Table of contents :

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Epidemiology Genomics Proteomics Transmission Pathogenesis Symptoms & signs Laboratory examinations Prognosis Prevention Therapy Web resources

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Epidemiology : prevalence = 3% of the world's population (180 million people worldwide ; 3 million Americans^ref) 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 : > 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
• 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.
• 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 2a^ref

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 eIF2a. 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.

• Hepatitis C virus type 1
• Hepatitis C virus type 1a
• Hepatitis C virus type 1b
• Hepatitis C virus type 1c
• Hepatitis C virus type 1d
• Hepatitis C virus type 1e
• Hepatitis C virus type 1f
• Hepatitis C virus type 2
• Hepatitis C virus type 2a
• Hepatitis C virus type 2b
• Hepatitis C virus type 2c
• Hepatitis C virus type 2d
• Hepatitis C virus type 2f
• Hepatitis C virus type 3
• Hepatitis C virus type 3a
• Hepatitis C virus type 3b
• Hepatitis C virus type 3g
• Hepatitis C virus type 4
• Hepatitis C virus type 4a
• Hepatitis C virus type 4c
• Hepatitis C virus type 4d
• Hepatitis C virus type 4f
• Hepatitis C virus type 4h
• Hepatitis C virus type 4k
• Hepatitis C virus type 5
• Hepatitis C virus type 5a
• Hepatitis C virus type 6
• Hepatitis C virus type 6a
• Hepatitis C virus type 10

Provisional classification of HCV is also possible using a partial sequence of the HCV genome :

• C gene : Okamoto's genotypes, 1992^ref : 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 C^ref
• I/1a
• II/1b
• genotype 1c appears to have evolved and remained in Indonesia^ref
• III/2a
• IV/2b
• V/3a^ref
• 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 Egypt^ref 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, ref2}reported 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 subtypes^{ref1, 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 year^{ref1, ref2}. Accumulation of nucleotide substitution in the HCV genome results in diversification and evolution into different genotypes^ref. 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 therapy^{ref1, 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 Japan^ref. 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 genotyping^{ref1, 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 priming^ref. 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 2b^ref. 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 studies^ref.
• E1 gene : 9 groups and 23 subgroups^ref
• NS5 gene : 6 major genotypes^ref

Okamoto's serotypes^ref 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 heterogeneity^ref.
• 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 cleavage^ref.
• E1 envelope glycoproteins
• nonstructural 1 (NS1) / E2 envelope glycoprotein binds to ...
• the large extracellular domain (EC2) of CD81 / TAPA-1^ref, which is expressed on ...
• hepatocytes
• lymphocytes
• the CD21/CD19/CD81/Leu13 costimulatory complex of B lymphocytes^ref, where reduces the threshold for B cell activation via the B cell receptor by bridging Ag specific recognition and CD21-mediated complement recognition^ref. 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 infection^ref : 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 MC^ref.
• 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 cells^{ref1, ref2}. Anti-CD81 can activate integrin a₄b₁ (VLA-4) on B cells, facilitating their adhesion to tonsilar interfollicular stroma. Similarly, anti-CD81 can activate a_Lb₄ (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 proteins^ref. 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 hepatocytes^ref. 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 cells^ref.
• the B cell receptor of HCV-associated lymphoma^ref, 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 line^ref 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 receptor^ref. The level of SR-BI needed for efficient infection varies between genotypes and subtypes^ref.
• LDLR^ref : very low density lipoprotein (VLDL) is selectively associated with HCV in type II cryoglobulins^ref. 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 patients^ref. Furthermore, it was demonstrated that LDLRs were up-regulated on keratinocytes in cutaneous vasculitis lesions compared with normal skin^ref. These observations and the finding that anti- lipoprotein precipitates HCV from infected serum^ref suggested that LDLR also may be a receptor for HCV complexed to VLDL or LDL^ref. 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 reasons^ref, 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 vitro^ref and the induction of HCV binding with transfection of the LDL receptor gene in COS-7 cells that were unable to bind HCV^ref 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 Donis^ref 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 study^ref 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 VLDL^ref, 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 cryoglobulinemia^ref. 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-1Rat^ref, which blocks the IL-1R-mediated stimulation by IL-1. Because IL-1 is known to increase LDLR activity^ref, 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 A^ref 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 response^ref.
• 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.
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
• NS3 helicase is essential for cytoplasmic RNA replication, and it is a representative member of helicase superfamily 2 (SF2)^ref . It transactivates HCV NS3-transactivated protein 1 (NS3TP1) and NS3TP2. It can induce reactive oxygen species^ref. It inhibits TBK1 and hence TLR3 STP^ref.
• NS3/4A serine protease causes specific proteolysis of the TLR3 adaptor protein TRIF / TICAM-1^ref and ablates retinoic acid-inducible gene I (RIG-I) signaling of downstream IRF3 and NF-kB activation^ref
• 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-Å resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond^ref. 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 prevalence^ref
• 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.
• 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 glands^ref
• HCV replicates in eccrine sweat glands cells and keratinocytes in healthy skin and is released into the sweat^ref

