ref2.
Accumulation of nucleotide substitution in the HCV genome
results in diversification and evolution into different
genotypesref.
There is increasing evidence that patients infected with
different HCV genotypes may have different clinical profiles,
severity of liver disease, and response to IFN-a therapyref1,
ref2,
ref3,
ref4,
ref5.
Hence, a convenient and reliable HCV genotyping system is
essential for large-scale epidemiological and clinical studies.
In this report, a new genotyping method, based on
genotype-specific primers for PCR of the core gene, by which HCV
isolates can be classified into genotypes 1a, 1b, 2a, 2b, 3a,
3b, 4, 5a, and 6a is described. So far, the genotyping system
described by Okamoto et al. is the most popular system used by
HCV investigators in Japanref.
This system is also used by a number of researchers worldwide.
However, for HCV isolates with HCV genotypes other than type 1
or 2, the typing system of Okamoto et al. may not suffice. In
Western patients with HCV type 2a infection, the system of
Okamoto et al. has also been reported to be less useful in
genotypingref1,
ref2.
Recently, it was also shown that the system designed by Okamoto
et al. had a higher number of mixed-infection designations, and
these samples were found to have non-type 1–non-type 2 isolates,
due to nonspecific primingref.
This observation is confirmed in this study, in which a
significant proportion of samples assigned to the
mixed-infection category were in fact nontype 1–non-type 2 HCV.
This is likely to result from nonspecific annealing of primers
to the sequences. It is well established that the matching of
the 2 to 3 nucleotides at the 39 end is one of the important
parameters for specific priming. Therefore, the lower the number
of HCV isolates employed in designing primers is, the lower the
specificity of the primers would be. The typing system of
Okamoto et al. was based on sequences of HCV isolates of
genotype 1a, 1b, 2a, or 2bref.
Since a number of new HCV genotypes were identified only
recently, the system designed by Okamoto et al. would be
insufficient to differentiate these newly identified genotypes.
In fact, Okamoto et al. have revised their assay to include HCV
type 3a. Our study was embarked upon before the publication of
their paper, and we did not have a chance to test their modified
system versus our system. However, we point out that the region
used by Okamoto et al. for their type 3a primer might not be a
suitable region for the design of primers if all the common
subtypes were to be detected. As our system is based on the
nucleotide sequences of genotype 1a, 1b, 1d, 2a, 2b, 3a, 3b, 4,
5a, and 6a HCV isolates, we believe that this system may have a
much broader application. However, the number of samples of HCV
types 3 to 6 tested was still not large enough for definitive
conclusions to be drawn. Certainly, this system should be
further tested in areas in which HCV types 3 to 6 are common to
further validate this genotyping method. If the accuracy of our
system is confirmed in these areas, we anticipate that this
convenient method will assist research workers in conducting
large-scale epidemiological studiesref.
- E1 gene : 9 groups and 23 subgroupsref
- NS5 gene : 6 major genotypesref
Okamoto's serotypesref
of
core protein : useful
in cases where serum samples were not stored under conditions to
preserve RNA or in infected hosts who have cleared the virus and
therefore have only antibodies remaining to identify the
infection.
- serotype 1 (IPKARRPEGRTWAQPGY core protein), conserved in
hepatitis C virus isolates with type I, II, and V genotypes
- serotype 2 (IPKDRRSTGKSWGKPGY core protein), conserved in
type III and IV genotypes.
Proteomics :
- core (C) protein
binds to aspartoacylase-3
(ACY-3) and translin,
partly explaining the molecular mechanism for hepatocellular
carcinoma and lymphoma. It can induce reactive
oxygen
species
ref
- F protein is a recently
described, frameshift product of HCV core encoding sequence
of genotype 1a. Its function and antigenic properties are
unknown. The F protein elicits specific antibodies in 62% of
individuals chronically infected with HCV; such anti-F
response does not seem to be affected by the F sequence
heterogeneityref.
- non-structural (NS) proteins :
- envelope glycoproteins that are released from HCV
polyprotein by signal peptidase cleavage. These proteins
assemble as a noncovalent heterodimer that is retained in
the endoplasmic reticulum. The transmembrane domains of E1
and E2 are multifunctional and play a major role in the
biogenesis of E1E2 heterodimer. Because HCV does not
replicate efficiently in cell culture, surrogate models have
been developed to study some steps of its life cycle.
Recently, infectious pseudotype particles (HCVpp) harboring
unmodified E1E2 glycoproteins onto retroviral core particles
have successfully been generated. They mimic the function of
native HCV particles, thus representing a model to study the
early steps of its lifecycle. The noncovalent E1E2
heterodimers present at the surface of the HCVpp, which
contain complex-type glycans indicating modification by
Golgi enzymes, are likely to mediate virus entry. Potential
structural homology with other fusion proteins from closely
related viruses suggest that HCV envelope glycoproteins
belong to class II fusion proteins, but contrary to what is
observed for other viral envelope proteins of this class,
they are highly glycosylated and are not matured by a
cellular endoprotease cleavageref.
- E1 envelope glycoproteins
- nonstructural 1 (NS1) / E2
envelope glycoprotein : HCV host cell entry is a
complex process that culminates in the
clathrin-dependent endocytosis of the virion and
low-pH-mediated fusion of viral and cellular
lipid membranes in an early endosome. The entry process
requires the two viral envelope glycoproteins,
E1 and E2, and many cellular factors, including
:
- glycosaminoglycans (GAGs)
- 2 tight junction proteins
- claudin-1 (CLDN1)
- occludin (OCLN)
- the large extracellular domain (EC2) of CD81 / tetraspanin / TAPA-1
ref,
which is expressed on ...
- hepatocytes
- lymphocytes
- the CD21/CD19/CD81/Leu13 costimulatory complex of
B lymphocytesref,
where reduces the threshold for B cell activation
via the B cell receptor by bridging Ag specific
recognition and CD21-mediated complement recognitionref.
In patients with chronic HCV, CD81 expression on
peripheral B lymphocytes is increased, while
antiviral treatment down-regulates peripheral B-cell
CD81 expression and CD5 expansion in chronic HCV
infectionref
: classification algorithms provide information
about specific E2 positions correlated with MC. CD81
expression and HCV core antigen levels in PBMCs are
increased in patients with MCref.
- the CD4/CD8/CD81 costimulatory complex on T
lymphocytes. In fetal
thymic
organ culture (FTOC)
, mAb to CD81 block maturation of
CD4-CD8- thymocytes, and expression of CD81 on CHO
cells endows those cells with the ability to support
T cell maturation. However, CD81-deficient mice
express normal numbers and subsets of T cells. These
mice do exhibit diminished antibody responses to
protein antigens. CD81 is also physically and
functionally associated with several integrins on
epithelial and hematopoietic cellsref1,
ref2.
Anti-CD81 can activate integrin a4b1 (VLA-4) on B
cells, facilitating their adhesion to tonsilar
interfollicular stroma. Similarly, anti-CD81 can
activate aLb4 (LFA-1) on
human thymocytes. CD81 can also affect cognate B-T
cell interactions because anti-CD81 increases IL-4
synthesis by T cells responding to antigen presented
by B cells but not by monocytes. The tetraspanin
superfamily (or TM4SF) includes CD81, CD9, CD37,
CD53, CD63, CD82, CD151, and an increasing number of
additional proteinsref.
Like CD81, several tetraspanins are involved in cell
adhesion, motility, and metastasis, as well as cell
activation and signal transduction. CD81 activates
Lck through lipid
raft
aggregation and thus leads to
enhanced costimulatory signaling in T lymphocytes :
this phenomenon may play a role in liver damage and
autoimmune manifestations associated with HCV
infection (see below)
E2 binds to human lymphoma and hepatocarcinoma cell lines,
chimpanzee mononuclear cells, whereas it does not bind to
rat, rabbit, or green monkey mononuclear cells or
hepatocytesref.
Whether virus binding to CD81 is followed by entry and
infection in all cell types is not clear, because it is
possible that additional factors are required for HCV
fusion or infectivity. CD81 is used by all the different
genotypes/subtypes analyzed to enter the cellsref.
- the tight junction protein occludin (OCLN)
- the B
cell receptor
of HCV-associated lymphomaref,
thus activating the intracellular signal transduction
pathway.
- links to atherosclerosis
??
- the hypervariable region 1 (HVR1) binds to scavenger receptor
class B type I (SR-BI)
on human hepatoma cell lineref
and primary tupaia hepatocytes (PTH)ref.
HDLs, the natural ligand of SR-BI, are able to
markedly enhance HCVpp entry, but HDL don't associate
with HCVpps, suggesting that HCVpps do not enter into
target cells using HDL as a carrier to bind to its
receptorref.
The level of SR-BI needed for efficient infection
varies between genotypes and subtypesref.
- LDLR
ref :
very low density lipoprotein (VLDL) is selectively
associated with HCV in type II cryoglobulinsref.
In studies on the cutaneous vasculitic lesions in type
II cryoglobulinemia using in situ
hybridization (ISH), the HCV RNA virion form (positive
strand) but not the putative replicative form
(negative strand) of the virus was detected in
keratinocytes in lesions but not normal skin of the
same patientsref.
Furthermore, it was demonstrated that LDLRs were
up-regulated on keratinocytes in cutaneous vasculitis
lesions compared with normal skinref.
These observations and the finding that anti-
lipoprotein precipitates HCV from infected serumref
suggested that LDLR also may be a receptor for HCV
complexed to VLDL or LDLref.
Several lines of evidence were presented supporting
the hypothesis that HCV and other members of
Flaviviridae are endocytosed via the LDL receptor.
Endocytosis of HCV was shown to correlate with cell
LDLR activity by experimental modulation of LDL
receptor activity and with the striking demonstration
that the CRIB cell line, resistant to infection with
BVDV for unknown reasonsref,
lacked detectable LDLR activity. Direct evidence for
the hypothesis was provided by inhibiting endocytosis
of HCV, BVDV, and GB virus C/HCV with anti-LDLR
antibody. Moreover, known biochemical inhibitors of
LDL endocytosis and the endocytosis inhibitor, PAO,
inhibited LDLR-mediated endocytosis of these viruses.
The reports that up-regulation of LDLRs facilitated
persistent HCV infection in vitroref
and the induction of HCV binding with transfection of
the LDL receptor gene in COS-7 cells that were unable
to bind HCVref
are consistent with these findings. Evidence also was
presented that endocytosis of HCV via the LDL receptor
was mediated by the complexing of HCV to VLDL or LDL.
Although, HCV also appeared to complex with HDL in the
serum to the same extent as VLDL and LDL, there was no
evidence of HDL-mediated endocytosis of HCV. It could
not be determined from the studies performed whether
the low level of endocytosis of HDL compared with
those of VLDL and LDL, as shown by cytometric studies
with DiI-labeled lipoproteins, was responsible for the
absence of detectable levels of HDL-mediated
endocytosis of HCV. The complete inhibition by
anti-LDL receptor antibody of HCV endocytosis by a
variety of cells in vitro suggests that the LDLR may
be the main mechanism for entry of HCV into cells.
