Genus: Hepacivirus

Distinguishing features

Hepatitis C virus (HCV) is transmitted between humans, principally via exposure to contaminated blood. There is no known invertebrate vector. Hepaciviruses differ from members of the genera Flavivirus and Pestivirus by their limited ability to be propagated in cell culture, since only a few adapted strains, including JFH1, efficiently infects the only susceptible cultured cell line, human hepatoma cell line (Huh7). In the HCV precursor protein, the NS2-3 junction is auto-catalytically cleaved by Zn-dependent NS2-3 protease activity.

Virion

Morphology

Virions are about 50 nm in diameter, as determined by filtration and electron microscopy. They are spherical in shape with a lipid envelope, as determined by electron microscopy and inactivation by chloroform. The viral core is spherical and about 30 nm in diameter. Detailed structural properties have not been determined.

Physicochemical and physical properties

Virion Mr has not been determined. Buoyant density in sucrose is predominantly about 1.06 g cm⁻³ for virus recovered from serum during acute infections while more dense forms (ca. 1.15–1.18 g cm⁻³) predominate when recovered from the serum of chronically infected individuals (Thomssen et al., 1993). Lower density banding results from physical association of the virion with serum very-low-density lipoproteins (VLDLs)(Agnello et al., 1999).  Higher density virions are those bound to serum antibodies. A buoyant density range in isosmotic iodixanol gradients of 1.01–1.10 g cm⁻³ has been measured for HCV recovered from hepatoma cells infected with HCV. The S_20,w is equal to or greater than 150S. The virus is stable in buffer at pH 8.0–8.7. Virions are sensitive to heat, organic solvents and detergents (Feinstone et al., 1983).

Nucleic acid

Virions contain a single positive-sense, infectious ssRNA (Figure 1.Hepacivirus). The genome is about 9.6 kb. The 5′-NCR a type IV IRES (Honda et al., 1999) of approximately 340 nt in length. . The 3′-NCR contains a sequence-variable region of about 50 nt, a polypyrimidine-rich region (average of 100 nt), and a highly conserved 98 nt  3′-terminal region with three stem-loop RNA secondary structures (Kolykhalov et al., 1996). There are at least two seed sites in the HCV 5′-NCR for the liver abundant microRNA miR-122; this virus-host interaction is required for efficient HCV replication (Jopling et al., 2005).

Proteins

The HCV virion comprises at least three proteins: the nucleocapsid core protein C (p19-21), and two envelope glycoproteins, E1 (gp31) and E2 (gp70). An additional protein, p7 (believed to have properties of an ion channel protein important in viral assembly) is incompletely cleaved from a precursor of E2 to yield E2-p7 and p7 (Shanmugam and Yi 2013) but it is not known whether these are virion structural components. In GB virus B, a corresponding protein, p13, is cleaved to p7 and p6 proteins (Takikawa et al., 2006). The two envelope glycoproteins can associate as non-covalent heterodimers; recent data, however, indicate that they are covalently linked in virions (Vieyres et al., 2010). Nonstructural proteins include NS2, a 21 kDa protein that, before cleavage, is part of a Zn-dependent cysteine protease that bridges NS2 and NS3 and mediates autocatalytic cleavage of the NS2/NS3 junction, and is involved with virus assembly and release, NS3, a 70 kDa protein with additional serine protease, helicase and NTPase activities; the NS3 protease cleaves the remaining junctions between nonstructural proteins, NS4A , a 6 kDa cofactor essential for trans NS3 serine protease activity, NS4B, a 27 kDa protein that induces a membranous replication complex at the endoplasmic reticulum, NS5A, a serine phosphoprotein of unknown specific function, but critical for viral replication and assembly, that exists in 56 and 58 kDa forms, depending on the degree of phosphorylation, and NS5B, a 68 kDa protein with RdRp activity.

Lipids

Virions have a lipid bi-layer envelope. Historically, based on the removal of the viral envelope and loss of infectivity following exposure to solvents or detergents (Feinstone et al., 1983), the presence of lipids was inferred. Recently, it has become apparent that the host lipid metabolism plays a critical role in the virus life cycle.

Carbohydrates

The E1 and E2 glycoproteins contain numerous N-linked glycosylation sites, and carbohydrate is associated with the products of these two HCV genes. E1 and E2 are transmembrane, type I glycoproteins, with C terminal retention signals that anchor them within the lumen of the endoplasmic reticulum. These signals are apparently masked when budding occurs allowing the virion to move through the secretory pathway. Recent data obtained in culture systems indicate that N-linked glycans of E1 remain in the high-mannose chains lacking complex carbohydrate, whereas those of E2 are modified (Op De Beeck et al., 2004). Glycosylation influences E1–E2 heterodimer formation, folding and assembly and the release of virions (Meunier et al., 1999).

