Hepatitis A-E Slide Set

HEPATITIS C VIRUS

 

For years, the nature of the agent(s) responsible for parenterally transmitted non-A, non-B hepatitis remained enigmatic, though the involvement of a virus appeared likely. Chimpanzee transmission studies carried out by Ian Bradley of the CDC Atlanta made available a pool of serum with a high titre of antibodies against an agent responsible for parenterally transmitted non-A, non-B hepatitis. The putative virus was pelleted from the serum and, because it was not known whether the genome was DNA or RNA, a denaturation step was included prior to the synthesis of cDNA so that either DNA or RNA can serve as a template. The cDNA was then inserted into lambda gt11 expression vector. Serum from a patient with non-A, non-B hepatitis was used to screen the cDNA library for clones which may be expressing the fusion protein antigen. Over 1 million clones were screened. This approach led to the detection of a clone (5-1-1) which was recognized by the sera of several infected patients. This clone was used as a probe to detect a larger overlapping clone in the same library. It was possible to demonstrate that these sequences hybridized to a positive-sense RNA molecule of around 100,000 nucleotides which was present in the liver of infected chimpanzees. Using gene walking techniques, additional clones with virus sequences in the library were identified and in due course, the complete nucleotide sequence of the virus became available. The organization of the genome closely resembled those of enveloped RNA viruses. To date, the virus has yet to be convincingly visualized by EM.
 

Properties

Genome resembled that of a flavivirus
positive stranded RNA genome of around 10,000 bases
1 single reading frame, structural genes at the 5' end, the non-structural genes at the 3' end.
enveloped virus, virion thought to 30-60nm in diameter
morphological structure remains unknown

 

Epidemiology

In America, since HBsAg screening was introduced, the incidence of post-transfusion hepatitis has a declined, but a significant number of cases remained. At least 95% of post-transfusion hepatitis was caused by non-A non-B. Worldwide, there are thought to be 100 million carriers of hepatitis C. It is particularly prevalent in Japan, where it is thought to be responsible for the majority of cases of hepatocellular carcinoma. There are 175,000 new cases of hepatitis C in the US per year. In the US, NANBH infection is more common than HBV infection amongst IVDA and those given blood transfusions. NANBH is usually transmitted parenterally. Sexual transmission can occur albeit considerable less efficient for HBV. There were recent reports of vertical infection.
 


   

Risk Groups for hepatitis C in the U.S.A.

1. IVDA 35%
2. Blood transfusion 5%
3. Promiscuity 7%
4. Household contacts 8%
5. Health care workers 20%
6. Unidentified sources 43%

The highest HCV antibody prevalence is found in haemophiliac patients who have received untreated blood or blood products where up to 85% of such patients in the UK have antibodies. A large proportion of cases occur in people with no known risk factors. The evidence for and against sexual spread is confusing. It is noteworthy though that 20% of non-drug using female partners of IV drug users with hepatitis C are positive for anti-HCV. In general, the mode of HCV transmission is similar to HBV.

Health care workers experiencing needlestick injuries are also at risk of acquiring HCV infection. In one study, 3% of health workers with a needlestick exposure in which the source patient was anti-HCV positive converted to anti-HCV. Accumulating evidence indicates that HCV is transmitted from mother to infant; however the actual risk is unknown. Anti-HCV seroconversion has only been rarely documented among infants born to anti-HCV positive mothers unless their mothers were also infected with HIV. However, in studies using PCR, HCV transmission apparently occurred without anti-HCV seroconversion.

In common with other RNA viruses, wide genetic variation of hepatitis C exist in nature. On the basis of phylogenetic analysis, HCV has been classified into six major genotypes (types 1 to 6) of which four (types 1 to 4) contain several more closely related subtypes (e.g. a, b and c). In West-ern Europe and the USA, the predominant geno-types are la, lb, 2b and 3a, with some variation in frequency. In Japan and Taiwan types lb, 2a and 2b are seen most frequently. Elsewhere in Asia, genotype 3 is the most common, and genotype 4 is found frequently in the Middle East and Africa. Type 5 is mainly found in Southern Africa and type 6 in SE Asia. There is increasing evidence demonstrating a link between HCV genotype and disease severity and re-sponsiveness to interferon therapy. Genotype lb was found significantly more often in cases of severe liver disease such as cirrhosis and hepatocellular carcinoma. It was also associated with a longer disease duration and a higher level of viraemia than other genotypes. Individuals infected with genotype 1b are less likely to respond to interferon therapy
   