However, the detection of small amounts of
intracellular HCV in LDL-deficient fibroblasts
inoculated with HCV that could not be inhibited by
anti-LDLR antibody suggested that receptors not
related to the LDL receptor mediate viral entry in
these cells. Furthermore, the finding that there was
only a delaying effect of anti-LDLR antibody on the
cytopathic effect of the NADL strain of BVDV indicates
that receptors for BVDV other than LDLRs or low
affinity LDLRs were present. This finding is
consistent with the observations of Flores and Donisref
that CRIB cells could be infected with high
concentrations of BVDV. Hence, the LDLR may be the
main but not exclusive means of entry of these viruses
into these cells. The evidence of HCV replication in
Hep G2 and Daudi cells, although transient, suggests
that endocytosis of HCV mediated by the LDLR can
result in infection. Whether this route of entry can
result in a productive infection in vivo and whether
all members of the Flaviviridae family of viruses use
this mechanism of cell entry remain to be determined.
The evidence presented in this study that VSV also may
be endocytosed by the LDLR appears to be contrary to
an earlier studyref
that found interferon- induces a soluble LDLR fragment
that inhibits VSV infection but concluded that the
LDLR was not a receptor for VSV. The latter conclusion
was reached because VSV was found to infect
LDLR-deficient fibroblasts. From our finding that
entry of HCV into LDLR-deficient fibroblast was
independent of the LDLRs, it is apparent that Fischer
et al.ref
did not present definitive evidence that VSV was not
endocytosed by means of the LDLR because receptors for
VSV other than LDLRs may have been present on those
cells. LDLR-mediated endocytosis may not be exclusive
to Flaviviridae. Rather, this mechanism may be used by
all viruses that can associate with VLDL or LDL in the
blood. The reported association of VSV with
lipoproteins in the blood, particularly VLDLref,
is consistent with this notion. Thus far, there only
has been indirect evidence of in vivo endocytosis of
HCV mediated by the LDL receptor, that is, the
apparent endocytosis of HCV by keratinocytes in the
inflammatory skin lesions of patients with type II
cryoglobulinemiaref.
In this study, evidence that BVDV contaminated media
from bovine serum is endocytosed by a wide variety of
human cells nonpermissive to BVDV infection suggests
that this mechanism may explain the presence of
positive-strand HCV that has been widely reported in
human nonhepatic cells, particularly peripheral blood
mononuclear cells. This mechanism must be considered
before assigning HCV tropism for tissues other than
the liver where clear evidence for HCV replication has
been demonstrated. The potential involvement of the
LDLR in HCV infection has implications for the current
therapy of HCV infection and also provides the
rationale for a new approach to therapyspecifically,
administration of anti-LDLR antibody or analogues of
the LDLR binding site of apolipoproteins B and E. The
effect of IFN, the current therapy for HCV infection,
may be mediated in part by the down-regulation of
LDLRs. IFN is known to induce IL-1Ratref,
which blocks the IL-1R-mediated stimulation by IL-1.
Because IL-1 is known to increase LDLR activityref,
IFN would indirectly cause a down-regulation of LDLR
activity by stimulating IL-1ra production, thereby
decreasing IL-1R-mediated stimulation by IL-1. It has
been reported that a minor group of human rhinovirus
(HRV2)ref
and Rous sarcoma virus Aref
enters cells via the LRP. In the latter report,
evidence was presented that a viral envelope moiety
binds to LDL receptor-related protein. An amino acid
change in exon 8 of LDLR was associated with severity
of liver fibrosis; a SNP in exon 10 correlated with
viral clearance and overall inflammation, and a SNP in
the 3'UTR appeared to influence treatment responseref.
- CD209L
/ L-SIGN
(largely expressed on endothelial cells in liver
sinusoids) and CD209
/
DC-SIGN
(expressed
on
dendritic cells)ref.
Capture of circulating HCV particles by these SIGN+
cells may facilitate virus infection of proximal
hepatocytes and lymphocyte subpopulations and may be
essential for the establishment of persistent infection.
The polarized nature of
hepatocytes and the
tight junction roles of OCLN and
CLDN1 suggest an entry pathway
similar to that of
the group B coxsackieviruses, where the virion
initially
binds readily accessible factors that then provide
a
mechanism for migration of the virion into the tight junction
region, just prior to internalization. Indeed, cellular
factors are utilized by the incoming HCV virion in a
temporal
manner. At least GAGs and LDL-R appear to
mediate virion binding. Conflicting evidence has shown that
SR-BI
acts as either a binding or postbinding entry
factor,
while CD81 and CLDN1 play postbinding
roles
in the HCV cell entry process.
CD81 and SR-BI are essential for HCVpp entry. However, these 2
proteins are not sufficient to provide entry functions in non
permissive cells, suggesting that additional unidentified
cellular factor(s) are necessary for HCVpp entry
ref.
- NS2-3 protease
mediates a single cleavage at the NS2/NS3 junction. The
crystal structure of the catalytic domain of the NS2-3
protease at 2.3 Ĺ resolution reveals a dimeric cysteine
protease with two composite active sites. For each active
site, the catalytic histidine and glutamate residues are
contributed by one monomer, and the nucleophilic cysteine by
the other. The carboxy-terminal residues remain coordinated
in the two active sites, predicting an inactive
post-cleavage form. Proteolysis through formation of a
composite active site occurs in the context of the viral
polyprotein expressed in mammalian cells. These features
offer unexpected insights into polyprotein processing by HCV
and new opportunities for antiviral drug designref.
- NS3/4A serine
protease cleaves at 4 downstream sites in the
polyprotein. NS3-4A is characterized as a serine protease
with a chymotrypsin-like fold. It causes specific
proteolysis of the TLR3
adaptor protein
TRIF / TICAM-1ref
and ablates retinoic acid-inducible gene I (RIG-I) signaling
of downstream IRF3 and NF-kB
activationref
- NS5A is an active component of
HCV replicase, as well as a pivotal regulator of replication
and a modulator of cellular processes ranging from innate
immunity to dysregulated cell growth. The RNA replication
machine of HCV is a multi-subunit membrane-associated
complex. NS5A is a large phosphoprotein (56-58 kDa)
with an amphipathic a-helix at
its amino terminus that promotes membrane association. After
this helix region, NS5A is organized into 3 domains. The
N-terminal domain (domain I) coordinates a single zinc atom
per protein molecule. Mutations disrupting either the
membrane anchor or zinc binding of NS5A are lethal for RNA
replication. However, probing the role of NS5A in
replication has been hampered by a lack of structural
information about this multifunctional protein. The
structure of NS5A domain I has been reported at 2.5-Ĺ
resolution, which contains a novel fold, a new
zinc-coordination motif and a disulphide bondref.
It can induce reactive
oxygen
species
ref
- NS5B interacts wih PLIC1
/ ubiquilin 1, a negative regulator of HCV RNA
replication
NS3. The serine protease, NS4A cofactor and RNA helicase domains
are shown in pink, green and blue, respectively. The serine
protease and RNA helicase active site residues are indicated in
red. b, NS5A domain I. Shown is a dimer, as seen in the crystal
structure. Individual subunits are shown in blue and green, with
their C termini (that is, leading into domain II) pointing
upwards. The N termini, which presumably face the membrane, are at
the bottom. The purple spheres represent Zn2+ ions. Disulphide
bonds are indicated in red. Brackets indicate highly conserved
surfaces. A basic groove, which may bind RNA, is also indicated.
c, NS5B. Shown is the typical 'right hand' model of the RdRP, with
palm, fingers and thumb domains in pink, blue and green,
respectively. The C-terminal region, which is not part of the
RdRP, is shown in yellow. Note the extensive interactions between
the finger and thumb domains. In addition, a -hairpin is shown in
purple, and active site residues Asp 220 and Asp 318 are shown in
red.
Transmission
:
- parenteral route
- blood transfusion. During the 1970s, the likelihood of
acquiring hepatitis after transfusion of voluntarily
donated, HBsAg-screened blood was approximately 10% per
patient (up to 0.9% per unit transfused); 90 to 95% of these
cases were classified, based on serologic exclusion of
hepatitis A and B, as "non-A, non-B" hepatitis. For patients
requiring transfusion of pooled products, such as clotting
factor concentrates, the risk was even higher, up to 20 to
30%. During the 1980s, voluntary self-exclusion of blood
donors with risk factors for AIDS and then the introduction
of donor screening for anti-HIV reduced further the
likelihood of transfusion-associated hepatitis to under 5%.
During the late 1980s and early 1990s, the introduction
first of "surrogate" screening tests for non-A, non-B
hepatitis [alanine aminotransferase (ALT) and anti-HBc, both
shown to identify blood donors with a higher likelihood of
transmitting non-A, non-B hepatitis to recipients] and,
subsequently, after the discovery of HCV, first-generation
immunoassays for anti-HCV reduced the frequency of
transfusion-associated hepatitis even further. A prospective
analysis of transfusion-associated hepatitis conducted
between 1986 and 1990 showed that the incidence of
transfusion-associated hepatitis at one urban university
hospital fell from a baseline of 3.8% per patient (0.45% per
unit transfused) to 1.5% per patient (0.19% per unit) after
the introduction of surrogate testing and to 0.6% per
patient (0.03% per unit) after the introduction of
first-generation anti-HCV assays. The introduction of second-generation
anti-HCV assays (tested by Procleix®)
reduced the frequency of transfusion-associated hepatitis C
to almost imperceptible levels, 1 in 100,000, and these
gains are being reinforced by the application of automated
PCR testing of donated blood for HCV RNA.
- hemodialysis patients are recognized as a group at
increased risk of infection with HCV. In a Multicenter
Spanish Study on HCV in dialysis, strict adherence to
universal infection control precautions proved to be
adequate to prevent nosocomial transmission of HCV. Time was
the most important factor (although interacting with the
isolation measures) and was independent of initial HCV
prevalenceref
- injection drug use : although new acute cases of
hepatitis C are rare, newly diagnosed cases are common among
otherwise healthy persons who experimented briefly with
injection drugs two or three decades earlier. Such instances
usually remain unrecognized for years, until unearthed by
laboratory screening for routine medical examinations,
insurance applications, and attempted blood donation.
- occupational exposure to blood
- sexual and perinatal routes are inefficient : although 10
to 15% of patients with acute hepatitis C report having
potential sexual sources of infection, most studies have
failed to identify sexual transmission of this agent. The
chances of sexual and perinatal transmission have been
estimated to be approximately 5%, well below comparable
rates for HIV and HBV infections. Moreover, sexual
transmission appears to be confined to such subgroups as
persons with multiple sexual partners and sexually
transmitted diseases; transmission of HCV infection is rare
between stable, monogamous sexual partners. Breast feeding
does not increase the risk of HCV infection between an
infected mother and her infant. Fisting and other rectal sex
practice was the only factor significantly associated with
contracting HCV during 4 years of follow-up amongst HIV+
gay men in a study conducted in Londonref
- infection of health workers is not dramatically higher
than among the general population; however, health workers
are more likely to acquire HCV infection through accidental
needle punctures, the efficiency of which is 3%
- infection of household contacts is rare as well
- organ transplantation
- those who require transfusions in the setting of cancer
chemotherapy
- transplacental route
- exocrine secretions :
- saliva : HCV replicates in salivary glandsref
- HCV replicates in eccrine sweat glands cells and
keratinocytes in healthy skin and is released into the sweatref
Environmental survival : RNA in plasma or serum has been found to
be stable at 4°C for 7 days
Resistance :
- HLA-DRB1*1101
- KIR2DL3
;HLA-C1 homozygous
genotype is observed at a higher frequency among Caucasians
and African Americans — individuals (indicating that KIR2DL3
and HLA-C1 have a synergistic and direct protective effect
rather than protection being a result of linkage
disequilibrium) who resolved infection with HCV than among
those with persistent infection after receiving a small viral
inoculum (through injection) but not a large inoculum, such as
that provided by blood transfusion, in whom the innate immune
response is likely overwhelmed. Although KIR2DL2 is also a
ligand for HLA-C1 allotypes, KIR2DL3 homozygosity was
essential for the association with HCV resolution. KIR2DL3 has
a lower affinity for HLA-C1 than KIR2DL2, so is associated
with protection because it provides weaker inhibitory signals
that allow NK cells to be more-easily activated by infected
hepatocytesref.
