This web text will specifically focus on Hepatitis C Virus.

1	Background / History

• Hepatitis C Virus, (HCV) was discovered in 1989 after major research into post-transfusion hepatitis (PTH).
• Hepatitis A, (HAV) and Hepatitis B, (HBV) can cause chronic hepatitis via blood and blood products.  However, another hepatitis causing agent was identified and named, non-A, non-B hepatitis, (NANBH) by the exclusion of the other main viral causes of hepatitis. NANBH was later named HCV.
• HCV is extremely difficult to culture and was classified into the Flaviviridae family. Molecular techniques gave rise to genome organisation as well as nucleic acid composition.

 

2	Classification

• HCV has a similar genome size to other Flaviviridae members. The HCV genome is 9379 bases in length, compared to the Yellow Fever Virus genome size of 10,862 bases.
• The HCV envelope contains viral encoded glycoprotein, (E1), as do other Flaviviridae.
• HCV polyprotein is cleaved by host cell and viral proteases which also occurs in other members of the Flaviviridae.
• There is no close HCV sequence similarity to other viruses in the family apart from two regions of conserved amino acid residues. The first is the NS5b protein which contains glycine-aspartate-aspartate, (GDD) which is the associated with the active site in virus RNA-dependent RNA polymersases, (RdRp) of positive strand RNA viruses. The second is the HCV NS3 similarity to virus helicase polypeptides.

Genotypes of HCV

• HCV is heterogeneous, a word derived from the Greek, heteros, meaning different, and genos, meaning kind. The sequences of HCV are heterogeneous and this consist of different elements or parts.
• Differences in nucleotide sequences for HCV were shown in studies where Japanese strains of HCV were compared to strains of HCV from the USA.
• All currently known isolates of HCV can be divided into six phylogenetically distinct groups. Some virologists claim that the HCV genome sequences can be classified into eleven genotypes, (websites 1-4), although this has not been universally adopted.
• Genome organisation - compared to nucleotide sequence differences between variants, the HCV genome shows small differences between genotypes.
• The HCV genome consists of positive polarity, single-stranded RNA.
• Approximately 96% of the HCV genome codes for virus proteins, equating to 9,033 bases and the remaining bases at the 5' and 3' ends of the genome are untranslated regions, (UTR's). Table 1 shows a schematic representation of the HCV RNA genome.
• UTR's are thought to be responsible for controlling and facilitating viral transcription and replication.
• The HCV genome has an open reading frame, (ORF) of 9,033 bases which codes for an individual precursor protein, 3,011 amino acids in length.
• The precursor protein is cleaved by both host and viral proteases to give rise to three structural proteins and six non-structural proteins.
• Structural proteins are processed from the precursor protein or gene by a host cell-encoded signal peptidase.
• The core protein, P22 is around 192 aa long, being the most conserved of all the structural proteins.
• E1 and E2 are both membrane associated glycoproteins and are 192 and 426-432 amino acids long, respectively. E1 is known as GP35 and E2 is known as GP76.
• E1 and E2 are variable but E2 is more so towards its N terminus which has thus been named hypervariable region 1, (HVR1).
• All the non-structural proteins are cleaved from the precursor protein by viral-encoded proteases. Functions are highlighted in Fig. 1.

Table 1: HCV genes and proteins

Nucleotide	0			~2,000		~4,000		~6,000		~8,000	9,379
Gene	5' UTR	Core	E1	E2/NS1	NS2	NS3	NS4a	NS4b	NS5a	NS5b	3' UTR
Protein product		P22	GP35	GP76	P23	P70	P8	P27	P58	P68
Protein function		Structural	Structural	Structural	Non structural	Non structural	Non structural	Non structural	Non structural	Non structural
Virion associated		Yes	Yes	Yes	No	No	No	No	No	No
Recombinant proteins (Ag)		C-22-3			C-200	C-33-C C-200	C-100-3 C-200	5-1-1 C-100-3 C-200	NS5 (2054-2995)	NS5 (2054-2995)

 

