| Abstract|| |
Hepatitis C Virus (HCV) has been identified as the major etiological agent of parenterally-transmitted non-A non-B hepatitis. The virus has been cloned recently and completely or partially sequenced. Molecular analysis of the HCV genome indicates that it is a small, enveloped RNA virus having a highly conserved structural region followed by five less conserved non-structural regions. Based on nucleotide sequences, it has been found that multiple types of HCV exist. Infection with HCV can now be diagnosed by using virus-specific antibodies. A first generation enzyme-linked immunosorbent assay (ELISA) was initially used. However, this test had many drawbacks and consequently a second generation ELISA has been developed. The inclusion of new antigens in a recombinant immunoblot assay has further increased the sensitivity of diagnosing HCV infection. Also, detection of the virus genetic material by using polymerase chain reaction has enabled detection of viremia within only a few days following exposure apart from offering a possibility to monitor anti-viral treatment, since a decrease in viral RNA level is noticed in patients with positive response to treatment.
Keywords: Hepatitis C virus, Non-A, non-B hepatitis, ELISA, Polymerase chain reaction.
|How to cite this article:|
Ramia S. Hepatitis C Virus: Molecular Virology and its Implications for Serologic Diagnosis. Saudi J Kidney Dis Transpl 1995;6:190-6
|How to cite this URL:|
Ramia S. Hepatitis C Virus: Molecular Virology and its Implications for Serologic Diagnosis. Saudi J Kidney Dis Transpl [serial online] 1995 [cited 2020 Jun 6];6:190-6. Available from: http://www.sjkdt.org/text.asp?1995/6/2/190/40865
| Introduction|| |
It is well established now that hepatitis C virus (HCV) is the major etiological agent of parenterally-transmitted non-A non-B hepatitis (NANBH) ,, . The virus has recently been cloned and completely, partially, sequenced by different investigators ,, . Since then, great interest has been evoked in understanding the molecular virology of the virus due mainly to its association with an increased frequency of chronic infection in a large number of infected individuals. It is estimated that at least 50% of HCV infections lead to persistent disease including chronic active hepatitis with or without concomitant cirrhosis ,,, . Furthermore, HCV have been implicated as one of the major causative agents of primary hepatocellular carcinoma in Japan  as well as other parts of the world ,,, . Another interesting aspect of HCV infection is that approximately 45% of cases have no apparent risk factors including parenteral exposure ,, , leaving unanswered the question of virus transmission via as yet unidentified routes of exposure. The aim of this article is to review the molecular biology of the HCV and discuss its implication in serologic diagnosis.
| Molecular Virology of HCV|| |
A complementary DNA (cDNA) clone, 5-1-1, that encoded at least one epitope which specifically identified antibodies in the serum from patients with blood-transmitted NANBH, was reported in 1989 by scientists at Chiron Corporation in California  . By using hybridization technique, the scientists constructed a larger overlapping clone, c 1003, producing a non-structural protein consisting of 363 amino acids. This clone was derived from a virus which was named HCV , . The HCV was subsequently shown to contain an approximately 9.4 kilobase (Kb) single-stranded, positive-sense RNA molecule with one single open reading frame, that encoded for a glycoprotein composed of structural and non-structural proteins  . Molecular analysis of the HCV genome strongly indicates that it is a small, enveloped RNA virus with similarities to the flavi and pesti viruses [Table - 1] , . A scheme of the genetic organization of HCV is shown in [Figure - 1]. Like its flavivirus and pestivirus relatives, HCV appears to encode a large polyprotein precursor from which individual viral proteins (both structural and nonstructural) are processed through the com bined action of host-encoded and viralencoded proteases. At the 5' end of the genome there is a highly conserved structural region encoding for the nucleocapsid(G) protein, which is followed by five less conserved non-structural regions, NS1-NS5, towards the 3' end. Between the structural and non-structural regions, there is a hyper-variable region encoding for the envelop (El, E2/NS1) [Figure - 1]. The non-structural proteins NS1-NS5 include a protease/heli-case (NS3) and the viral RNA-dependent RNA-polymerase. No function has yet been assigned to NS2 or NS4.
| Classification|| |
In addition to the molecular properties of HCV, physicochemical properties of the virus ,,, also indicate that it is enveloped like flavi/pesti virsuses with a diameter of < 50 nm. It is worth mentioning that HCV has not yet been visualized by electron microscopy or immune electron microscopy. Based on the general similarities of HCV to portions of both flavivirus and pestivirus genomes, including the presence of characteristic consensus sequences as well as the spacing between the hydrophobic NS2 and NS4 regions, investigators now believe that HCV should be included within the family Flaviviridae as a separate genus along with another genus encompassing the pestiviruses [Figure - 2].