Pathogenesis
: primed CD8 T cells are critical for antiviral immunity and
subsets of circulating CD8 T cells have been defined in blood but
these do not necessarily reflect the clonality or differentiation
of cells within tissue. Current models divide primed CD8 T cells
into effector and memory cells, further subdivided into central
memory (CCR7
+, L-selectin
+), recirculating
through lymphoid tissues and effector memory (CCR7
-,
L-selectin
-) mediating immune response in peripheral
organs. D8 T cells derived from organ donors and patients with
end-stage HCV infection showed that: 1) all liver-infiltrating CD8
+
T cells express high levels of CD11a, indicating the effective
absence of
naive CD8 T cells in the liver. 2) The liver
contains distinct subsets of primed CD8
+ T cells
including a population of CCR7
+ L-selectin- cells,
which does not reflect current paradigms. The expression of CCR7
by these cells may be induced by the hepatic microenvironment to
facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are
present on lymphatic, vascular, and sinusoidal endothelium in
normal liver and in patients with HCV infection. The recirculation
of CCR7
+/L-selectin
- intrahepatic CD8 T
cells to regional lymphoid tissue will be facilitated by CCL19 and
CCL21 on hepatic sinusoids and lymphatics. This centripetal
pathway of migration would allow restimulation in lymph nodes,
thereby promoting immune surveillance in normal liver and renewal
of effector responses in chronic viral infection
ref.
Compared with peripheral cells, intrahepatic T cells from patients
with chronic hepatitis C were selectively enriched with CD45RO
+
memory T cells but had a lower percentage of CD4
+ T
cells expressing the differentiation markers CD27 and CD28. The
percentage of intrahepatic CD45RO
+ and CD28
+
T cells correlated with the degree of liver inflammation, which
suggests that memory T cells at relatively early stages of
differentiation are directly involved in liver inflammation.
Despite their memory phenotype, intrahepatic T cells were
defective in proliferation capability, produced less IFN-
g in response to stimulation by TcR, and
contained less perforin but expressed higher levels of Fas and
FasL, compared with their counterparts in peripheral blood. The
distinct characteristics of intrahepatic T cells suggest that they
play an important role in the immunopathogenesis of chronic
hepatitis C
ref.
Incubation 15÷160 days =>
=>
asymptomatic in babies
=>
acute
or protracted hepatitis
C (a.k.a.
non-A, non-B hepatitis) from
immune-mediated damage
- very rarely fulminant
hepatitis

- 15-30% : self-limiting without treatment (sometimes
endogenous reactivation may occur as antibody-mediated
immunity is not protective)
- 85% of HCV-infected individuals (expecially those with an
inadequate CD4+ T-lymphocyte response and HLA-DR5
haplotype) become carriers and develop chronic
hepatitis
C
(CHC) (a.k.a. non-A, non-B hepatitis) : increase
in SGPT up to 4,000 U/L at 37°C.
- 80% of chronic carriers remain stable
- 20% (increases with increasing duration of infection. In
Asian patients infected at birth, infection for over 60
years causes cirrhosis in 71% of infected individuals.
Because relationship between the severity of fibrosis and
age in Asian patients is similar to that seen in Caucasian
patients it is likely that similar rates of cirrhosis will
be seen in other patients who are infected for > 60 yearsref)
develop liver
cirrhosis
(the heterozygous ArgPro of codon 25 of TGF-b1 predicts significantly
faster fibrotic progression than the homozygous (25)ArgArg
genotype. The homozygous LeuLeu genotype of codon 10 showed
a slow progression of fibrosis)
- 50% die (eventually due to hepatocellular
carcinoma
(HCC)
).
In
the absence of PEG-IFN-a,
activated CD3-56+69+ NK
cell turnover may be enhanced in SVR compared with NR
patients : activated NK cells may play a role in liver
inflammationref.
Some HCV-associated HCCs have mutations in the tumor
suppressor p53, the protooncogene b-cateninref1,
ref2
and several other genes. However, the long latency period
of HCV infection makes it difficult to demonstrate the
causal association between protooncogene mutations and HCV
infection. Furthermore, many HCV-associated HCCs do not
have detectable HCV RNAref1,
ref2,
suggesting that HCV-induced tumorigenesis may employ a
hit-and-run mechanism. HCV infects not only hepatocytes,
but also B cells in vitro and in vivoref1,
ref2.
Iron
accumulation in chronic
hepatitis C relates to hepatic iron distribution, HFE genotype,
and disease courseref
Associated diseases : although HCV is
a hepatotropic virus, the HCV genome and its replicative
intermediates have also been detected in peripheral blood
mononuclear cells (PBMCs) and in lymphoid tissues of chronically
infected patients
ref1,
ref2,
ref3,
ref4,
ref5,
ref6,
ref7.
This evidence, however, has been questioned, as commonly used
techniques are limited in their ability to discriminate between
positive and negative RNA strands, the presence of the latter
being regarded as a direct evidence of viral replication
ref.
Importantly, in several studies that used assays carefully
optimized for strand specificity, HCV RNA negative strand was not
detected in PBMCs from infected patients
ref1,
ref2,
ref3.
Similarly, although the presence of active replication in bone
marrow (BM) was suggested by
in situ detection of viral
RNA and viral antigens
ref,
it was not confirmed by an investigation using strand-specific
assay
ref.
However, the latter study was relatively small, as it included
only 6 patients. It was found in 38% of serum and 31% of bone
marrow in a US clinic
ref
- cholestasis

- siderosis

- porphyria
cutanea tarda

- immune disorders :
- autoimmune diseases :
- lichen
planus
(13.6% in Japanref)
- Sjogren
syndrome
(21-26% in Japanref)
represents a pathological model of the evolution from
polyclonal B lymphocyte activation to
oligoclonal/monoclonal B cell expansion, which may
culminate in the development of a malignant
lymphoproliferative disease. The different phases of this
process are usually marked by the appearance of
antigen-driven activated B cell clones, which are commonly
IgM+ and with rheumatoid factor (RF) activity.
There are remarkable homologies between the antigen
combinatory regions of the IgR expressed by SS-associated
monoclonal non-neoplastic lymphoproliferations and
HCV-associated NHLs, concerning : a) the specific
combinations of heavy and light variable region genes; b)
the limited length of CDR3; c) the homology with
antibodies with RF activity; d) the amino acid sequences
of CDR3 in which common somatic mutations are present that
possibly determine the antigen-binding specificity.
Although there are significant differences between SS and
HCV-associated lymphoproliferative diseases, they share
many molecular characteristics, which suggest an
immunological cross-reactivity or molecular mimicry among
the agents that underlie these disordersref.
- lichen
planus
+ Sjogren
syndrome
(8.6% in Japanref)
- essential
mixed
cryoglobulinemia (EMC)
(36%; 20% of all chronic HCV infectedref)ref1,
ref2,ref3,
ref4,
a benign monoclonal lymphoproliferation that sometimes (7%
in Italyref)
evolvesref1,
ref2
to overt B-cell NHL. Antibodies to HCV (anti-HCV) and HCV
RNA have been found in up to 98% of patients with mixed
cryoglobulinemiaref.
In type
III cryoglobulins
,
immunoglobulin immune complexes contain both polyclonal
IgM with rheumatoid factor activity and polyclonal IgGref.
In type
II cryoglobulins
,
immunoglobulin immune complexes contain both monoclonal
IgM and polyclonal IgGref.
The monoclonal IgM has rheumatoid factor (RF) activity and
is encoded mostly by a restricted set of variable (V)
region genes, specifically VH1-69 (also
known as 51p1) and Vk3-A27
(also known as kv325)ref1,
ref2,
ref3,
ref4.
In non-HCV-infected individuals approximately 1.7% of
peripheral blood B cells express the VH1-69
generef,
as expected for a random use of the total repertoire of
functional VH gene regions. Restricted V gene
usage combined with restricted CDR3 length has been seen
in inbred mice strains responding to experimental
vaccination protocols. B cells derived from such immunized
mice and selected by reactivity with specific haptens such
as 4-hydroxy-3-nitrophenylacetyl (NP)ref,
2-phenyloxazoloneref,
and p-azophenylarsonateref
exhibit a V gene restriction bias. In human volunteers who
were vaccinated
with
the Haemophilus influenzae type b capsular
polysaccharide (Hib PS) antigen
approximately half the VH gene rearrangements
are of the VH3b subfamily, while the VL
gene response to the Hib PS antigen was less restricted,
as seen in the anti-E2 response, suggesting that in both
cases the VH gene segments are the ones that
play a more dominant role in antigen bindingref.
> 60% of monoclonal RFs from patients with type II MC
express the Wa cross-reactive idiotype (CRI)ref,
which, in turn, in > 70% of the cases is associated
with the light chain CRI 17.109 and the heavy chain CRI G6ref.
The latter 2 CRIs are characteristic for VL and
VH regions encoded by germline kv325 and 51p1 genes, respectively.
Antibodies against E2
envelope glycoprotein (E2) were detected in 88%
of chronically infected and in 49% of acutely infected
HCV patientsref1,
ref2.
V region genes from human anti-E2 antibodies,
derived from B cells of HCV-infected individuals, show
similar V gene bias to that observed in HCV-associated
MC and NHLref1,
ref2,
ref3,
namely a strong bias for VH1-69 and Vk3-A27ref1,
ref2.
A possibility exists that cognate B cells, which bind E2
via their specific B-cell receptor (BCR) could engage 2
signaling complexes simultaneously, the BCR and the
CD19/CD21/CD81 complex (CD81 is the putative E2 receptorref),
which
reduces the threshold of B-cell activationref.
Anti-E2 antibodies that block E2 binding to cells have
been referred to as neutralization of binding (NOB)
antibodiesref.
Subsequent studies characterizing 10 human anti-E2 mAbs
showed that they could be either NOB-positive or NOB-negativeref,
the later do not block E2 binding to CD81. The rescued
lymphoma immunoglobulin exhibited properties similar to
those of one of the NOB-negative mAbs. The
lymphoma-derived immunoglobulin and the anti-E2 mAb
recognized a similar spectrum of E2 glycoproteins and
CD81-bound E2 molecules. Both immunoglobulins bound to E2
glycoproteins of multiple viral genotypes,
implying reactivity with a conserved E2 epitope. Because
of the biased V gene usage in HCV-associated
lymphoproliferative disorders, it is likely that a conserved
epitope is involved in the process.