Pathogenesis

Each clinical form of viral hepatitis with HBV can also occur with HCV. Acute disease may lead to recovery, fulminant hepatitis, relapsing hepatitis with intervening periods of normal liver function, inapparent chronic infection, chronic active hepatitis and cirrhosis have been documented. The incubation period lies between that of HAV and HBV. HCV infection is particularly associated with high ALT levels and it was suggested that ALT levels could be used as surrogate marker for the screening of NANBH before the availability of a test for anti-HCV.

Chronic Infection - It is thought chronic infection occurs in 50- 75% of all patients infected with HCV, in contrast to 10% for adults infected with HBV. In a cohort study where patients with HCV chronic disease had been followed up for 10 years, 20% have cirrhosis, 30% made improvements, 45% remained stable, 25% have shown progression (including the development of cirrhosis).

Association with hepatocellular carcinoma - Several recent studies have established a strong association between HCV and hepatocellular carcinoma. anti-HCV antibodies, in the absence of HBV markers, were detected in 44.5% of HCC patients in Spain, 43% in Japan, 16% in Italy, 7% in South Africa. Since HBV carriers often have chronic HCV infection, it is conceivable that the 2 viruses may act together to cause HCC and epidemiological data linking HBV to HCC may require reassessment in terms of the magnitude of the risk involved with the availability of testing for chronic HCV infection. HCV may prove to be as important as HBV in the causation of hepatocellular carcinoma worldwide. It has recently been suggested that the risk of developing HCC is approximately 5% year in cirrhotic patients with chronic hepatitis C; this is greater than the risk from hepatitis B.

In chimpanzees and to a lesser extent in humans, cytoplasmic tubules (cylindrical confronting cisternae) are regularly seen under EM. The nature of these structures are unknown in particular to the part they may play in HCV replication. In HCV hepatitis, direct cytopathic damage to the hepatocytes is thought to be the mechanism of pathogenesis, in contrast to the immunopathological mechanisms in HBV.

                                HBV                            HCV

Symptomatic             50                              10-35

Chronicity                 10-15                         50-75

Hepatocyte injury CMI direct CPE

In general, hepatitis C is a slower disease than hepatitis B. It takes 10 years to develop chronic hepatitis, 20 years to develop cirrhosis, and 30 years to develop hepatocellular carcinoma. There are certain nonhepatic disorders which are sometimes associated with chronic hepatitis C infection. eg. mixed cryoglobulinaemia, membranoproliferative glomerulonephritis, Sjogren's syndrome, sporadic porphyria cutanea tarda, and lichen planus.
   

Laboratory Diagnosis
 

1. Serology - the first generation ELISA kits relied on a fusion protein antigen produced from a original 5-1-1 clone with a few adjoining clones. The antigen was denoted as the C100 antigen. The problem with the first generation kits was that the antigen used was non-structural and thus may not pick up all cases of HCV. Furthermore, antibodies against this antigen could not be detected until 15 weeks after the onset of hepatitis. Therefore, HCV infection cannot be excluded in those whose serum is antibody-negative up to 6 months after the onset of symptoms. The first generation kits had also been demonstrated to have a poor specificity. Second and third generation assays are now used for serological diagnosis. Second generation assays incorporate C22 core antigen as well as NS4 (C-100-3) and NS3 antigens. The newer third generation assays incorporate NS5 as an additional antigen. However this increase in sensitivity is offset by a slight decrease in specificity. They reduce the "diagnostic window" down to 4 weeks after initial infection.

Recombinant Immunoblot assays (RIBA) were developed by Ortho whereby nitrocellulose strips were coated with discrete bands of E. coli  and yeast  cloned antigens. It is thought that this test carried a much higher specificity with equal sensitivity. Another method to increase sensitivity was to use "blocking tests". Sera positive for anti-HCV were blocked by C-100-3 antigen. Inability to be blocked is interpreted as non-specificity. Recombinant antigens are being developed based on structural proteins which should lead to serological tests with higher sensitivities. Also it is feasible that antibodies to these antigens may appear earlier and thus diagnosis of acute infection may become possible.

