Structure

HCV is a small, enveloped virus with a single stranded RNA genome of positive (coding) polarity (Fig. 52.1). HCV has been visualized in the plasma of HCV-infected individuals as small (50 nm), round particles. The surface of the virus particle contains a number of small projections thought to be formed from complexes of the virally encoded envelope glycoproteins, E1 and E2. The RNA genome is approximately 9400 bases in length, of which over 98% contains protein-coding sequence. In common with many other small RNA viruses, the gene sequences of HCV are translated in a single block to produce a large (>3000 amino acid) polyprotein. During and after translation, proteases cleave this precursor into a total of 9 mature proteins, which are involved in virus replication (NS2–NS5B), or form structural components of the virus particle (core, E1 and E2).

Fig. 52.1 Organisation of HCV genome, showing (A) the structural (core, E1 and E2) and non-structural genes (NS2–NS5B), and (B) the proteins produced from them by proteolytic cleavage of the translated polyprotein. Properties and functions of the HCV proteins, where known are summarised below. ER, endoplasmic reticulum; UTR, untranslated terminal region.

Replication

Information on the replication of HCV and its assembly into infectious virus particles remains limited because it still remains problematic to culture the virus in vitro. However, investigation of individual HCV-encoded proteins and comparison with related viruses have allowed the functions of most to be ascertained (Fig. 52.1). For example, NS5B is the RNA polymerase required by HCV for replication of its genetic material through a negative-stranded intermediate. NS3 contains protease and helicase activities; the former is required for the majority of cleavage reactions in the processing of the polyprotein after translation. The three proteins at the left hand end of the genome are structural proteins. Multiple copies of the core protein assemble to form a nucleocapsid that packages the viral RNA, while E1 and E2 are synthesized on internal membranes within the infected cell, become heavily glycosylated and form the HCV envelope as the nucleocapsid buds out of the cell.

The extreme ends of the HCV genome are non-coding, and play a number of roles in the transcription and translation of the virus genome. Details of the factors that initiate and regulate HCV transcription remain unclear at present, although the non-coding ends are likely to be involved in protein complexes with NS5B, NS3 and other HCV and cellular proteins to mediate binding and initiation of RNA copying. The 5′ untranslated region (5′UTR) plays an important role in directing the translation of the HCV polyprotein from an internal (methionine) codon. RNA secondary structure formation allows direct binding of the host cell ribosome to internal sequences in the 5′UTR (known as the internal ribosomal entry site, or IRES), bypassing the conventional attachment to the (capped) end of the RNA molecule, as is found in the translation of cellular mRNAs.

Hepatocytes are the principal sites of HCV replication in vivo, although there may be extrahepatic sites of replication including haemopoietic cells such as B lymphocytes, and stem cells in the bone marrow. The entry mechanism of HCV into hepatocytes (and other cell types) is complex and incompletely understood. A variety of cellular proteins are involved in the initial attachment of HCV to the cell (e.g. the LDL receptor, heparin sulphate, DC-SIGN, L-SIGN). Interaction with other host factors such as CD81, the scavenger receptor B-1 (SRB-1), claudin 1 and occludin leads to viral internalisation through endocytosis of the bound virion in a clathrin-coated endosome, and fusion of viral and endosomal membranes. In vivo, this likely occurs on the basolateral membrane surface of hepatocytes to allow concentration of the virion. On entering the cytoplasm, the RNA genome is released from the viral capsid and translated. The newly synthesized RNA replicating and unwinding enzymes, such as NS5B, NS5A and NS3, subsequently initiate transcription of the genomic RNA into a full-length negative-polarity copy, which in turn acts as the template for the production of multiple positive-stranded RNA copies. These can be used for further rounds of transcription, or used for the production of further viral proteins. Factors that regulate negative- and positive-strand synthesis and the use of the latter transcripts for translation remain undetermined, and are likely to be complex.

Replication of HCV takes place in membranous webs associated with the NS4B protein and cellular endoplasmic reticulum (ER). The core protein, however, remains within the cytoplasm after cleavage from E1, E1 and E2 are embedded in the ER membrane, and their extracellular domains are glycosylated. Details of the subsequent stages of capsid assembly and maturation, the insertion of HCV RNA, and the budding of HCV through the ER into extracytoplasmic space and release from the cell await further studies.

Classification

HCV shows the greatest similarity in structure, size and genome organization with the flaviviruses. Together, these viruses are classified as members of the Flaviviridae (Table 52.1), currently divided into four named genera. The Hepacivirus genus contains HCV, a recently discovered canine homologue of HCV (canine hepacivirus; CHV) and GB virus-B, originally isolated from a captive tamarind, in which it causes acute hepatitis and liver disease similar to that of HCV in humans. Both GBV-B and CHV are currently under evaluation as possible experimental models for HCV vaccines and antiviral treatment.