Genome organization and replication

The genome contains a single large ORF encoding a polyprotein of about 3000 aa (Figure 1.Hepacivirus). The gene order is 5′-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3′. Immediately downstream of the three structural proteins (C, E1, E2) is a small protein, p7 in HCV, p13 in GB virus B, followed by the nonstructural proteins in the 3′ portion of the ORF. Replication occurs in association with intracytoplasmic membranes. Replicative forms of viral RNA have been detected in liver tissue. The genomic RNA is translated into a polyprotein that is rapidly processed both co- and post-translationally by host and viral proteases. Translation initiation occurs via an IRES within the 5′-NCR. Translocation of the structural glycoproteins to the endoplasmic reticulum probably occurs via internal signal sequences. Cleavage of the structural proteins is mediated by host cell signal peptidases, and signal peptide peptidase. With the exception of the p7/NS2 signalase cleavage, viral proteases cleave all non-structural protein junctions. Virus assembly is believed to occur by budding into vesicles from the endoplasmic reticulum.

Figure 1.Hepacivirus. Hepacivirus genome organization (not to scale) and polyprotein processing. For members of the species Hepatitis C virus, the RNA is about 9.6 kb. The 5′-NCR is about 340 nt, the 3′-NCR is about 250 nt, and the ORF is about 9 kb. HCV has a p7 protein between E2 and NS2. The host and viral proteases involved in cleavage of the polyprotein are indicated. The cleavage by host signal peptide peptidase (at the C-terminus of the core protein) is indicated by a green arrow; the cleavages by host signal peptidase (remaining sites) are indicated by filled arrows. The locations of the NS2-3 protease, NS3 protease, NS3 RNA helicase and NS5B RdRp are indicated by P′, P″, H and R, respectively.

Antigenicity

Virus-specific antibodies to recombinant-expressed structural proteins (C, E1 and E2) and nonstructural proteins (principally NS3, NS4 and NS5) have been detected in individuals infected with HCV. Both linear and conformational epitopes are believed to be involved in the humoral immune response of the host to infection. Significant antigenic diversity throughout the genome is reflected in heterogeneity in the humoral immune response. In HCV, high variability is found in the N-terminal 27 aa of E2 (hypervariable region 1; HVR1). The HVR1 contains an HCV neutralization epitope and escape variants of HVR1 are positively selected by the host humoral immune response (Mondelli et al., 2003). Other neutralization epitopes have been identified in E2 outside of HVR1 (Keck et al., 2004, Johansson et al., 2007), and at least one neutralization epitope has been identified in E1 (Keck et al., 2004). Cell-mediated immune responses to all HCV proteins have been detected (Klenerman and Thimme 2012); it is believed that such responses are associated with amelioration or resolution of infection. With the development of intra- and intergenotypic genotype 1-7 JFH1-based recombinant viruses with strain-specific structural proteins, it is now possible to carry out in vitro virus neutralization assays to address the antigenic diversity of HCV (Gottwein et al., 2011).

Biology

Host range

Humans are the natural host and apparent reservoir of hepatitis C virus although the virus can be transmitted experimentally to chimpanzees. No other natural host has been identified. The natural host for GB virus B is not known. Other hepaciviruses have been detected in Old World primates (Sibley et al., 2014), cows (Baechlein et al., 2015), horses (non-primate hepacivirus; NPHV)(Burbelo et al., 2012) and a range of rodent and bat species (Firth et al., 2014, Kapoor et al., 2011, Kapoor et al., 2013, Drexler et al., 2013, Quan et al., 2013). The host specificity of these variants for their mammalian hosts is undetermined, although at least one rodent species, the bank vole (Moyodes glareolus) can be infected with two highly divergent hepacivirus variants (RMU10-3382/GER/2010 and NLR07-oct70/NEL/2007)(Drexler et al., 2013), indicative of a degree of cross-species transmission.

Transmission

Hepatitis C virus is transmitted almost exclusively by parenteral exposure to blood, blood products and objects contaminated with blood. Effective screening of blood donors and implementation of inactivation procedures have virtually eliminated the transmission of HCV via blood and blood products, but other routes of exposure, principally via blood-contaminated syringes, are now the most important recognized risk factors. Sexual and mother-to-child transmission has been documented but is relatively uncommon. Other routes of transmission are suspected for hepaciviruses infecting non-human primates, rats, bats and horses.