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 hepatocytes^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 years^ref) 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 inflammation^ref. Some HCV-associated HCCs have mutations in the tumor suppressor p53, the protooncogene b-catenin^{ref1, 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 RNA^{ref1, 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 vivo^{ref1, ref2}.
Iron accumulation in chronic hepatitis C relates to hepatic iron distribution, HFE genotype, and disease course^ref

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 Japan^ref)
• Sjogren syndrome (21-26% in Japan^ref) 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 disorders^ref.
• lichen planus + Sjogren syndrome (8.6% in Japan^ref)
• essential mixed cryoglobulinemia (EMC) (36%; 20% of all chronic HCV infected^ref)^{ref1, ref2,ref3, ref4}, a benign monoclonal lymphoproliferation that sometimes (7% in Italy^ref) evolves^{ref1, 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 cryoglobulinemia^ref. In type III cryoglobulins, immunoglobulin immune complexes contain both polyclonal IgM with rheumatoid factor activity and polyclonal IgG^ref. In type II cryoglobulins, immunoglobulin immune complexes contain both monoclonal IgM and polyclonal IgG^ref. The monoclonal IgM has rheumatoid factor (RF) activity and is encoded mostly by a restricted set of variable (V) region genes, specifically V_H1-69 (also known as 51p1) and V_k3-A27 (also known as kv325)^{ref1, ref2, ref3, ref4}. In non-HCV-infected individuals approximately 1.7% of peripheral blood B cells express the V_H1-69 gene^ref, as expected for a random use of the total repertoire of functional V_H 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-phenyloxazolone^ref, and p-azophenylarsonate^ref 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 V_H gene rearrangements are of the V_H3b subfamily, while the V_L gene response to the Hib PS antigen was less restricted, as seen in the anti-E2 response, suggesting that in both cases the V_H gene segments are the ones that play a more dominant role in antigen binding^ref. > 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 G6^ref. The latter 2 CRIs are characteristic for V_L and V_H 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 patients^{ref1, 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 NHL^{ref1, ref2, ref3}, namely a strong bias for V_H1-69 and V_k3-A27^{ref1, 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 receptor^ref), which reduces the threshold of B-cell activation^ref. Anti-E2 antibodies that block E2 binding to cells have been referred to as neutralization of binding (NOB) antibodies^ref. Subsequent studies characterizing 10 human anti-E2 mAbs showed that they could be either NOB-positive or NOB-negative^ref, 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 NHL^{ref1, 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 areas^ref. However, unlike the HCV-associated immunocytomas, the 51p1 V_H sequences in CLL are almost exclusively unmutated and usually have significantly longer CDR3 regions^{ref1, 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 hypothesis^ref. 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 al^{ref, (Bahler DW, 375a)} the 51p1 gene was found in 10 of the 18 V_Hsequences and was associated with a kv325 V_L region in all cases in which the V_L 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 MC^ref. Moreover, the histologic presentation of many HCV-associated NHLs is typical of germinal center (GC) and post-GC B cells^ref. 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 NHL^ref. 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 process^{ref1, ref2, ref3}. Moreover, the CDR3 regions of this case and of several other lymphoma immunoglobulins showed homology to the CDR3 regions of anti-E2 antibodies^{ref1, 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 V_H and V_L 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 V_H and/or V_L 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 antibody^ref. 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 protein^ref. 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 pathway^{ref1, ref2,} has been shown to bind more avidly to cell surface-expressed CD81 than does secreted E2₆₆₁^{ref1, 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 complex^{ref1, 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 V_H1-69 gene^ref, whereas neither of the lymphoma cases expressed this V_H gene. In contrast, both V_k 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 lymphomas^ref. 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 deleted^ref, 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 complexes^ref. 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 V_H 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 pressure^ref. A high R:S ratio which was significantly different from the one expected for random mutations was seen only in the V_H 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 V_L domains. A low number of R mutations was also observed in the FWRs of most of the V_H and V_L 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 combination^ref, 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 mutations^ref. 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 MC^ref. 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 counterparts^ref. 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 mechanisms^{ref1, ref2, ref3}. Because HCV RNA can be detected in the peripheral blood mononuclear cells of patients with chronic hepatitis C^ref, 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 minks^ref
HCV RNA is found in :
• 23.8% in Hungary^ref
• 20.8% in Sicily, Italy^ref
• 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 lymphomas^ref
• 0% in West of Scotland^ref
• 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 subjects^ref
• ^ref
• ^ref
• ^ref