Table 52.1 Members of the Flavivirus family

Human pegivirus (HPgV, originally described as GB virus C or hepatitis G virus) is classified as a member of the recently described Pegivirus genus. This virus is widely distributed in humans and was originally thought to be a further agent involved in post-transfusion and chronic hepatitis of unexplained aetiology. This association has subsequently been disproved, and infection appears to be entirely asymptomatic, despite being persistent in a significant proportion of those it infects. Its genome shows a number of similarities to that of HCV, including a 5’UTR that has similar ribosomal binding and internal initiation (although a different RNA secondary structure), while the coding region contains homologues to HCV structural and non-structural proteins. However, it lacks a protein corresponding to the core protein of HCV that forms the nucleocapsid. Viruses similar to HPgV are widely distributed in a range of non-human primate species (simian pegiviruses; formerly described as GBV-A) and in bats (bat pegivirus; originally described as GBV-D). The pestivirus genus contains viruses which are structurally similar to HCV and infect a number of domestic and wild species of ruminants such as pigs, sheep and cows.

Genetic variation

Nucleotide sequences of HCV frequently show substantial differences from each other. This has led to the current genotypic classification of HCV, in which variants from a variety of geographical locations can be classified into 6 main genotypes and a very rare genotype 7, and a number of subtypes (Fig. 52.2). Genotypes show approximately 30% sequence divergence from each other, differences that greatly modify their antigenic properties, and their biology (see Treatment).

Fig. 52.2 Comparison of complete genome sequences of the common genotypes of HCV plotted as a phylogenetic tree, showing the 6 main genetic groups (genotypes 1–6) and a number of the commoner subtypes (A, B, C). The distribution of the different genotypes in the principal risk groups for HCV infection is indicated.

Some HCV genotypes (types 1a, 2a, 2b) show a broad worldwide distribution, while others such as type 5a and 6a are only found in specific geographical regions (South Africa and South East Asia respectively). HCV infection in blood donors and patients with chronic hepatitis from countries in Western Europe and the USA frequently involves genotypes 1a, 1b, 2a, 2b and 3a. The relative frequencies of each may vary geographically, such as the trend for more frequent infection with type 1b in southern and Eastern Europe, and the association of genotype 1a and 3a infection with infection through drug use. HCV genotypes can be identified by analysis of sequences from the 5′UTR or from coding regions. Methods for rapid genotyping of HCV have been developed, and play a role in the pre-treatment assessment of patients receiving antiviral treatment (See Treatment).

Virus stability

HCV is inactivated by exposure to chloroform, ether and other organic solvents and by detergents. The effectiveness of a number of virus-inactivating procedures have been demonstrated by studies of the infectivity of products manufactured from plasma such as the factor VIII and IX concentrates used to treat haemophiliacs. For example, dry-heat treatment at 80°C or wet-heat treatment at 60°C, organic solvents (n-heptane) and detergents have been shown to efficiently remove HCV infectivity.

Direct detection methods

While current screening and supplementary serological tests will detect the vast majority of infections, there remains a considerable window period in HCV infection between exposure and development of antibody detectable by the best current ELISAs (Fig. 52.3). Also, the presence of antibodies to HCV does not allow distinction between past, cleared infection and current infection. For these reasons, further direct methods for the detection of HCV antigens or RNA sequences are required for the effective diagnosis of HCV infection in acute hepatitis, for diagnosis of HCV infection in immunosuppressed individuals who do not mount a detectable antibody response, and to identify current, as opposed to past, infection. Genome detection methods have also been widely adopted in addition to serological tests for the routine screening of blood donors for HCV in most Western countries.

Fig. 52.3 Virological and biochemical markers of acute HCV infection. HCV RNA (red line) and abnormal ALT levels (blue line) appear approximately 4–6 weeks after exposure to HCV in a typical individual. The subsequent development of chronic hepatitis is indicated by persistent viraemia and by fluctuating abnormal ALT levels. Antibody to HCV first appears after the onset of acute hepatitis, in this example leading to ‘window periods’ of around 100–150 days for the first generation serological assay (containing only NS3 and NS4 proteins) and approximately 60–80 days for second and third generation assays (containing additional NS3, NS5 and core proteins).

Direct detection methods are based upon either the detection of HCV RNA sequences by the polymerase chain reaction (PCR) or other nucleic acid amplification methods, or of viral antigens by ELISA. Reverse transcriptase PCR is the most commonly used method for direct detection of HCV. Diagnostic PCR methods are commercially available and are capable of high sensitivity and specificity for the detection of HCV RNA sequences in plasma (or liver biopsy) specimens. Alternative, non-PCR-based methods, such as transcript-mediated amplification, have recently been developed and provide comparable sensitivity. An alternative to nucleic acid detection is the use of ELISA-based methods for the detection of HCV core protein in plasma, and more recently combined antibody/antigen detection methods.