Fig.1: HCV protein functions

Protein	Function
Core	Associated with formation of virus nucleocapsid showing RNA-binding activity
E1	Membrane-anchored glycoprotein with a number of N-linked glycosylation sites
E2/NS1	Membrane-bound glycoprotein showing hypervariable region. Homologous region in other Flaviviruses could be non-structural so denoted as E2/NS1
NS2	Protease dependent upon zinc
NS3	Multi-functional protein with protease and possibly helicase activity
NS4a	Co-factor for NS3 protease
NS4b	Function not known
NS5a	Function not known
NS5b	RNA-dependent RNA polymerase necessary for gene replication

 

3	Morphology

• Obtaining electron micrographs of HCV is difficult, although HCV has been visualised by electron microscopy by staining with 2% phototungstic acid at pH 6.5. HCV shows fine spike-like projections around the edge, 6nm in length. A diagrammatic representation of the proposed structure of HCV is shown in Fig.2
• The virus is 55-65nm in diameter.

Fig.2: The proposed structure of HCV

• Electron micrographs so far taken of HCV do not show internal structure in acceptable detail, however, it can be assumed with some confidence, that the nucleocapsid containing the RNA genome closely resembles other viruses in the Flaviviridae family.
• The HCV is surrounded by a lipid envelope containing the E1 and E2 glycoproteins.
• E1 and E2 show numerous sites for N-linked glycosylation, four sites in E1 and eleven sites in E2. Possibly with the aid of large CHO groups viral infectivity can be neutralised.

 

4	Other Properties of HCV

• HCV density - unknown owing to HCV's association with beta-lipoprotein and antibody in serum.
• Inactivation - organic solvents, for example, ether and chloroform render HCV inactive. Dry heat at 80^oC and wet heat treatment at 60^oC are also effective against HCV.
• HCV culture - HCV is extremely difficult to culture and shows no cytopathic effect, (CPE), plus liberates very small number of virus from cells.
• Cellular receptor - The HCV cellular receptor still has not clearly been clearly identified.
• Cell entry - It is also unclear how HCV enters the host cell.
• Replication - This occurs in the cell cytoplasm with viral assembly in the rough endoplasmic reticulum, (RER) and Golgi apparatus.
• Viral release - It is not well documented how this happens although the nucleocapsid is likely to be released from the host cell by budding through the lipid membrane.

 

5	Clinical Disease

Acute hepatitis

• Up to 95% of patients infected with HCV are asymptomatic.
• Jaundice is not normally associated with HCV.
• It can take up to eight weeks to develop hepatitis after exposure to HCV, whereupon alanine aminotransferase, (ALT) levels can be raised.
• Hepatitis caused by HCV is clinically indistinguishable from hepatitis caused by other viruses.
• Non-specific symptoms of hepatitis caused by HCV include nausea, anorexia and fatigue (jaundice can sometimes occur).
• PCR - used to detect viraemia in the early stages of disease.
• Antibody to HCV can take from seven weeks to a number of months to appear after the onset of hepatitis.
• Histology - liver biopsy cannot really be used to diagnose HCV as histological patterns are normally the same for hepatitis A, (HAV) and hepatitis B, (HBV).

Chronic hepatitis

• Symptomatic primary infection and Post Transfusion Hepatitis, (PTH) may be indicative of chronic HCV infection.
• Up to 80% of persons infected with HCV by blood transfusion or haemophiliacs receiving infected clotting factor tend to have persistent HCV infection.
• Where exposure to HCV is much less, chronic HCV is not so apparent in the population.
• Viraemia is also associated with chronic HCV although no link between viral load and severity of chronic HCV has been established.
• Histology - Chronic HCV sufferers can show the presence of lymphoid follicles within the portal tracts of the liver. Other causes of viral hepatitis rarely show lymphoid follicles. A dense periportal inflammatory process can be seen linked with the migration of lymphocytes and plasma cells giving rise to bile duct damage in the form of enlarged epithelial cells lining the bile ducts.
• Lobular hepatitis - where lymphocytes amass in sinusoids around hepatocytes.

Cirrhosis

• HCV sufferers may go on to develop cirrhosis which can lead to liver failure. However, HCV infection cannot be distinguished clinically from other forms of cirrhosis.
• A patient with cirrhosis tends to progress to forming hepatocellular carcinoma, (HCC) although HCC is nearly always associated with cirrhosis.
• HCV infection is normally asymptomatic. When symptoms present they are specific features of liver failure.