Oligoclonal lymphoproliferative disorders^ref and chromosomal translocation^ref 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 cells^ref. 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 V_H 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 V_H genes exhibited most of the features characteristic of the somatic hypermutation of the Ig gene in normal B cells^ref. 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 V_H gene probably represents the enhancement of the normal somatic hypermutation mechanism of the V_H gene, which typically affects genomic sequences within 2 kbp downstream from the transcription initiation sites of the Ig gene^ref. Area B of BCL-6 had dG·dC-biased mutations, which preferentially targeted the RGYW motif, similar to the pattern seen for V_H. 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 V_H 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 V_H and area B of BCL-6. These results suggest that HCV infection activates both the normal hypermutation mechanism of the V_H 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 DSBs^{ref1, ref2} or ssDNA breaks^ref, possibly mediated by error-prone DNA polymerases^{ref1, 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 dC^ref with a strong bias toward dG·dC base pairs^ref) (but not mismatch repair enzymes (pms1, pms2, mlh1, msh2, msh3, and msh6), which may play a role in the error-prone repair process^ref), 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 cerevisiae^ref. These mutations depend on the error-prone polymerase z, which has the DNA-damage bypass activity^ref. 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 fidelity^ref. Polymerase i also induces somatic hypermutation in Ig genes in the BL2 cell line^ref. Both error-prone polymerases z and i could be activated as a result of B cell receptor stimulation^{ref1, 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 information^ref. 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 virus^ref. 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
• Italy^ref : a profound regional variation in findings that parallels levels of HCV endemicity : 32% in Pisa^ref, 22.3^ref-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 Rotondo^ref, 22.4% in B-LPD (B-NHL, MM, MGUS, B-CLL) in Naples (61.5% in WM)^ref, 37% in Piacenza^ref, 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 Pisa^ref, 37% in Modena^ref2, 22.4% in Naples^ref3)
• 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 subjects^ref.
In contrast, the majority of studies from nonendemic areas elsewhere in Europe (4.3% in Ulm, Germany^ref, 2.0% in France^ref, ?% in The Netherlands^ref, 0% in Newcastle upon Tyne, UK^ref) or from North America (0% in Columbia, USA^ref, 0% in Toronto, Canada^ref) 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 comparison^{ref1, 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 population^ref. 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 cohort^ref
• 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 tumors^ref
• because incident HCV infection is uncommon after age 40^ref, 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 life^ref.
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 association^{ref1, 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 HCV^ref.
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-J_H recombination characteristic of follicular lymphoma^ref, and successful antiviral therapy may decrease recombination frequency^ref
• immunocytoma (IC)–lymphoma (CD5^-19⁺IgM⁺) / lymphoplasmacytoid lymphoma^{ref1, ref2, ref3, ref4} (40-76%^ref) : in 3 studies^{ref1, 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 MC^ref. In the USA study^ref, 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 report^ref 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 lymphoma^ref.
• primary hepatosplenic diffuse large-B-cell lymphomas
• marginal-zone lymphomas :
• nodal MZL^{ref1, ref2}
• splenic MZL/splenic lymphoma with villous lymphocytes (SLVL)
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 lymphomas^ref.
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 malignancy^ref
• Hodgkin's lymphoma (4% in Italy^ref; 9% = 12-fold higher prevalence of infection in Hungary^ref)
• no HCV reactivation was found in malignant hematologic diseases, such as NHL or HD^{ref1, 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 infection^ref. 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 apoptosis^ref. Recently, an increased rate of clonal proliferation of B cells and bcl-2 translocation and overexpression in PBMCs of patients with HCV infection was demonstrated^{ref1, 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 immunocytomas^ref, 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 stimulus^ref.
• 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 hypothesized^ref. 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 protein^ref or core protein^ref 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 MC^ref. 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 V_H1–69 in patients with lymphoproliferative disorders^ref. Sequence analysis for these regions demonstrated somatic hypermutation, evidence for affinity maturation under constant antigen stimulation^ref. 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 V_H1–69^ref and B-cell receptor from one HCV-associated lymphoma patient bound to E2^ref. CD81 is a cellular ligand for E2 envelope on the surface of lymphocytes^ref. CD19/CD21/CD81 complex along with B-cell receptor stimulation is considered to activate proliferation of lymphocytes^ref. 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 NHL^ref. CD81-E2 cross-linking activates Lck through raft aggregation and thus leads to enhanced TCR signaling^ref. Although further clarification is required, the positive association we observed for TNHL may be explained in part by this mechanism^ref
• 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 results^ref. In Sweden the risk of NHL and MM was significantly increased in the HCV stratum with more than 15 years of infection^ref
• anti-HCV positivity in 69%, 11% and 4.3% of the EMC, MM and B-CLL samples tested^ref. 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%, respectively