- lymphoproliferative diseases (LPD) :
- non-Hodgkin's
B-cell
lymphomas
: the same set of V region genes is expressed by
the majority of HCV-associated NHLref1,
ref2
:
- 10-20% of patients with CD5+
B cell chronic lymphocytic leukemia
ref1,
ref2,
ref3.
Preferential use of the 51p1 gene has also been observed
in CLL, with a prevalence of > 20% in particular
geographic areasref.
However, unlike the HCV-associated immunocytomas, the
51p1 VH sequences in CLL are almost
exclusively unmutated and usually have significantly
longer CDR3 regionsref1,
ref2.
- 61% of patients with salivary
gland
mucosa-associated lymphoid tissue (MALT) lymphomas
.
Interestingly, MALT lymphomas that develop in the
stomach do not show this bias. Taken together, these
observations suggest immune stimulation and selection by
an antigen that may be located only in the salivary
gland for those lymphomas that arise in the salivary
gland. The finding that the length of CDR3 is restricted
in salivary gland MALT lymphomas, but not in other MALT
lymphomas, strengthens this hypothesisref.
Anti-HCV antibodies and HCV RNA sequences were
documented in 50% of the MALT lymphoma patients
examined, without elevation of serum transaminases : 2
patients with parotid and conjunctival MALT lymphomas,
respectively, with a previous history of Sjogren's
syndrome
,
were HCV+ref.
In 2 recent studies by Bahler et alref,
(Bahler DW, 375a) the 51p1 gene was found in 10
of the 18 VHsequences and was associated with
a kv325 VL region
in all cases in which the VL sequence was
reported. Salivary gland MALT lymphomas are typically
associated with Sjogren's syndrome, which is frequently
characterized by the presence of RFs and cryoglobulins
in patients' sera. Somatic hypermutation, intraclonal
diversity, and selection against R mutations in the CDRs
was also observed, indicating similar events in the
pathogenesis of HCV-associated immunocytomas and
salivary gland MALT lymphomas.
..., suggesting a malignant progression from type II
MCref.
Moreover, the histologic presentation of many HCV-associated
NHLs is typical of germinal center (GC) and post-GC B cellsref.
Therefore, it is likely that lymphomagenesis occurs when B
cells proliferate in response to antigen. The same B-cell
clone, present in an HCV-infected MC patient early in the
course of the disease, was later detected as a NHLref.
The V regions expressed by this B cell showed significant
intraclonal diversity and accumulated multiple somatic
mutations, indicative of an antigen-driven clonal selection
and expansion processref1,
ref2,
ref3.
Moreover, the CDR3 regions of this case and of several other
lymphoma immunoglobulins showed homology to the CDR3 regions
of anti-E2 antibodiesref1,
ref2.
The V genes in these lymphomas undergo changes typical of a
T-cell-dependent antibody response, indicating a role for
chronic antigen stimulation by HCV-containing immune
complexes in the clonal evolution of HCV-associated
immunocytomas. The VH and VL gene
sequences from all of the HCV-associated immunocytomas
showed a number of nucleotide differences with respect to
their germline counterparts. In addition, substantial
intraclonal VH and/or VL gene
diversity was evident in each case, consistent with an
ongoing somatic hypermutation process in the tumor cells
subsequent to the neoplastic transformation. A similar
phenotype has been observed in a number of other B-cell
malignancies such as follicular
lymphoma
ref,
gastric
and salivary
gland
MALT lymphoma
ref1,
ref2,
Burkitt's
lymphoma
ref,
and monoclonal
gammopathy
of undetermined significance
ref,
indicating that the malignant events in these neoplasms
might have occurred at a similar stage of B-cell
differentiation. Clonally related transcripts of the IgM and
IgG H isotype were detected in one of the HCV-associated
immunocytomas, providing evidence for isotype switching in a
subset of the malignant B cells. CSR and SHM are processes
which typically occur in the germinal centers of lymphoid
tissues during a T-cell-dependent antibody response, and are
consistent with the hypothesis that the E2 antigen
drives B-cell expansion, leading to a population at risk
for malignant transformation. The immunoglobulin
from one of 2 HCV-associated lymphomas bound the E2 protein
in a manner identical to a bona fide human anti-E2
antibodyref.
Recombinant soluble E2 proteins, truncated to remove the
hydrophobic C-terminus at amino acids 661 (E2661)
or 715 (E2715), have been used as soluble
surrogates for viral particles in immune assays and
functional studies. For example, the identification of CD81
as the putative cellular receptor for HCV is based on its
binding to a truncated form of the E2 proteinref.
Several different E2 forms were tested in binding studies
with the lymphoma-derived immunoglobulins. Intracellular E2661,
which lacks many of the complex sugars acquired by secreted
E2661 during transit through the host-cell
secretory pathwayref1,
ref2,
has been shown to bind more avidly to cell surface-expressed
CD81 than does secreted E2661ref1,
ref2.
Nevertheless, the rescued lymphoma immunoglobulin from
patient 2 bound both the secreted and the intracellular
forms of E2661 and also the noncovalently linked
E1-E2 heterodimer, which is believed to be the prebudding
form of the HCV envelope glycoprotein complexref1,
ref2.
Moreover, the level of binding of the rescued immunoglobulin
and the reactivity pattern with multiple E2 glycoforms was
comparable to that of a well-characterized anti-E2 mAb.
Interestingly, the characterized mAb expresses the VH1-69
generef,
whereas neither of the lymphoma cases expressed this VH
gene. In contrast, both Vk
chains expressed by the lymphoma cases have been frequently
reported in immunoglobulin derived from HCV-associated NHLs
and in MC. To ascertain whether patient 2 could have evolved
from a nonapparent MC with RF activity, sequence homologies
were analuzed and no similarity was found between the
rescued CDR3 regions to those of sequenced RF-encoding
immunoglobulins. In addition, the rescued immunoglobulin
of patient 2 did not cross-react with human IgG,
suggesting that its reactivity is limited only to the
viral antigen. Thus, in this case there is no evidence
of progression from MC to overt B-cell lymphoma.
HCV-infected patients may also develop lymphomas that are
either independent of viral infection or are the result of
an indirect activation of B cells by the virus. It remains
to be determined whether reactivity of HCV-associated
lymphoma immunoglobulin with the E2 envelope protein is a
rare or more prevalent event. The results are consistent
with the receptor-mediated lymphomagenesis hypothesis that
was proposed nearly 2 decades ago. In that model, chronic
antigen stimulation by retroviral env protein was suggested
to be both necessary and sufficient to induce virus-specific
T-cell proliferation. In the absence of immune regulatory
mechanisms, proliferating T-cell clones with high-affinity
receptors for env could eventually give rise to frank T-cell
lymphomasref.
The hypothesis of B-cell activation has the additional
feature of dual binding of E2 to a cognate BCR and to the
CD81 molecule, which is a component of a signaling complex.
This is the first identification of a cognate antigen for
a human lymphoma BCR. The RF activity of the Igs from
most of the HCV-associated immunocytomas suggests that the
proliferation of the neoplastic B cells was driven by immune
complexes composed of polyclonal IgG and HCV. T cells
specific for IgG Fc are normally deletedref,
but the RF-producing B cells may obtain T-cell help from
HCV-specific T cells while presenting peptides derived from
HCV proteins present in the immune complexesref.
Interestingly, however, the pattern of somatic mutations in
the HCV-associated immunocytoma V genes is not suggestive
for selection of changes which could increase the affinity
for the antigen. Rather, the low number of replacement
mutations in the CDRs is indicative of selection against
mutations which could generate high-affinity antibodies.
This is most clearly seen in the case of the 51p1-encoded VH
regions which have an overall R:S ratio in the CDRs of only
1.2 (12:10), which is substantially lower than the R:S ratio
of 3.6 expected from random accumulation of nucleotide
changes in the absence of selective pressureref.
A high R:S ratio which was significantly different from the
one expected for random mutations was seen only in the VH
CDRs of the lymphoma immunoglobulin that lacked RF activity.
The CDRs of the light chains similarly lacked high R:S
ratios, which was especially evident in the case of the kv325-encoded VL domains.
A low number of R mutations was also observed in the FWRs of
most of the VH and VL domains.
Negative selection against R mutations in the FWRs is
consistent with selective pressure for maintenance of
functional Ig molecules, and further indicates the
requirement for a functional BcR in the clonal evolution of
the HCV-associated immunocytomas. Rheumatoid factor
antibodies that are induced after immunization of healthy
donors are frequently encoded by the 51p1/kv325 combinationref,
indicating a common cellular origin with the monoclonal RFs
of type II MC and HCV-associated immunocytoma. Nucleotide
sequence analysis of 51p1-encoded RFs from healthy immunized
donors also shows a strong selection against R mutations in
the CDRs, and moreover, no increase in the affinity for the
Fc region of IgG with the accumulation of mutationsref.
Intraclonal diversity and absence of significant clustering
of R mutations in the CDRs has also been observed in a 51p1/kv325-encoded RF from a patient with
type II MCref.
Also in this case the affinity for the Fc region of IgG did
not change after substituting the mutated 51p1 and kv325
genes with their germline counterpartsref.
Thus, although all of the above data indicate that antigen
stimulation can lead to the proliferation, somatic mutation,
and isotype switching of RF-expressing B cells, it also
seems that B cells expressing high affinity RF receptors are
either not selected or are eliminated by peripheral
tolerance mechanismsref1,
ref2,
ref3.
Because HCV RNA can be detected in the peripheral blood
mononuclear cells of patients with chronic hepatitis Cref,
the persistence of HCV in these cells may chronically
stimulate B-lymphocytes. This may cause clonal expansion of
these immunoglobulin-secreting cells and eventually results
in malignant B-cell lymphoproliferative diseases. Consistent
with this hypothesis, chronic infection of B lymphocytes by
a DNA parvovirus was shown to induce polyclonal and, later,
monoclonal immunoglobulin production in minksref
HCV RNA is found in :
- 23.8% in Hungaryref
- 20.8% in Sicily, Italyref
- 8.9% in Italy : 95.4% among the patients with,
and 4.6% among the patients without production of
cryoIg. The most common histotype among the HCV+,
cryoIg-producing cases, was the immunocytoma (16/21,
76%). Among the HCV+, non cryoIg-producing
cases, the marginal zone and the follicle center
lymphomas were the commonest. Close association between
HCV infection and cryoIg production, already described
in mixed cryoglobulinemia, is confirmed also among
B-cell NHL. Nevertheless, 50% of HCV-related lymphomas
are non-cryoIg producers. Low-grade lymphomas (in
particular the immunocytoma) are the most frequent
HCV-related lymphomasref
- 0% in West of Scotlandref
- 30% of primary cutaneous B-cell lymphomas (CBCLs)ref
- current, heavy smokers (> or = 20 cigarettes/day)
in Italy had an odds ratio (OR) of NHL of 2.10 (95%
confidence interval, CI: 1.07-4.12) compared to never
smokers, consistent across strata of sex and age.