Hepatocellular Carcinoma (HCC)

• HCC is common in long-term HCV infections. 60-90% of cases of HCC show HCV present.
• HCC can appear up to thirty years after HCV infection and usually manifests itself in patients who already have cirrhosis.
• HCV is considered to be an indirect cause of HCC after prolonged damage to liver tissue.

Immune Reaction to HCV

• Humeral and cell mediated immunity (CMI) are stimulated by HCV although there is a weak response.
• Antibody response is also unpredictable giving no real clue to prognosis.
• Those infected with HCV can show a good cellular immune response to HCV. T-helper cells taken from peripheral blood multiply when mixed with recombinant HCV proteins, for example, E1 and E2. However, humoral and cell mediated responses do not clear viral load very effectively.

Persistence

• In chronic HCV, humeral and cellular responses are not adequately able to clear virus or protect against reinfection, even with the same strain.
• The HCV envelope proteins convey heterogeneity giving rise to HCV variants. High envelope protein sequence divergence makes it difficult for the humeral immune responses to inactivate HCV.
• The HCV E2 gene shows sequence variability in specific hypervariable regions close to the area where antibodies attach to viral receptors.
• HCV may also remain in the body owing to T cell epitope changes in NS3 which assists HCV avoid being recognised by cytotoxic T lymphocytes (CTL's).
• It has been proven that E1 and E2 variants of HCV form as an infection progresses, backing up the theory that immune pressure drives sequence change.
• HCV viral structure can allow persistence owing to extensive N-linked glycosylation of E1 and E2 which hide epitopes from antibodies to avoid viral neutralisation.
• HCV can persist by binding to lipid. Lipid uptake by hepatocytes may facilitate HCV entry into the liver. 

 

6	Epidemiology

• There are in excess of 170 million chronic carriers of HCV globally.
• HCV is most commonly transmitted via the parenteral route, (shared needles used by intravenous drug abusers - IVDA's).
• IVDA's are the group most at risk of being infected with HCV. Up to 80% have been found to be infected.
• Another group at risk of HCV infection is those persons receiving blood and blood products. Patients that are being transfused, dialysis patients and health care workers are all at risk of contracting HCV.

Table 2: World Health Organisation HCV global prevalence rates (1999)

Region	Population (millions)	Number Infected (millions)	Prevalence rate %
Africa	602	32	5.3
Eastern Mediterranean	466	21	4.6
Western Pacific	1600	62	3.9
South East Asia	1500	32	2.2
Americas	785	13	1.7
Europe	858	9	1.0

Fig 3: Global incidence of HCV

7	Laboratory Diagnosis

• After the molecular structure of HCV was characterised, laboratories were still unable to culture HCV or view it using EM.
• Both HAV and HBV could be effectively screened for in the early eighties but PTH caused by Non-A, Non-B known as, (NANB) as already briefly stated.
• NANB was associated with >90% of PTH. NANB was modified to NANBH, the 'H' standing for hepatitis. The first tests for HCV were tests for surrogate markers.

Surrogate Markers

• Surrogate tests were used by blood transfusion centres before specific HCV tests were developed.
• Such tests relied on the fact that 20% of blood donors who had PTH presented with raised levels of alanine aminotransferase (ALT) and up to 10% shared Anti-HBc antibodies.
• The problem was that many persons who had an HCV infection showed normal levels of ALT. However, despite this, many carriers of HCV were picked up by using surrogate marker tests in conjunction with preventing donors in high-risk groups, for example, IVDA's, from giving blood. It was estimated that PTH was lowered by up to 50%.
• ALT levels can be affected by gender, age, alcohol, etc and so were deemed to be an unreliable test which led to the development of specific tests for HCV.

HCV Tests

• The NANBH agent was obtained from chimpanzee plasma inoculated with human cases of NANB. The extracted nucleic acid from this plasma was cloned in a suitable vector to produce a series of clones. The recombinant antigens from these clones were used to detect patients antibody. The initial reactive clone was 5-1-1 and then C-100-3 was developed as a result of overlapping clones. These clones were expressed in yeast to produce antigens for commercial assay development.
• The C-100-3 antigen was used to detect complementary antibodies and later became one of the first generation tests for HCV antibody screening.

