With the introduction of sensitive assays for screening blood for hepatitis B virus in the late 1970s, it was anticipated that posttransfusion hepatitis would be virtually eliminated, but this was not to be. There remained a substantial residue of cases which were called non-A, non-B hepatitis (NANBH). The causative agent remained frustratingly elusive for over a decade and has still not been convincingly cultured in vitro nor visualized by electron miscro-scopy. Nevertheless, in 1989, a team of molecular biologists in the United States succeeded in an ambitious assignment which seemed to many to be unachievable. The ingenious protocol they devised serves as a prototype which could be applied in the future to the discovery not only of additional NANBH viruses but also of many other currently unknown noncultivable infectious agents.
Bradley and colleagues had previously demonstrated that hepatitis could be transmitted to chimpanzees by inoculation of factor VIII known to have been contaminated with an agent that had caused non-A, non-B hepatitis in hemophiliacs. Filtration studies had indicated the size of the causative agent to be of the order of 40-50 rim, while a buoyant density of 1.1 g/cm3 and sensitivity to chloroform suggested it to be an enveloped virus. The reasonable assumption was made that plasma from chimpanzees that had developed chronic hepatitis following inoculation with factor VIII would constitute a good source of the putative virus, and that virions could be concentrated from the plasma by ultracentrifugation. Total DNA and RNA was extracted from the pellet, denatured to single strands, and reverse-transcribed using random primers that prime the transcription of any single-stranded nucleic acid. The resulting complementary DNA was cloned into the bacteriophage Xgtll expression vector, which was then used to infect bacteria. Bacterial colonies were then screened for production of any antigenic polypeptide sequence recognizable in an immunoassay by serum taken from chronically infected patients, assumed to contain antibody against the putative virus. After screening about a million such random cDNA clones, a single clone capable of binding antibodies from several infected individuals was found! This DNA was then used as a hybridization probe to derive a larger overlapping clone from the cDNA library, which in turn was used to identify the full-length (9.5 kb) plus sense ssRNA hepatitis C viral genome. Eventually the complete nucleotide sequence of the genome was determined by isolating overlapping cDNA clones using hybridization probes based on the sequences of previous clones.
The genome of hepatitis C virus (HCV) is a single 9.5 kb molecule of ssRNA of positive polarity, with a gene order characteristic of the family Flaviviridae. A single long ORF encoding a polyprotein of about 3000 amino acids is flanked by untranslated 5' and 3' sequences, each containing short direct repeats and taking the form of a hairpin. The 3' terminus is not polyadenylated. The structural proteins occupy the 5' quarter of the ORF, and the nonstructural proteins the remainder. The gene order, namely, 5'-C, El, E2/NS1, NS2, NS3, NS4, NS5-3', closely resembles that of other members of the Flaviviridae. Structural protein C is highly basic and presumably represents the core (cap-sid) of the virion; El and E2 are glycoproteins, presumably both membrane proteins as in the genus Pestivirus, although it is possible that E2 may be the equivalent of the nonstructural protein NS1 of the genus Flavivirus, which is otherwise missing from hepatitis C virus. Of the four other nonstructural proteins, it can be deduced from characteristic motifs that, as for other members of the Flaviviridae, NS3 carries serine protease activity in its amino-terminal half and helicase activity in its carboxy-terminal half, whereas NS5 has RNA-dependent RNA polymerase activity, and NS2 and NS4 may be comparable with the membrane-binding proteins postulated to be required by other flaviviruses during membrane-associated replication.
Analysis of PCR products after reverse transcription of the genome of hepatitis C viruses from around the world reveals significant heterogeneity in nucleotide sequence, indicating the existence of at least a dozen genotypes. Whereas differences occur in all genes, there are hypervariable regions within the glycoproteins El and E2, suggesting that these envelope proteins are subject to immune selection in vivo. However, the relationship between genotypes and serotypes has yet to be sorted out.
Little is currently known about the replication cycle, as no simple cell culture system has yet been discovered. Replication probably occurs exclusively in the cytoplasm. Dense reticular cytoplasmic inclusions and convoluted membranes are conspicuous by electron microscopy in hepatocytes from infected chimpanzees. Immunofluorescence reveals viral proteins, mainly NS3 and NS4, confined to the cytoplasm. Full-length plus and minus RNA strands as well as a full-length double-stranded RNA (replicative form) have been found by in situ hybridization in the cytoplasm of infected liver, and apparently also in T lymphocytes. Consistent with the transcription strategy of other flaviviruses, no subgenomic RNAs have been detected by northern blot analysis of hepatocytes; it is reasonable to assume that a single poly-protein translated from the single long ORF is systematically cleaved at the appropriate motifs by viral and cellular proteases.
In many developed countries today hepatitis A, B, and C are about equally common. Acute hepatitis C is clinically similar lo hepatitis A and B, and the reader is referred back to Chapters 22 and 23 for descriptions. The major differences are as follows. The incubation period of hepatitis C, though ranging up to several months, averages 6-8 weeks. About 75% of infections are subclinical. Clinical infections are generally less severe than hepatitis B, having a shorter preicteric period, milder symptoms, absent or less marked jaundice, and somewhat lower serum alanine aminotransferase (ALT) levels, which often fluctuate widely. The case-fatality rate from fulminant hepatitis is 1% or less. However, HCV leads much more commonly lo chronic liver disease than does HBV. At least 50% of all patients with hepatitis C remain continuously or erratically viremic with moderate elevation of ALT levels for at least a year or two, and often much longer. Most of these are asymptomatic carriers or mild cases of chronic persistent or chronic active hepatitis which spontaneously resolve, but up to 20% progress to cirrhosis. Indeed, one U.S. study suggests that hepatitis C may be as important a cause of cirrhosis in that country as alcoholism, and more common than hepatitis B.