Compared to never smokers, current smokers of > or =
20 cigarettes/day had ORs of 1.14 for B-cell-low-grade,
2.10 for B-cell-intermediate and high-grade, and 25.84
for T-cell NHL. The effect of tobacco smoking and HCV
were independent on the relative risk, leading a 4-fold
elevated risk in current smokers HCV positive subjectsref
- ref
- ref
- ref
Oligoclonal lymphoproliferative disordersref
and chromosomal translocationref
have frequently been observed in B lymphocytes, suggesting
that HCV may cause chromosomal instability. To examine the
possible association between HCV infection and somatic
mutations of cellular genes, it was taken advantage of the
recently established systems of in vitro and in
vivo HCV infection of B cellsref.
Acute and chronic HCV infection caused a 5- to 10-fold
increase in somatic
hypermutation
frequency in Ig heavy chain, BCL-6
(which also hypermutates in normal germinal-center
B
lymphocytes
ref
as well as diffuse
large B-cell lymphoma
ref,
by the same somatic hypermutation mechanism as for the VH
gene), p53
, and b-catenin
(and the b-globin gene) genes of in vitro HCV-infected
B cell lines and HCV-associated peripheral blood mononuclear
cells, lymphomas, and HCCs. The mutations in VH
genes exhibited most of the features characteristic of the
somatic hypermutation of the Ig gene in normal B cellsref.
These features include the preferential dG·dC mutations over
dA·dT mutations, preferential mutations in RGYW and WRCY [R,
purine (A/G); Y, pyrimidine (C/T); W, A/T]ref
motifs and a high replacement/silent mutation ratio. Thus,
the enhanced mutation frequency of the VH gene
probably represents the enhancement of the normal somatic
hypermutation mechanism of the VH gene, which
typically affects genomic sequences within 2 kbp downstream
from the transcription initiation sites of the Ig generef.
Area B of BCL-6 had dG·dC-biased mutations, which
preferentially targeted the RGYW motif, similar to the
pattern seen for VH. In contrast, the nucleotide
substitution pattern of p53, -catenin, and area A of BCL-6
showed that mutations preferentially occurred on dA·dT over
dG·dC (most commonly, from dA to dG or dT to dC) and that
there was no RGYW preference. Furthermore, the ratio of
replacement/silent mutation was substantially lower for p53
and -catenin than for VH and the ratio of
transition/transversion mutations in the former was
relatively higher. Thus, the mechanism of the enhanced
mutations within p53, b-catenin,
and area A of the BCL-6 gene sequence is likely to
be different from that of VH and area B
of BCL-6. These results suggest that HCV infection
activates both the normal hypermutation mechanism of the VH
gene and the general mutation mechanism of other somatic
genes. In addition, the mutated protooncogenes were
amplified in HCV-associated lymphomas and HCCs, but not in
lymphomas of nonviral origin or HBV-associated HCC. To rule
out the possibility that the high mutation frequency was due
to growth stimulation by HCV, we determined the growth rate
of HCV-infected cells. HCV infection was found to retard the
cell growth in comparison with the uninfected counterparts,
indicating that HCV-infected cells undergo a comparably
lower number of divisions. Therefore, cell growth rate could
not explain the higher frequency of mutations in
HCV-infected cells. These results indicate that HCV
infection induces DSBs, the repair process of which may be
associated with the introduction of mutations. Furthermore,
these DSBs could not be examined by apoptosis alone.
Mutations occur while homologous recombination repairs DSBsref1,
ref2
or ssDNA breaksref,
possibly mediated by error-prone DNA polymerasesref1,
ref2,
ref3,
ref4.
In particular, error-prone polymerases z,
h, i,
and µ have been postulated to be the mutagenic polymerases
for somatic hypermutation. HCV induced error-prone DNA
polymerase z, polymerase i, and AID
(which plays a role in the hypermutation of Ig, probably by
deaminating dCref
with a strong bias toward dG·dC base pairsref)
(but not mismatch repair enzymes (pms1, pms2, mlh1, msh2,
msh3, and msh6), which may play a role in the error-prone
repair processref),
which
together, contributed to the enhancement of mutation
frequency, as demonstrated by the RNA interference
experiments. These results indicate that HCV induces a
mutator phenotype and may transform cells by a hit-and-run
mechanism. Repair of DSBs by homologous recombination
has been reported to result in an 100-fold increase in the
rate of point mutations in the vicinity of the breaks in Saccharomyces
cerevisiaeref.
These mutations depend on the error-prone polymerase z, which has the DNA-damage bypass
activityref.
Polymerase i behaves as a dA·dT
mutator in the middle of DNA templates but as a dG·dC
mutator at their ends, when acting on a primer terminus with
a long template overhang, with extraordinarily low fidelityref.
Polymerase i also induces
somatic hypermutation in Ig genes in the BL2 cell lineref.
Both error-prone polymerases z
and i could be activated as a
result of B cell receptor stimulationref1,
ref2.
It is interesting that error catastrophe and lethal
mutagenesis reported in several RNA viruses may extend to
DNA error catastrophe, which has been described as "melting"
of genetic informationref.
On the other hand, HCV-induced DNA breaks might be the
result of reactive oxygen species; it has been reported that
core, NS3 and NS5A
proteins can induce reactive
oxygen
species
ref.
The enhanced DSBs may also explain the occurrence of apoptosis
associated with hepatitis and chromosomal instability found
in the B cells and hepatocytes of HCV-infected individuals.
This finding provides a mechanism of oncogenesis for an RNA
virusref.
The association of HCV with B-cell neoplasia comes
primarily from
- case-control studies : a disadvantage of
case-control studies in establishing causation is their
inability to determine whether the exposure of interest
occurred before or after onset of disease
- Italyref
: a profound regional variation in findings that
parallels levels of HCV endemicity : 32% in Pisaref,
22.3ref-42%ref
in Modena, 28% in Pordenone (38.4% in low-grade, 11.4%
in intermediate, and 15.2% in high-grade)ref,
39.7% in S.Giovanni Rotondoref,
22.4% in B-LPD (B-NHL, MM, MGUS, B-CLL) in Naples
(61.5% in WM)ref,
37% in Piacenzaref,
8.9% in Udine (95.4% if cryoIg+ and 4.6%
among cryoIg-)ref1,
ref2,
compared with a 2% to 13% prevalence in patients with
other blood malignancies, and a 1% to 5.4% prevalence
in healthy controls. In several of these studies, antibody-negative
viremia has reportedly accounted for a
substantial fraction of HCV-associated tumors, in
apparent contrast to the usually robust serologic
response to this infection (30% in Pisaref,
37% in Modenaref2,
22.4% in Naplesref3)
- Japan : prevalences among NHL cases of 8.1%
(but only in males)ref-16%ref-22%ref.
- Los Angeles, southern California :
prevalence of 22% in B-cell NHL case subjects, 4.5% in
other hematologic malignancies, and 5% in general
medicine clinic control subjectsref.
In contrast, the majority of studies from nonendemic areas
elsewhere in Europe (4.3% in Ulm, Germanyref,
2.0% in Franceref,
?% in The Netherlandsref,
0% in Newcastle upon Tyne, UKref)
or from North America (0% in Columbia, USAref,
0% in Toronto, Canadaref)
have generally failed to find an association. The marked
discrepancies among studies from different geographic
areas might be explained by the following:
- the studies in favor of a relationship between HCV
infection and B-NHL originated from areas with high
endemicity for HCV. However, because of the higher HCV
prevalence in older subjects in these areas,
age-matched control groups should have been used
systematically for comparisonref1,
ref2
- in contrast, the studies suggesting a lack of
association originated from countries with a lower
prevalence of HCV infection. Thus, HCV appears to be
associated with B-NHL only in areas where HCV is
highly prevalent, suggesting that HCV is not
essentially involved in the pathogenesis of B-NHL.
- prospective cohort studies :
- Japan : among 2162 patients with HCV and chronic
liver disease, the incidence of NHL was not
significantly increased. On the basis of 4 observed
cases in 12 400 person-years of observation, the
relative risk was 1.9 (95% confidence limits, 0.6-5.4)
compared with the general populationref.
Blood transfusion, which conferred risk of HCV
infection in the past, has also been a risk factor for
NHL in some studies
- southern Sweden : blood recipients had standard
morbidity ratios for malignant lymphoma of 2.7 in a
hospital-based cohort and 3.1 in a population-based
cohortref
- UK : a neonatal transfusion cohort had a 2-fold NHL
excess at 15 to 49 years of age, although the increase
was not statistically significant (P = .12)ref
- USA :
- in contrast, blood transfusion was not a
significant risk factor for NHL in a case-control
study from Olmstead County, MN, with an odds ratio
of 0.84 (95% confidence limits, 0.50-1.41)ref
- in the Iowa Women's Study cohort, a history of
blood transfusion was associated with an NHL
relative risk of 1.6, with stronger associations for
low-grade tumorsref
- because incident HCV infection is uncommon after
age 40ref,
the HCV infection status in young adulthood was
examined as a predictor of subsequent B-cell
malignancy in a prospective cohort study in northern
California : these data are inconsistent with a
relative risk of B-cell neoplasia for HCV infection
> 6.2. The corresponding maximum fraction of
B-cell neoplasia in the general population
potentially attributable to chronic HCV infection is
2.5%. Nevertheless, these data do not address a
possible risk from shorter-term infection, as
researchers did not have follow-up sera to rule out
seroconversion in later liferef.
Posttransplant lymphoproliferative disease, which includes
B-cell NHL as its most extreme manifestation, has been
variably associated with HCV infection. Patient series
from several countries report increased risk of this
complication in HCV+ transplant recipients,
although one (from the USA) failed to find an associationref1,
ref2.
However, HCV infection does not seem to further increase
the B-cell NHL risk associated with HIV infection.
Hemophilic AIDS patients, who are nearly all HCV infected,
are not at higher risk of lymphoma than other
HIV-transmission categories, and homosexual male AIDS
patients with NHL do not have an increased prevalence of
HCVref.
Regardless of these inconclusive epidemiologic data,
there is some molecular evidence in support of a
possible causal role for HCV in the etiology of B-cell
lymphoproliferative disorders. HCV infection is
also associated with an increased frequency of circulating
DNA with the bcl-2-JH recombination
characteristic of follicular lymphomaref,
and successful antiviral therapy may decrease recombination
frequencyref
- immunocytoma (IC)–lymphoma (CD5-19+IgM+)
/ lymphoplasmacytoid
lymphoma
ref1,
ref2,
ref3,
ref4
(40-76%ref)
: in 3 studiesref1,
ref2,
ref3,
HCV was principally found in patients with
lymphoplasmacytoid lymphoma/immunocytoma associated with
type II MC, with 30% to 45% of these patients being
HCV-RNA+. The lymphoplasmatocytoid
lymphoma/immunocytoma originates from a CD5-peripheral B
lymphocyte able to differentiate into a plasma cell.
Sites involved include bone marrow, lymph nodes, spleen
and, less frequently, peripheral blood or extranodal
sites. This condition is commonly associated with type
II MCref.