Initial Test for HCV Antibody Screening (1991)

• The C-100-3 antigen was used to detect complementary antibodies. Later came the first generation HCV antibody tests.

First Generation Tests for HCV Antibody Screening

• First generation HCV tests relied on the use of non-structural protein antigens constituting less than 5% of the genone. Coupled with this fact antibody development to non-structural proteins takes place much later on in the infection and some patients with NANBH did not even develop antibodies.
• The first generation tests missed up to 45% of the infectious donors and were unable to detect HCV seroconversion until 3-6 months after becoming infected.

Fig.4: First generation antibody screening assays - recombinant antigens

Recombinant Protein	Encoding Gene
5-1-1	NS4b Region of HCV genome
C-100-3	NS4a/NS4b Region of HCV genome

 

Second Generation Tests for HCV Antibody Screening (1992)

Fig.5: Second generation antibody screening assays - recombinant antigens

Recombinant Protein	Encoding Gene
C-200	NS3/NS4 Region of HCV genome
C-22-3	Recombinant Ag from the core region of HCV genome

 

• Second generation HCV tests had the advantage of including both structural and non-structural antigens from stable regions of the HCV genome.
• Sensitivity improved and more positives were identified. Where synthetic peptides were used lower cut off readings were seen compared to kits containing recombinant antigens. In general, seroconversion could be picked up earlier.

Third Generation Tests for HCV Antibody Screening

Fig.6: Third generation antibody screening assays - recombinant antigens

Recombinant Protein	Encoding Gene
C-200	NS3/NS4 Region of HCV genome
C-22-3	Recombinant Ag from the core region of HCV genome
NS5	Non-structural antigen

 

• In addition to the c-200 and c22-3 antigens, the recombinant antigens from the NS5 region was added to the third generation antibody detection ELISAs. Other modifications lead to further improvement in sensitivity.
• Seroconverters were picked up at a much earlier stage. NS3 antigens were added because NS3 antibodies have been shown to be the first to appear.
• Some false positives were found with the third generation kits owing to the stickiness of patients' serum as well as other factors.
• HCV antibodies from a donor can be passed onto the recipient so care has to be taken not to mistake this for an acute infection.
• IgM is not screened for and no IgA activity has been seen.

 

PCR

• HCV RNA can be detected within 1-2 weeks after infection has occurred.

Current Confirmation Tests for HCV

• RIBA-3 - Chiron: Core, NS3, NS4, NS5
• ImmunoBlot HCV - Mikrogen: Western Blot
• Liatek-III - Organon Technika: Core, E2/NS1, NS3, NS4, NS5
• Matrix Dot Immunoblot - Abbott Labs: Core, NS3, NS4 (yeast), NS4 (E.coli) 
• DB-2-Blot - Diagnostics Biotechnology: Core, NS3, NS4
• Murex Blot - Murex Diagnostics: Core, NS3, NS4, NS5
• HCV rapid device (serum/plasma) - Biotec Laboratories Ltd: Protein A coated particles and recombinant HCV proteins to selectively detect antibody to HCV.

Diagnostics

Product	Product Code	Details
ImmunoBlot HCV Western Blot	CB55/4302	Immunoblot test with antigens produced by recombinant techniques for the detection of IgG antibodies against the hepatitis C virus (HCV)
HCV rapid device (serum/plasma)	CB55/8006	A rapid chromatographic immunoassay for the qualitative detection of antibody to Hepatitis C Virus in serum or plasma. The test utilises a combination of protein A coated particles and recombinant HCV proteins to selectively detect antibody to HCV. The recombinant HCV proteins used in the device are encoded by the genes for both structural (nucleocapsid) and non-structural proteins.

Molecular Assays

• Antibody testing has its limitations, so molecular techniques for HCV detection are necessary. RTPCR is used to detect HCV RNA viral load. Using a reverse transcription step, viral RNA is converted into cDNA then amplified in a nested PCR using primers from the 5' region of the genome. The products of amplification can be used for determination, should RIBA give an indeterminate result.
• Antibody tests and PCR may produce conflicting results. PCR can give false negative results if the HCV infection does not give rise to viral load. It is, therefore, good practice to use Western Blot assays alongside PCR for confirmation of results.
• The advantages of PCR are:
  • HCV RNA may be present in early infection whilst patients are seronegative.
  • Even in cases of chronic HCV, viral RNA persists which can be detected by PCR.
  • Because PCR is used to quantify viral load, it may be helpful to gauge the effectiveness of interferon alpha or ribovirin treatment.
  • Negative PCR indicates no HCV-RNA in the liver.