Direct virus detection is the only reliable method of monitoring the effect of treatment with antiviral drugs in patients with chronic infection since normalization of liver biochemistry is not always associated with virus clearance, and chronic HCV infection with associated disease may arise in patients with normal liver functions tests. Quantitation of the virus genome titre is now increasingly possible with commercial tests based on real-time PCR, other amplification methods or hybridization, and is used as a predictor for response to antiviral therapy (see Treatment).

Acute hepatitis

Exposure to HCV usually results in an asymptomatic infection without jaundice, with only 25–50% patients clearing the infection spontaneously – the remainder become chronically infected. Most studies have reported an interval of around 8 weeks to the development of abnormal liver function tests (such as alanine aminotransferase (ALT) levels) (Fig. 52.3). Clinically, hepatitis caused by HCV is indistinguishable from that caused by other hepatitis viruses; jaundice may develop, but more usually symptoms are non-specific such as fatigue, anorexia and nausea. Viraemia can be detected in the early stages of acute hepatitis, appearing at the same time or slightly earlier than abnormal ALT levels, while seroconversion for antibody may be delayed for several weeks or months after the onset of hepatitis. Histologic features of acute HCV are similar to those associated with acute HAV and HBV infection; liver biopsy is rarely indicated to make a diagnosis of acute HCV infection.

Chronic hepatitis

The frequency of chronic infection following exposure to HCV is between 50 and 75% (Fig. 52.4). Chronic infection with HCV is generally associated with persistent and progressive hepatitis, with fluctuating or continuously abnormal ALT levels. Although viraemia is invariably detected in patients with chronic hepatitis, there is no correlation between level of viraemia and the severity of liver disease, ALT levels or other biochemical abnormalities associated with hepatitis.

HCV infection causes a range of characteristic histological changes in the liver, although few allow a specific diagnosis of HCV infection to be made. These include lymphoid follicles within the portal tracts, a dense periportal inflammatory process, bile duct damage, and lobular hepatitis, with lymphocyte infiltration within sinusoids surrounding the hepatocytes. Chronic infection with genotype 3 HCV is particularly associated with the development of fatty change in the liver (steatosis). There are a variety of ‘scoring systems’ or histological activity indices (HAI) available (e.g. Knodell, Ishak, Metavir), designed to assess the degree of liver damage as seen on biopsy.

The percentage of chronically infected individuals who progress to cirrhosis and liver failure is not known. When chronic hepatitis does progress to clinically significant liver damage, then progression is almost invariably very slow, although faster progression may be associated with risk factors such as alcohol ingestion, older age, and immunodeficiency. Particularly aggressive HCV-associated liver disease has been observed in immunosuppressed organ transplant recipients, and in patients with inherited immunodeficiency states. Cirrhosis is rarely observed within 10 years of infection, but around 20% of infected patients have cirrhosis after 20 years follow-up. Cirrhosis may be complicated by liver failure (decompensated cirrhosis), manifested as jaundice, portal hypertension and variceal bleeding; these manifestations of liver failure are shared with other forms of cirrhosis. Hepatocellular carcinoma (HCC) frequently complicates chronic hepatitis C, although HCC is rare within 15 years of initial infection. In many Western countries such as Spain and Italy, and, in Japan, HCV infection is found in 60–90% cases of HCC.

Extrahepatic manifestations

In a minority of infected patients, HCV may be responsible for extrahepatic clinical manifestations and disease. These include certain types of vasculitis and glomerulonephritis caused by immune complex deposition. Associations between HCV infection and Sjogren’s syndrome, essential mixed cryoglobulinaemia and membranoproliferative glomerulonephritis type 1 have been suggested.

Prevention

Screening of blood donors has proved to be effective at preventing transmission of HCV infection through blood transfusion. A combination of blood donor screening and virus inactivation has virtually eliminated HCV transmission by blood products such as clotting factor concentrates and immunoglobulins.

The main continuing risks for HCV transmission are injecting drug use and the use of unsterile needles for medical and dental procedures, tattooing and other percutaneous exposures. Much of this could be prevented by education, greater availability of disposable needles, and for drug users by needle exchange programmes. Many of the public health measures adopted to prevent transmission of HIV by parenteral routes will assist efforts at controlling HCV.

Immunization

The development of a vaccine for HCV faces a series of formidable obstacles, amongst which viral heterogeneity, and the difficulty in evaluating candidate vaccines in suitable animal models, are the most acute. Recombinant envelope proteins (E1 and E2) have been shown to induce a short-lived specific anti-E1 and E2 response in immunized chimpanzees, and transient protection from challenge with the same virus strain. There is, however, little prospect of an effective vaccine for human use in the coming decade.

Infection with HCV is a growing medical problem worldwide. A combination of public health preventative measures, improved diagnosis, screening, antiviral treatment and immunization will undoubtedly all be required to combat its spread in the future.