In the USA studyref,
monocytoid nodal B-cell lymphoma accounted for 23% of
all lymphomas and 67% of all low-grade lymphomas among
the HCV+ patients. Furthermore, another Italian reportref
pointed out an additional subset of overt B-NHL in
HCV-infected individuals, diffuse large B-cell lymphoma
(23 of 83, 27.7% of cases were HCV+). The
overrepresentation of lymphoplasmatocytoid lymphoma in
several Italian studies (14% to 33%)ref1,
ref2
might result from a selection bias of the patients, due
to the follow-up of a significant number of patients
with MC in these centers. This could account for the
high prevalence of HCV infection in B-NHL patients in
these series. The lower prevalence of
lymphoplasmatocytoid lymphoma in our series of
unselected patients with B-NHL (4.5%) is in agreement
with the 1.2% prevalence, as estimated by R.E.A.L.ref,
and could explain the low prevalence of HCV markers
observed. Furthermore, the low prevalence of HCV
infection (2 of 118, 1.7%) in the large cohort of our
patients with diffuse large B-cell lymphoma does not
confirm the recently suggested association between HCV
and diffuse large B-cell lymphomaref.
- primary hepatosplenic diffuse
large-B-cell lymphomas

- marginal-zone
lymphomas
:
16 studies where an anti-viral regimen was administered to
65 HCV-infected patients with lymphoproliferative disorders
were identified. Complete remission of the
lymphoproliferative disorder was achieved in 75% of the
cases. In contrast, HCV- subjects did not respond
to interferon, indicating that the response in the
HCV-infected patients is not merely due to the
antiproliferative effect of interferon. Remission after HCV
eradication was maintained, provided that infection did not
reappear. In HCV-infected patients with non-Hodgkin's
lymphoma treated with corticosteroids/chemotherapy liver
function tests deterioration did not occur. The addition of
interferon to standard chemotherapy may decrease hepatic
side-effects of chemotherapy. Although it is evident that
larger therapeutical trials of anti-viral therapy are needed
to determine the role of this strategy in HCV-infected
patients with lymphoproliferative disorders, encouraging
data emerge from recent studies showing that interferon (+
ribavirin) is an attractive therapeutic option for some
HCV-related low-grade lymphomasref.
Clonal B lymphocytes are frequently detected in the blood
(26%) and liver (32%) of patients with chronic HCV
infection, in the absence of overt B cell malignancy. These
clones are usually, but not always, associated with the
presence of type II cryoglobulins. A high percentage of
patients with B cell clonality in both the blood and liver
are finally diagnosed as having a definite B cell malignancyref
- Hodgkin's
lymphoma
(4% in Italyref;
9% = 12-fold higher prevalence of infection in Hungaryref)
- no HCV reactivation was found in malignant hematologic
diseases, such as NHL or HDref1,
ref2
- while t(14;18)(q32;q21) involving fusion of IGH with
MALT1 occurs frequently in MALTmas, the classical form of
t(14;18)(q32;q21) involving fusion of IGH with bcl-2
can be detectable in a
subset of MALT lymphomas in patients with HCV infectionref.
B cells from patients with type II mixed cryoglobulinaemia
(MC), strongly express the antiapoptotic bcl-2 oncogene
product. Therefore, we investigated a possible mechanism
of lymphomagenesis, the occurrence of bcl-2 and
immunoglobulin gene rearrangement (IgH) in HCV-infected
patients. Three groups of patients were studied: (1) 44
patients with HCV and MC (anti-HCV and HCV RNA positive);
(2) 59 patients with chronic HCV infection without MC; (3)
50 patients with chronic liver disease (CLD) not related
to HCV infection. The t(14;18) translocation (MBR
bcl-2-JH) and IgH rearrangement (FR3/JH) were detected by
polymerase chain reaction (PCR) in peripheral mononuclear
cells. bcl-2 translocation was detected in 17/44 (39%),
7/59 (12%) and in none of the patients of groups 1, 2 and
3 respectively (P < 0.01). Monoclonal IgH rearrangement
was detected in 15/44 (34%), 5/59 (8.5%) and 2/50 (4%)
patients of groups 1, 2 and 3 respectively (P < 0.05).
HCV-infected patients had a higher prevalence of
monoclonal IgH rearrangement and bcl-2 translocation than
patients with CLD of other aetiologies. These data suggest
that HCV may play a role in the multistep mechanism of
lymphomagenesis by inducing clonal proliferation of B
cells and inhibition of apoptosisref.
Recently, an increased rate of clonal proliferation of B
cells and bcl-2 translocation and overexpression in PBMCs
of patients with HCV infection was demonstratedref1,
ref2,
ref3.
Because chronic antigenic stimulation by HCV has been
shown to play a role in the development of B-cell
expansion and malignant transformation of immunocytomasref,
it is possible that eradication of the persistent
infection by antiviral treatment may possibly lead to
regression of the proliferating clone. 15 of 29 patients
(8 with IgH rearrangement, 6 with t(14;18) translocation,
and 1 with both) were treated with either IFN-a or by combination therapy with
interferon and ribavirin for 6 to 12 months. IgH
rearrangement became negative in 7 of 9 treated patients
compared with only 1 of 8 of nontreated patients (P
<.02). The t(14;18) translocation became negative in 6
of 7 treated patients compared with 1 of 6 nontreated
patients (P =.03). Disappearance of IgH rearrangement or
t(14;18) translocation was strongly associated with
virologic response to treatment. 2 t(14;18)+
patients developed B-cell lymphoma during follow-up.
Antiviral treatment appears to be effective in eliminating
the clonal proliferation of B cells in patients with
chronic HCV infection and may prevent the subsequent
development of lymphoma. The mechanism can be related to a
direct effect of interferon-alpha on the proliferating
clone or to an indirect effect by eradicating the
antigenic stimulusref.
- In a meta-analysis, a strikingly positive association
between anti-HCV positive status and risk of NHL was
confirmed, especially with B-NHL. Contrary to expectation,
endemic status of HCV did not change the significance of
the association. In addition, a possible selection bias
was identified owing to the use of blood donor controls. 2
possible biological mechanisms for lymphomagenesis,
particularly for B-NHL, can be hypothesizedref.
The first is the direct phenotypical change of lymphocytes
by HCV. Although a complete mechanism is not available for
HCV-induced carcinogenesis of hepatocytes, viral
replication in the cell is known to be important. E1/E2
envelope proteinref
or core proteinref
from the replicated virus induces transformation of
hepatocytes. The same mechanism can be hypothesized for
lymphomagenesis by HCV. HCV can be detected in
B-lymphocytes, but evidence for HCV replication in
lymphocytes is controversial. Therefore, the direct
lymphomagenic effect of HCV is not clear at this point. A
second possible mechanism is that HCV antigen stimulates
the expansion of mono- or oligoclonal B-cells. Oligoclonal
B-cell expansion in the peripheral blood was observed in
100% of HCV-infected patients with type II MCref.
A similar phenomenon was reported for intrahepatic
lymphocyte infiltrates of HCV-infected liver. Analysis of
the Ig heavy/light chain usage revealed involvement of the
specific VH1–69 in patients with
lymphoproliferative disordersref.
Sequence analysis for these regions demonstrated somatic
hypermutation, evidence for affinity maturation under
constant antigen stimulationref.
Envelope protein E2 is now considered as a candidate
antigen based on several lines of evidence: anti-HCV E2
B-cell clones from an HCV-infected patient preferentially
used VH1–69ref
and B-cell receptor from one HCV-associated lymphoma
patient bound to E2ref.
CD81 is a cellular ligand for E2 envelope on the surface
of lymphocytesref.
CD19/CD21/CD81 complex along with B-cell receptor
stimulation is considered to activate proliferation of
lymphocytesref.
The strong association we observed for B-NHL warrants
further study regarding this issue. A similar CD81-E2
mediated mechanism is supported for T-cell NHLref.
CD81-E2 cross-linking activates Lck through raft
aggregation and thus leads to enhanced TCR signalingref.
Although further clarification is required, the positive
association we observed for TNHL may be explained in part
by this mechanismref
- non-B NHLs and MPD : in Italy the prevalence of
HCV infection was not higher in patients with HD (3.2%, 5
out of 157 cases) or MM (4.7%, 5 out of 107) than in
controls. On the other hand, it was consistently higher in
T-NHL (13.8%, 4 out of 30), CLL (9.0%, 9 out of 100), ALL
(7.6%, 5 out of 54), AML (7.9%, 11 out of 140), and CML
(12.2%, 6 out of 49) patients. These patient groups were
not, however, large enough to render statistically
significant resultsref.
In Sweden the risk of NHL and MM was significantly
increased in the HCV stratum with more than 15 years of
infectionref
- anti-HCV positivity in 69%, 11% and 4.3% of the EMC, MM
and B-CLL samples testedref.
Although very similar, this figure is slightly lower than
that of other reports, which may be explained by false
negative results. Serologic tests have been demonstrated to
give a lower positive results when compared with the more
sensitive PCR technique. The overall prevalence of HCV RNA
among patients with type II cryoglobulinemia was estimated
to be 84% and 96%, whereas the frequency of anti-HCV Ab in
the same groups of patients was 42% and 77.4%, respectivelyref1,
ref2.10,13
False
negative results may also be attributed to the presence of
immune complexesref10
or
production of low levels of normal immunoglobulinsref,13
usually
observed in many hematologic malignancies as a result of
immunosuppression and infiltration of the malignant cells in
the bone marrow. Presence of some HCV genotypes such as HCV3
or HCV4 which may not be detected by the commercial ELISA
kitsref29
and/or
extensive mutations within the E2/NS1 region with subsequent
loss of some B-cell epitopesref1,
ref230,31
also
explain failure of detection of HCV infection by serologic
techniques. False positive results have also been reported
using commercial anti-HCV ELISA kitsref.12
This
limitation has paved the way for introduction of the
immunoblotting technique as a confirmatory tool for the
ELISA positive samples. In this study, though all the EMC
samples which were positive by ELISA, also reacted in RIBA,
anti-HCV reactivity of only 5/8 (62%) and 1/2 (50%) of the
ELISA positive MM and B-CLL sera was confirmed by RIBA,
respectively. This seems to be largely due to weak
reactivity of some of the samples, since strongly reactive
samples also gave positive results by RIBA. Non-specific
interaction of some charged paraproteins with the pre-coated
recombinant viral proteins could have lead to weak anti-HCV
reactivity in the MM samples. Despite the fact that only 62%
of the ELISA reactive MM samples were confirmed by RIBA, HCV
infection was found to be substantially higher in the MM
patients (11%) compared to the RA control group (p<0.06)
and normal population. Although prevalence of HCV infection
in MM patients was substantially higher than in RA patients,
(11% vz 0), however, due to the small sample size, the
difference was not statistically significant. < 1% of the
normal adult population of Iran (0.3%) have been reported to
be anti-HCV antibody positiveref,32
unpublished
data from the Blood Transfusion Center of Iran). Our
results, in line with othersref33
indicate
that HCV infection is not necessarily associated with
cryoglobulinemic activity. Most of the data reported so far,
however, suggest a strict association between HCV infection
and monoclonal gammopathies with cryoglobulinemic activityref1,
ref2,
ref3,
ref4.