2. Detection of HCV-RNA - A PCR test had been developed where the level of HCV viraemia is roughly quantified and thus infectivity of the blood. A 5' conserved non-coding region had been identified. PCR has enormous potential value in support of the anti-HCV ELISA and in the study of HCV. It can be used to diagnose acute infection as HCV viraemia occurs well before the development of anti-HCV antibodies. Evidence provided by PCR for fluctuated level of infectivity in certain individuals may play an important role in the consideration of infectivity of seropositive donors. RIBA-2 positivity appears to correlate well with PCR positivity. A branched DNA assay has also been developed for the detection of HCV-RNA. Quantitation of HCV-RNA is important in monitoring the response to antiviral therapy.

3. Hepatitis C Antigen - Recent reports described the identification of hepatitis C antigen (HCAg) in hepatocytes of patients suffering from chronic HCV infection using a FITC polyclonal IgG obtained from patients with hepatitis C. A test for hepatitis C antigen in serum is being developed for the detection of HCV viraemia, rather like PCR.

4. HCV Genotype - There are six known genotypes and more than 50 subtypes of hepatitis C. Determining the HCV genotype is useful in epidemiological research and also in making recommendations regarding therapy.  Patients with genotypes 2 and 3 are two to three times more likely to respond to interferon-based therapy than patients with genotype 1. Furthermore, when using combination therapy, the recommended dose and duration of treatment depend on the genotype. For patients with genotypes 2 and 3, a 24-week course of combination treatment using peginterferon and 800 milligrams (mg) of ribavirin daily is adequate, whereas for patients with genotype 1, a 48-week course and full dose of ribavirin (1,000 to 1,200 mg daily) is recommended. For these reasons, testing for HCV genotype is clinically important.
   

Treatment

Early studies indicate that interferon and ribavirin are effective of cases of acute and chronic hepatitis C. A combination of interferon and ribavirin may be useful. There is more experience in the use of interferon for the treatment of hepatitis C. The current recommendation is that interferon treatment may be considered in those with chronic active hepatitis who are at risk of progression to cirrhosis and HCC. The recommended regimen is 3 MU tds sc or im for 6 months. The response rate is around 50%. However, approximately 50% of responders relapse upon cessation of treatment. At present, it is not clear what factors predict response to interferon therapy. There is some data to suggest that older patients and those with established cirrhosis respond less well. There is also growing evidence that the genotype of the infecting HCV determines the response to IFN. Type 1, in particular is associated with poor response to IFN. Therefore, a 48 week course of therapy is recommended for these patients instead of 24 weeks for genotypes 2 and 3. A newer preparation of interferon (peginterferon) is now available that allows for weekly injections. However, it must be given for 48 weeks irrespective of the genotype.

It also appears that the level of viraemia at the commencement of treatment predicts the response. Most responders will have significant reduction of SGPT level within 2 months of interferon therapy. One may try a higher dose such as 5 or 10 MU in non-responders although it is not certain whether the higher doses work. At present, it is not clear what factors predict relapse after treatment. For those who relapse after treatment, they may be offered a second course and then put on maintenance therapy for 6 to 12 months.  It is important to monitor the response to antiviral therapy virologically during the treatment period. Commercial quantitative PCR and bDNA assays are usually used for this purpose.

Prevention

The introduction of HCV screening in the blood transfusion service should reduce significantly the number of cases of post- transfusion hepatitis C. However, the extra cost is high as the current screening ELISA is 5 times more expensive than the anti-HIV screen test. Inactivation procedures should eliminate the risk of HCV spread via blood products. However, the above measures would only contribute to the prevention of a small proportion of cases of hepatitis C. Therefore, further preventive measures would depend on a better understanding of the epidemiology of hepatitis C infection. The efficacy of immunoglobulin for pre- or post-exposure prophylaxis is controversial. Vaccine development is going on and animal trails are being carried out.

CDC hepatitis branch; "Epidemiology and Prevention of Hepatitis A-E: An Overview"

Hepatitis A-E Slide Set