 

• Patients may be RNA negative, RIBA-3 positive as the virus may have been cleared and therefore below the detection limit of PCR.
• HCV may reside outside the liver in other areas of the body as well as serum.
• HCV is heterogeneous and PCR has been used to identify nucleotide sequence homology which can specifically show variation, divergence and from where HCV isolates have been derived.
• HCV has been divided into up to 11 genotypes. Molecular sequencing has established that it is the actual RNA that varies hence the classification into genotypes.
• Epidemiological work concerning HCV relies specifically on genotype sampling.
• The genotype may be important for disease management owing to the variation in genotype pathogenesis where some genotypes respond better to antivirals than others.
• Others factors affecting pathogenesis of HCV include age and gender, viral load and how long the patient has had the disease.
• Genotype 1 subtype 1bis associated with HCV chronicity and poor response to IFN alpha.
• Viral load and subtype analysis can indicate the possible outcome of IFN alpha treatment regimes.
• PCR can show a reduction in viral load and hence a positive outcome of antiviral treatment.

 

8	Prevention / Control

• All blood donors are screened for anti-HCV.
• Dry heat treatment of blood products, i.e. 80^oC for 72 hours.
• Prevention of IVDA's needle sharing plus education about the risks and consequences of contracting HCV is necessary.
• Provision of clean needles and condoms, although HCV is not necessarily transmitted sexually.
• Care has to be taken by healthcare professionals to avoid needlestick injuries after taking blood from high-risk patients.

Vaccine

• It has been difficult to develop a vaccine to HCV owing to the heterogeneity of the virus. However, vaccines are now in various stages of clinical trails and a vaccine should be available in 2007. Any vaccine still has to take into consideration multiple serotypes.

 

9

Treatment

• HCV is treated with IFN alpha on its own or in combination with Ribovirin.
• Approximately 50% of HCV-infected patients benefit from antiviral treatment. However, the disease can recur when treatment stops.
• Owing to the asymptomatic nature of HCV infection, the disease may be missed in the acute phase, only being identified in the chronic phase when it may be too late or inappropriate to treat.

 

10	References

• Ball, et al., (1999). Viral Hepatitis - A National Masters Distance Learning Package. Coventry: Jones Sands Publishing: pp 43-57.
• Collier, L., Oxford, J., (2000). Human Virology. New York: Oxford University Press. pp717-744.
• Collier, L., (1998). Topley & Wilson's Virology. 9th ed. New York: Oxford University Press. pp 717-744.
• Desselberger, U., (1995). Medical Virology: A Practical Approach. New York: Oxford University Press. pp 182-186.
• Murray, P. R., (2002). Medical Microbiology. Fourth Edition. USA: Mosby inc. pp 591-608.
• White, D., Fenner, F. J., (1994). Medical Virology. 4th ed. San Diego, California: Academic Press. pp 358-380.

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Websites

1. The hepatitis c virus - http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index2.html
2. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses : proposals for standardization - http://cat.inist.fr/?aModele=afficheN&cpsidt=1692872
3. HCV Genotype and Quasispecies - http://www.hcvadvocate.org/hepatitis/factsheets_pdf/genotype_FS.pdf
4. Hepatitis C information centre - http://hepatitis-central.com/hcv/genotype/explained.html

12	Disclaimer

The information contained in the Education Series text was obtained through collaboration with laboratories identifying and diagnosing HCV as well as scientific publications and relevant text books. We believe the information specified in this text to be correct although it is the responsibility of the participant to confirm information validity. Cosmos Biomedical Ltd claim no responsibility for misinterpretation of information. Copyright ^© Cosmos Biomedical Ltd, 2006.

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Copyright

The MES Scheme, which includes question cards, crosswords and web text is Copyrighted and Trademarked and should not be reproduced under any circumstances in any way and at any time now or in the future without the express permission of Cosmos Biomedical Ltd. Only persons registered by Cosmos Biomedical Ltd are legally allowed to participate in the MES Scheme and use this information. 

 

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