13,33-35 The fact that about 80% of the anti-HCV+
MM samples contain paraproteins of the IgG isotype, suggests
that anti-HCV reactivity may also be found in gammopathies
with heavy chain isotypes other than IgM and devoid of
cryoglobulin activity. The frequency of anti-HCV Ab was
similar, though slightly higher in the B-CLL patients (4.3%)
compared to the control group. However, due to the small
sample size of the B-CLL patients studied and the fact that
only one of these samples gave a positive result, firm
conclusion can not be drawn. High frequency of HCV infection
in haematologic malignancies could also be attributed to
frequent transfusion of blood or blood products to such
patients due to suppression of haematopoisisref.8
Our
clinical records, regarding transfusionof blood to the
patients particularly the EMC patients, are not convincingly
enough to allow us to address the argument, though some of
available data does not support involvement of blood
transfusion. Apart from 2 of the BCLL patients who were in
the stage 4 of the disease with anemic manifestations
(without a history of blood transfusion at the time of blood
sampling) the rest of the patients were mostly at stage 0 or
1 of the disease. Of the MM patients studied from whom a
complete clinical records is available none show a history
of blood transfusion. Our findings regarding similar
distribution of the anti-HBs Ab and HBs Ag (i.e. past and
present HBV infection) within the subject and control groups
suggest that the relative risk for acquisition of a
blood-born viral infection is similarly represented in all
groups, regardless of their particular diseases. This
reiterates the significance of the role of HCV in
pathogenesis of EMC and to a lesser extent MM.
In this study, the presence of HCV RNA negative strand in BM
was demonstrated in 4 of 24 (17%) patients who were HCV
positive and the titer of positive strand was 1 to 2 logs
higher than the titer of the negative strands, which is a
proportion similar to that expected for a flavivirus at its
replication site. However, the actual prevalence of active
infection could be higher, as the replication in other
subjects could have been below the sensitivity limit of our
strand-specific assay. Extrahepatic HCV replication is more
likely to be detected in the presence of immunosuppression:
The presence of HCV RNA negative strand was found in lymph
nodes and BM from HIV-coinfected subjectsref,
as well as in hematopoietic cells derived from patients who
were HCV positive and transplanted into the severe
immunodeficiency mouseref.
However, even in these studies, the presence of viral
negative strand was by no means universal and the titers
were very low. This contrasts with the reported common
detection of HCV RNA and viral antigens in BM and PBMCs by in
situ techniquesref1,
ref2,
ref3.
Because the strand-specific assays are relatively sensitive,
our RT-PCR was capable of detecting approximately 103
viral genomic eq in 1 µg of total RNA, this suggests a very
low rate of replication or rapid degradation of negative
strand RNA. HCV invasion of hematopoietic cells may not be
benign; HCV infection was associated with such
lymphoproliferative disorders as non-Hodgkin's lymphoma and
cryoglobulinemiaref.
Although the effect of HGV on BM is unclear, one study
reported a possible link between HGV infection and low-grade
non-Hodgkin's lymphomaref.
Interestingly, in our previous study, which used the same
Tth-based assay, we did not detect HCV replication in PBMCs
from HIV-negative subjectsref.
Similarly, other studiesref1,
ref2
failed to demonstrate the presence of HCV RNA negative
strands in PBMCs when using highly strand-specific RT-PCR
assays. One possible explanation for this discrepancy is
that HCV may be particularly apt at infecting CD34+
hematopoietic progenitor cellsref.
It is also possible that HCV replication becomes more
efficient in proliferating cells. In support of this
concept, our recent observations are that PBMCs from
subjects with chronic hepatitis C occasionally become
positive for the presence of HCV RNA negative strand after
stimulation with phytohemagglutin (PHA) mitogenref.
HCV circulates as a number of closely related but not
identical genomes, referred to as quasi-species, and the
presence of this dynamic mutant reservoir could facilitate
viral adaptation to replication in various secondary cells.
HGV does not seem to be as variableref,
and BM cells may represent the primary, not the secondary,
site of replication. BM replication may not be that unusual
for flaviviridae, for example, the hog cholera virus was
found in megakaryocytesref,
whereas dengue virus was shown to replicate efficiently in
BM progenitors and hematopoietic cell linesref.
Interestingly, we identified 3 patients in whom HGV RNA was
detectable in the BM but not in the serum; 2 of them were
anti-HGV negative in serum. The latter 2 patients were
probably in the early phase of the infection when antibodies
have not yet developedref.
Although it supports the notion of HGV replication in BM, it
also demonstrates that studying HGV RNA and specific
antibodies in serum may underestimate the true prevalence of
the infection. However, this may also be occasionally true
for HCV infection as one of the patients studied was HCV RNA
positive in BM but not in serum. This could probably be
explained by the fact that viral levels in serum can
fluctuate during chronic infection, becoming occasionally
undetectable by RT-PCRref.
Whether this was the case is unclear, as follow-up serum
samples were unavailable for analysis.
- B-cell acute
lymphoblastic leukemia (ALL)
ref
- a high prevalence of HCV and Helicobacter
pylori
infections in paediatric NHL patients at NCI. Concerning
the hypothesis of their pathogenetic role in
ymphomagenesis, it is still unclear whether these agents
have a direct role in malignant transformation in
pediatric lymphoma because a typical NHL
clinico-histological feature associated with HCV and H.
pylori is lackingref.
- membranoproliferative
glomerulonephritis

- patients without severe liver dysfunction have anyway
increased risk of carotid
atherosclerosis
ref1,
ref2
Laboratory
examinations :
- ELISA-3 looking for Igs : in immunosuppressed individuals,
levels of anti-HCV may be undetectable, and a diagnosis may
require testing for HCV RNA.
- IgM
- < 1 index => negative
- 1-1.5 index => mildly positive
- > 1.5 index => positive
- total Igs
- < 10 mIU/mL => negative
- > 10 mIU/mL => positive
... confirmed by testing for HCV RNA :
- RIBA-3
- qualitative or quantitative real-time PCR

1 international unit equals to 2.7 copies of viral genome.
Research on HCV has been hampered by the lack of a virion-capable
cell culture system : most existing models rely on strains
isolated from chronic carriers and only a few HCV strains can
replicate in this system and these strains need adaptive mutations
in their viral genome to replicate efficiently in cultured cells.
Since 2005 several strains have been cultivated in the human
hepatoma cell line
Huh7 :
- a construct containing the entire cDNA genome of an
infectious HCV strain, CG1b, was engineered and placed
between 2 ribozymes designed to generate the 5' and 3' ends of
the virus. The construct was transfected into Huh7 cells, and
positive- and negative-strand HCV RNAs were found by RNase
protection assays. HCV core, E2, and NS5A proteins were
detected in the transfected cells by immunofluorescence and
Western blot. To test for virion production, the cell culture
medium was fractionated by sucrose density gradient
centrifugation. Fraction 5, with a density of 1.16 g/ml,
coincided with the published density of free HCV virions, and
HCV particles were visualized by EM : virion RNA matched HCV
RNA from infected individualsref.
- subgenomic replicons of the JFH1 genotype 2a strain
cloned from an individual with fulminant hepatitis replicate
efficiently in cell culture. The JFH1 genome replicates
efficiently and supports secretion of viral particles after
transfection into a human hepatoma cell line (Huh7). Particles
have a density of about 1.15-1.17 g/ml and a spherical
morphology with an average diameter of about 55 nm. Secreted
virus is infectious for Huh7 cells and infectivity can be
neutralized by CD81-specific antibodies and by immunoglobulins
from chronically infected individuals. The cell
culture-generated HCV is infectious for chimpanzee, although
viral RNA was no longer detectable in vivo beyond 5
weeks after infection. This system provides a powerful tool
for studying the viral life cycle and developing antiviral
strategiesref.
A simple yet robust HCV cell culture infection system based on
the HCV JFH-1 molecular clone and Huh-7-derived cell lines
(Huh7.5.1, more efficient than Huh7) has been established and
allows the production of virus that can be efficiently
propagated in tissue culture. This system provides a powerful
tool for the analysis of host-virus interactions that should
facilitate the discovery of antiviral drugs and vaccines for
this important human pathogenref.
A full-length chimeric HCV genome made from JFH1's
nonstructural genes with structural gene sequences derived
from other strains (such as the 2a genotype strain J6)
replicates and produces virus particles that are infectious in
cell culture (HCVcc) (unless full-length J6). Replication of
HCVcc was robust, producing nearly 105 infectious
units/ml within 48 hours. Virus particles were filterable and
neutralized with a monoclonal antibody against the viral
glycoprotein E2. Viral entry was dependent on cellular
expression of a putative HCV receptor, CD81. HCVcc replication
was inhibited by interferon-alpha and by several HCV-specific
antiviral compounds, suggesting that this in vitro system
will aid in the search for improved antivirals. By swapping
genomic components between virulent and nonvirulent strains,
it will become possible to identify genetic determinants of
viral pathologyref
Prognosis :
HCV RNA may persist and replicate in the liver (genomic strand :
83%; antigenomic strand : 100%) and PBMCs (genomic strand : 50%;
antigenomic strand : 83%) of healthy, anti-HCV antibody–positive,
serum HCV RNA–negative patients who have persistently normal ALT
levels. These patients should be followed up, because they have an
ongoing viral infection
ref.
Prevention
:
Therapy :
- IFN-a was approved as a therapy
for hepatitis C in 1991. However, the overall rate of
sustained virologic response, defined as the absence of HCV
RNA in serum > 6 months after the discontinuation of
therapy, was low (generally <20%) with interferon alfa
monotherapyref.
IFN-a2a
, IFN-a2b
, IFN-an-1
, IFN-an-3
is
successful in 40-60%. IFN-a is not
effective on genotype 1b and its usage is not recommended in
individuals with autoimmune
diseases
, pregnancy
, liver
cirrhosis
, hepatocellular
carcinoma
,
severe heart
, kidney
or
lung
diseases, severe thrombocytopenia
or
leukopenia
, epilepsy
, diabetes
mellitus
.
- IFN-b

- IFN-g was detectable in the
livers of the chimpanzees that cleared or controlled the
virus, raising the possibility that IFN-g
might perform antiviral effector functions during HCV
infection. Based on these observations, therapeutic induction
of intrahepatic IFN-g by adoptive
immunotherapy might be able to control chronic HCV infectionref.
- ribavirin
alone lowered serum enzyme levels but had little effect on HCV
RNA levelsref
- combination therapy with pegylated interferon and
ribavirin for :
- acute hepatitis C : although neither standard nor
pegylated interferons are approved, several prospective
studies have shown excellent responses when therapy is
started within 6 months after the onset of infection, with
rates of sustained virologic response of 85% or greaterref1,
ref2.
Left untreated, acute hepatitis C progresses to chronic
hepatitis in up to 75% of adults. Unfortunately, treatment
of acute hepatitis C is associated with high rates of side
effectsref.
The optimal doses have yet to be defined, but a 24-week
course of peginterferon at standard doses and ribavirin at a
dose of 800 mg daily is more than adequate
- chronic hepatitis C : improvement in rates of
sustained virologic response (40 to 45%)ref1,
ref2.
With its increased half-life, peginterferon can be given as
a weekly doseref.
2 peginterferon formulations are currently approved for the
treatment of hepatitis C: a2a
(Pegasys, Roche) and a2b
(Peg-Intron, Schering-Plough). In 2 large trials of these
agents, the rates of sustained virologic response to a
48-week course of peginterferon and ribavirin were 54 and
56%, as compared with 44 and 47% with standard interferon
and ribavirin and only 29% with peginterferon aloneref1,
ref2.