There is also a clear correlation between chronic HCV infection and the development of hepatocellular carcinoma (HCC), more than 90% of HBV-negative cases in some countries being HCV antibody positive, but proof of etiological association may have to await controlled prospective studies. Because the HCV genome is RNA, and reverse transcriptase is not involved in its replication, it is unlikely that integration of cDNA is involved as with HBV-associated HCC.
Little is known of the pathogenesis of HCV infection. Using (he limited range of assays currently available to study the progress of infection in chimpanzees and humans, certain facts have emerged. The major target cell seems to be the hepatocyte, but there is also evidence suggesting viral replication in leukocytes and perhaps other cells. Viremia is detectable by PCR within days of infection and lasts for weeks or months before resolution in most cases, but it may persist for years in chronic carriers, often fluctuating erratically. Whether these swings reflect reactivation of virus, with or without accompanying relapses in clinical hepatitis, and if so what triggers them have yet to be resolved. Although antibodies continue to be made, and most of the virus is bound in virus-antibody complexes, the infection is not eliminated. Indeed, chimpanzees that have recovered from HCV infection can be reinfected with the homologous (or heterologous) strain of virus. Humans also frequently experience multiple episodes of acute hepatitis C, but it is currently unclear whether these are exogenous reinfections with the same or another strain or reactivation of the original infection.
In 1990 the first specific diagnostic test for hepatitis C virus was licensed for the screening of blood donors in the United States. A cDNA clone representing part of the HCV genome was expressed in yeast to produce a recombinant antigen (fusion protein) corresponding to a large portion of the nonstructural protein, NS4, and this antigen was employed in an enzyme immunoassay to detect antibodies to that particular protein in the serum of potential blood or organ donors. However, this first-generation EIA was not sufficiently sensitive or specific, and it was replaced by a second-generation version based on a recombinant yeast chimeric protein comprising the three most conserved HCV proteins, C, NS3, and NS4. Although this cloned antigen, which lacks the hypervariable El and E2 proteins, fails to distinguish between putative serotypes or strains of the virus, it does identify hepatitis C antibody in up to 95% of posttransfusion non-A, non-B hepatitis cases and has replaced the first-generation EIA in blood banks and elsewhere. Because of the slow and variable development of antibodies postinfection, there is a window period of about 3 months before the test registers positive.
Of course, assays for antibody do not distinguish between acute, chronic, and past infection. Because about 50-60% of HCV infections progress to the chronic carrier state it is currently prudent for blood banks to discard all HCV antibody-positive blood, but for the purposes of clinical management it is important to develop additional tests. Assays for IgM antibody have not proved to be particularly useful. Immunoassays probing for antigen are not sufficiently sensitive to pick up the low titer of virus present in serum. The PCR is much more sensitive and successfully detects the viral genome from about 2 weeks postinfection but may miss some chronic carriers because of the fluctuating levels of viremia in any given patient over the years. Immunofluorescence and in situ nucleic acid hybridization are invaluable tools for studying pathogenesis in vivo but are of diagnostic value only on biopsy and autopsy specimens. There is an obvious need for a reproducible system for isolation of HCV in cultured ceils.
In most Western countries the prevalence of antibodies to hepatitis C virus is around 1% in blood donors, but this may underestimate the prevalence in the community as a whole. While blood transfusion and factor VIII have been major sources of infection in the past, they have already declined as a result of more rigorous selection of donors and can be expected to fall off much more rapidly with the introduction of second-generation (and later) EIAs for screening in blood banks. Today the most clearly identifiable infected cohort are intravenous drug users, the majority of whom are infected. Whereas these three parenteral risk groups account for about half of all HCV infections, the route of infection of the remainder is a mystery. There is some evidence that, like HBV, HCV may be shed in genital secretions and saliva, as well as in blood. Heterosexual promiscuity seems to correlate with HCV seropositivity; however, presumably because of the lower circulating virus titer, sexual transmission does not appear to be as important as with HBV. The same applies to perinatal transmission: it appears to be relatively uncommon except when the mother is infected with HIV as well as HCV.
The surprisingly poor homologous immunity that has been reported to follow infection of chimpanzees does not augur well for prevention of hepatitis C by vaccination. Nevertheless, experimental vaccines based on molecularly cloned envelope proteins are in the pipeline. Provided the preparation is highly immunogenic and attention is given to any antigenic variation evident in natural strains, there is some hope of success.
Interferon a injected subcutaneously at a dosage of 2-3 million units thrice weekly for 6 months reduces ALT levels to normal in about half of chronic hepatitis C patients, but all except about 20-25% of these relapse after withdrawal of the drug. Higher doses are no advantage, but patients with less severe disease or those treated before cirrhosis sets in appear to have a better chance of long-term response. Ribavirin may have a comparable effect. HCV-induced cirrhosis is one of the commonest indications for liver transplantation, but endogenous recurrence of infection is a major problem.
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