Geographical distribution

HCV has a worldwide distribution with about 3% of the world population infected with HCV, equivalent to 170 million chronic infections, with 3-4 million new infections each year. Antibody prevalences are 0.1–2% in developed countries but as high as 20% in some developing countries, possibly reflecting the use of contaminated needles and syringes.  Horses infected with non-primate hepacivirus have been reported from four continents with viraemia frequencies ranging from 3-10%, indicative of a wide geographical distribution (Burbelo et al., 2012, Lyons et al., 2012, Figueiredo et al., 2015, Lu et al., 2016, Matsuu et al., 2015, Pfaender et al., 2015). 

Pathogenicity

HCV infections range from subclinical to acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Persistent infection occurs in 60–80% of cases and in about 20% of the cases, the infection progresses over many years to chronic active hepatitis and cirrhosis. Patients with liver cirrhosis have an approximately 5% risk per year of developing hepatocellular carcinoma.

Persistent HCV infection has been epidemiologically linked to primary liver cancer, cryptogenic cirrhosis and some forms of autoimmune hepatitis. Extrahepatic manifestations of HCV infection include mixed cryoglobulinemia with associated membrano-proliferative glomerulonephritis and, possibly, porphyria cutanea tarda, Sjögren’s-like syndromes and other autoimmune conditions.

Similarly to HCV infections in humans, GB virus B causes hepatitis and replicates in the liver of tamarins and owl monkeys, but infection is self-limited and has not been demonstrated in humans or chimpanzees. Only one strain of GB virus B has been identified to date, in contrast to hundreds or thousands of often quite divergent variants of HCV. The pathogenicity of other hepaciviruses infecting non-human primates, rodents, bats and horses is poorly characterized although the presence of miR-122 sites in several of these predicts hepatotropism. Recent evidence suggests that infection of horses with NPHV may be associated with mild inflammatory liver disease (Pfaender et al., 2015).

Cell tropism

HCV has been reported to replicate in several cell lines derived from hepatocytes and lymphocytes, but virus growth has only been sufficient for practical application of these systems in a human hepatoma cell line, Huh7 cells and derivatives thereof. In vivo, HCV replicates in hepatocytes and possibly lymphocytes. The cellular or tissue tropism of other hepaciviruses is poorly characterized although there is evidence that GB virus B and NPHV are hepatotropic. The presence of binding sites for miR-122 (Jopling et al., 2005) in most described hepaciviruses is suggestive of hepatotropism, given the restriction of expression of this miRNA to liver tissue.

Species demarcation criteria

Not applicable.

Member Species

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.
Download GenBank/EMBL query for sequences listed in the table here.

The genus Hepacivirus comprises a single species, Hepatitis C virus. However, members of the species can be classified into seven genetic groups (termed genotypes; see table above), based upon the genome-wide heterogeneity of isolates recovered throughout the world (Smith et al., 2014). These differ from each other by about 30–35% at the nt level (Simmonds et al., 2005). Within each genotype, there are a number of subtypes, differing from each other by about 15–25% at the nt level. Although genotypes are distinct genetically, discrimination of subtypes is less clear, particularly in areas of high diversity such as sub-Saharan Africa and Southeast Asia. Because systematic serological typing by virus neutralization has not been performed to date, and because major genotypes do not have any other taxonomic characteristics except, in some cases, geographic distribution and differences in treatment response, the seven genetic groups of HCV comprise one species. Complete or near complete genomic sequences have been obtained from each of the seven genotypes of HCV, and replication competence has been demonstrated by intrahepatic chimpanzee inoculation of RNA transcripts from cDNA clones of genotypes 1a [strains H77 (AF011751 and AF009606), SF9 A (AF271632), HC-TN (EF62149)], 1b [Con1 (AJ238799), HCV-N (AF139594)], 2a [HC-J6CH (AF177036) and JFH1 (AB047639)], 3a [S52 (GU814264)] and 4a [ED43 (GU814265)].

Related, Unclassified Viruses

GBV-B and other hepaciviruses infecting non-human hosts are highly divergent in sequence from each other and from HCV and will likely justify further species assignment within this genus. GB virus B comprises one isolate, and its replication competence has been demonstrated by intrahepatic inoculation of RNA transcripts from a cDNA clone into tamarins (AF179612). Recently, the replication competence of NPHV has been demonstrated by intrahepatic inoculation of RNA transcripts from a cDNA clone into horses (Scheel et al., 2015). Phylogenetic analysis of other hepaciviruses demonstrates the diversity of viruses assigned to this genus and the existence of further candidate species corresponding to the numbered lineages shown in Figure 1.Flaviviridae. The ICTV Executive committee has recently approved proposals for the creation of additional species in the genus as described in (Smith et al., 2016). The chapter will be updated to include these new assignments once the proposals are ratified by the ICTV (estimated March 2017).

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.
Download GenBank/EMBL query for sequences listed in the table here.