Response rates were higher among patients with genotype 2 or
3 than among those with genotype 1. A subsequent trial of
different regimens of peginterferon a2a
and ribavirin showed that patients with genotype 2 or 3
could be treated with a lower dose of ribavirin (800 mg
rather than 1000 to 1200 mg daily) and that the rates of
sustained virologic response after 24 weeks of therapy (81
and 84%) were similar to the rates after 48 weeks of therapy
(79 and 80%)ref.
Therapy is recommended for adults with chronic hepatitis C
who have detectable HCV RNA in serum, transaminitis,
histologic evidence of progressive liver disease, and no
other serious coexisting conditions or contraindicationsref1,
ref2,
ref3.
Specific qualitative assays for HCV RNA are available that
have a lower limit of sensitivity of 10 to 50 IU
(approximately 40 to 100 genome equivalents)/mlref.
In addition, HCV RNA can be quantified; most patients with
chronic infection have HCV RNA levels between 0.2 and 5
million IU (approximately 1 to 20 million genome
equivalents)/ml. An elevated serum ALT level is one of the
criteria for instituting therapy because most persons with
normal ALT levels have mild, nonprogressive diseaseref.
However, the degree of elevation does not always reflect the
severity of disease and is not predictive of a patient's
response to therapy. Thus, normal aminotransferase levels
should not exclude patients from therapyref1,
ref2.
A liver biopsy is helpful in determining the severity of the
inflammation and necrosis (the disease activity) and the
stage of illness (the extent of permanent injury or
fibrosis)ref.
In the 2 commonly used histologic scoring methods, the
Metavirref
and Ishak Scoringref
systems, fibrosis is scored as none, portal fibrosis only,
portal fibrosis with septa formation, bridging hepatic
fibrosis, or cirrhosis. Therapy usually is recommended for
patients with more than portal fibrosis only (an Ishak score
3 or a Metavir score 2)ref.
However, a liver biopsy has the major limitations of being
costly, invasive, potentially harmful, and prone to sampling
errorref1,
ref2.
For these reasons, a liver biopsy cannot be considered to be
a prerequisite for therapy. For patients with genotype 2
or 3 infection, rates of response to therapy are high, and
a liver biopsy is not generally needed to make a decision
regarding treatment. In patients with genotype 1
infection, a liver biopsy is helpful if there is little
clinical evidence of advanced disease and if the patient is
uncertain about whether to undergo treatment. For patients
who want therapy regardless of the severity of the illness
or for those with clinical evidence of fibrosis or
progressive disease, therapy can be initiated without a
liver biopsyref.
In patients infected with HCV genotype 1, the rates of
sustained virologic response and tolerability does not
differ significantly between the 2 available (a2a and a2b)
peginterferon–ribavirin (800 to 1400 mg per day) regimens
or between the 2 doses of peginterferon a2b (1.0 or 1.5 mg/kg)ref.
Absolute contraindications to therapy with peginterferon and
ribavirin include pregnancy, breast-feeding, and a known
allergy to either drug. Relative contraindications because
of the potential for side effects include decompensated
liver disease (bilirubin level >1.5 mg/dl [25.6 µM];
prothrombin time >15 seconds or INR >= 1.7; albumin
level <3.4 g/dl; ascites; bleeding esophageal varices; or
hepatic encephalopathy), major neuropsychiatric disease,
coronary or cerebrovascular disease, renal failure, and a
history of solid organ transplantationref1,
ref2.
Patients with active substance or alcohol abuse may also not
be candidates for therapy, and those with recent substance
abuse should receive counseling during treatment because of
the risk of relapse. Patients with anemia, thrombocytopenia,
or leukopenia should be treated with caution, because these
abnormalities are worsened by treatment. In patients with
renal dysfunction, the ribavirin must be adjusted, and such
patients are at increased risk for ribavirin-induced
hemolytic anemia. Hepatitis C is more rapidly progressive in
patients with HIV coinfection, particularly as the
immunodeficiency worsensref.
Thus, it is appropriate to treat hepatitis C in all
persons with HIV coinfection as early in the course of HIV
infection as possible and regardless of the ALT level
or the histologic features of the liver, as long as there
are no other contraindications to treatmentref.
The currently recommended regimen for the treatment of
chronic hepatitis C is the combination of weekly
subcutaneous injections of peginterferon and twice daily
oral doses of ribavirinref1,
ref2,
ref3,
ref4,
ref5,
ref6,
ref7.
The recommended dose of peginterferon a2a is 180 µg per weekref
and that of peginterferon a2b
is 1.5 µg/kilogram of body weight per weekref.
The optimal duration of therapy and dose of ribavirin vary
according to the HCV genotype. Patients with genotype 1
should receive ribavirin for 48 weeks at a daily dose of
1000 mg (if their body weight is < 75 kg) or 1200 mg
(if their weight exceeds 75 kg). Patients with genotype 2
or 3 infection should receive 24 weeks of combination
therapy with a dose of 800 mg of ribavirin dailyref.
There is little information on the treatment of hepatitis
C in patients with genotypes 4, 5, and 6ref,
and the regimen for genotype 1 is usually recommended for
such patients. The cost of a 48-week course of
peginterferon and ribavirin ranges from $30,000 to $40,000,
depending on local charges and the dose and brand of drugs
used. The expenses for monitoring and physician visits also
need to be considered in weighing the costs of therapy. 3
general patterns of response occur with therapy:
- a sustained virologic response : HCV RNA levels usually
fall rapidly with the initiation of therapy, becoming
undetectable within 4 to 24 weeks and remaining
undetectable throughout the course of treatment and
follow-up. Alanine aminotransferase levels fall within a
few weeks after the decline in HCV RNA levels, usually
become normal during treatment, and remain normal during
follow-up. If a liver biopsy is performed, the histologic
findings show marked improvement. With the use of current
regimens, overall sustained virologic response rates are
75 to 80% among patients with HCV genotype 2 or 3
infection and 40 to 50% among those with genotype 1ref1,
ref2,
ref3,
ref4,
ref5.
Among patients with genotype 1, the rates of sustained
virologic response are lower among blacks (28%) than among
whites (52%)ref.
Other factors associated with a lower response rate are
higher initial levels of HCV RNA (>600,000 IU per
milliliter), male sex, higher body weight, and more
advanced liver fibrosis. Not all patients with HCV RNA
that becomes undetectable during treatment have a
sustained virologic response
- a transient virologic response and relapse or
breakthrough (10%) : HCV RNA reappears in serum later
during the course of treatment (as a breakthrough), and in
20% it reappears when therapy is stopped (as a relapse).
In patients with a relapse, HCV RNA usually reappears
within a few weeks after the discontinuation of therapy,
and alanine aminotransferase levels rise toward
pretreatment levels thereafter. Relapses are more common
with shorter courses than with longer ones, with
interferon monotherapy than with combination therapy, and
with a delayed loss of HCV RNA during treatmentref
- nonresponse : HCV RNA remains detectable during
treatment, although the level may decline. In these p
ersons, alanine aminotransferase levels usually remain
elevated, and treatment provides little clinical benefit.
Nonresponse is uncommon among patients with genotype 2 or
3, but occurs in at least 30% of patients with genotype 1.
Improved regimens or formulations of peginterferon and
ribavirin are needed to prevent or control disabling side
effects and reduce costs. One practical approach is the use of
abbreviated courses of treatment. Therapy can be discontinued
early if there is no virologic response, making further
therapy futile, or if there is a rapid virologic response,
making prolonged therapy unnecessary. Thus, further therapy is
probably futile after 24 weeks in patients with genotype 1 who
remain HCV RNA+, since they are unlikely to have a
sustained responseref1,
ref2,
ref3.
Furthermore, if the level of HCV RNA does not decrease by more
than 2 log10 IU/ml or does not become undetectable
after 12 weeks of treatment, nonresponse can be predicted in
98 to 100% of instances, and therapy can be discontinuedref1,
ref2.
Therapy can also be curtailed if there is a rapid response.
Several recent studies have shown that treatment can be
abbreviated in patients who become HCV RNA– within
4 weeks of starting therapy. Among such patients, therapy can
be discontinued at 12 to 16 weeks in those with genotype 2 or
3ref1,
ref2,
ref3
and at 24 weeks in those with genotype 1 and low baseline
levels of HCV RNAref.
Current indications for the treatment of hepatitis C are based
on statements from a Consensus
Development
Conference Panel of the National Institutes of Healthref,
which are supported by treatment guidelines published by 2
academic societiesref1,
ref2.
Persons with chronic hepatitis C who are 18 years of age or
older, are willing to be treated, and do not have
contraindications to treatment are candidates for therapy if
they have detectable HCV RNA in serum and evidence of chronic
hepatitis (either elevated serum alanine aminotransferase
levels or the presence of considerable necroinflammatory
activity and fibrosis on liver biopsy).
Side effects of peginterferon and
ribavirin affect virtually all patients who receive treatmentref.
The most common adverse effects of peginterferon are muscle
aches and fatigue, but more difficult to manage are the
psychological side effects such as depression, anxiety,
irritability, sleep disturbance, and difficulty concentrating.
These side effects typically are managed with counseling,
antidepressant drugs, or anxiolytic agents, with variable
success. The most common side effect of ribavirin is
hemolysis, and anemia is the major reason for dose reduction.
The stress of the sudden onset of anemia can induce myocardial
infarction in persons with preexisting coronary artery disease
or stroke in those with cerebrovascular disease. Ribavirin is
also teratogenic, and strict adherence to an effective means
of birth control is mandatory for both women and men who
receive this drug. Serious side effects of combination therapy
occur in 1 to 2% of patients, and permanent injury and death
can occur. Among patients who receive treatment for 48 weeks,
dose reductions (usually of ribavirin) are necessary in 30 to
40% of patients and early discontinuation is necessary in up
to 20%ref1,
ref2,
ref3,
ref4.
Ribavirin should be reduced in increments of 200 mg daily and
can be temporarily withheld in the event of severe anemia. The
dose of peginterferon a2a
can be reduced from 180 to 135 µg per week, and the dose of
a2b can be reduced
from 1.5 to 1.0 µg per kilogram per week. Further reductions
in the dose of peginterferon are associated with reductions
in rates of sustained virologic responseref.
Peginterferon is not yet approved for use in children,
although prospective controlled trials are under way. Pilot
studies suggest that the rate of response to combination
therapy in children is similar to that in adults and that
children may have better tolerance of both interferon and
ribavirinref.
Peginterferon + ribavirin elicits long-term responses in only
about 50% of patients treated. No effective alternative
treatments exist for non-responders. Consequently, significant
efforts are continuing to maximize response to combination
therapy. However, rational therapy optimization is precluded by
the poor understanding of the mechanism(s) of ribavirin action
against HCV. Ribavirin alone induces either a transient early
decline or no decrease in HCV viral load, but in combination
with interferon it significantly improves long-term response
rates. A model of HCV dynamics in which, on the basis of growing
evidence, it is assumed that ribavirin decreases HCV infectivity
in an infected individual in a dose-dependent manner. The model
quantitatively predicts long-term response rates to interferon
monotherapy and combination therapy, fits observed patterns of
HCV RNA decline in patients undergoing therapy, reconciles
conflicting observations of the influence of ribavirin on HCV
RNA decline, provides key insights into the mechanism of
ribavirin action against HCV, and establishes a framework for
rational therapy optimizationref.
- direct-acting antivirals (DAAs) :
Web resources
:
LANL HCV Databases (LANLHCV)