Nucleotide sequences of different HCV isolates have been made in many laboratories. Comparison of a Japanese isolate (H.CV-J1) with the nucleotide sequences of the original isolate derived in the United States (HCV-1), ,, revealed that HCV-Jl differed in both nucleotide and polypeptide sequence in the NS3 and NS4 regions  as well as in the NS5  and envelop regions  . This suggested that, as in the case for the flavi and pestiviruses, multiple types of HCV exist. It is now established that there are at least three basic groups and possibly a fourth group of HCV. The groups are distinguished by the nucleotide and amino acid homologies observed between different viral isolates. Group I, II and III viruses have been observed in Japan , whereas so far only group I viruses have been reported from the United States  . Recently, HCV has been classified into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region  . There is little doubt that this viral diversity among HCV isolates deserves close attention with respect to virus-host interactions, evolution of chronicity and development of vaccine. Another important clinical aspect is the possibility of occurrence of infection with different HCV types and whether the prognosis of this group of patients is different from those infected with a single agent.
| Diagnosis of HCV Infection|| |
First generation anti-HCV tests
Infection with HCV, once a diagnosis of "exclusion" based on the absence of markers of acute infection with hepatitis A virus (HAV) or hepatitis B virus (HBV), can now be specifically diagnosed by using serodiagnostic assays for virus-specific antibodies. A first generation anti-HCV enzyme-linked hnmunosorbant assay (ELISA) was developed by using recombinant c 100-3, (derived from the NS3/NS4 region of the genome) as antigen [Figure - 1]. Generally speaking, very high prevalence rates of anti-HCV were found among patients with chronic post-transfusion NANBH (70-100%) ,, , intravenous drug addicts (50-90%) , , hemophiliacs (64-83%) , and patients on hemodialysis (1-46%) , . Among blood donors anti-HCV c 100-3 differed with geographic locations and varied' from 0.20.5% in Scandinavian countries  to 0.92.0% in some European , and Asian countries ,, .
The test however, had some major drawbacks which included failure to discover all patients with HCV infection , , long "window-phase" before seroconversion (up to 12 months)  and inability to differentiate an ongoing infection from a resolved one. Furthermore, the test gave false-positive reactions in patients with hypergammaglobulinemia and autoimmune type-I chronic active hepatitis (CAH) , , primary biliary cirrhosis  , rheumatoid arthritis  , malaria  , and paraproteinemia  . Some of the falsepositivity was also due to cross-reactive antibodies to superoxide dismutase, a component of the assay  .
The Second generation anti-HCV tests
To circumvent the drawbacks of the first generation anti-HCV assay, recombinant or synthetic antigens derived from other regions of the HCV genome were included and this test is referred to as second generation ELISA anti-HCV test. The additional antigens c 22 and c 33 were derived from the structural region (core) I and NS3 regions respectively , [Figure - 1]. In patients with posttransfusion NANBH the seroconversion rate increased from 54% with the first generation to 82% with the second generation test. Also, the time lag to seroconversion decreased from a mean of 6.1 weeks from the onset of hepatitis with the first generation test to a mean of 2.3 weeks with the second-generation test  . Thus, the second generation test reduces the "window-phase" to seroconversion( and also increases the sensitivity in diagnosing HCV infection. There is also a reduction in the number of falsepositive reactions. Earlier diagnosis of HCV infection is now possible with the availability, of an ELISA test for detecting anti-HCV-IgM from Abbott Laboratories.
The inclusion of the new antigens (c 22 and c 33) in a second generation recombinant immunoblot assay (RIBA) also increased the sensitivity of diagnosing chronic HCV infection ,, . Blood donors who were anti-HCV c 100-3 positive and RIBA-positive seemed to transmit NANBH while transmission of the disease was not seen with donors who were only anti-HCV c 100-3 positive  .
Detection of viral RNA
For a more accurate way of diagnosing HCV infection and to be able to differentiate infectious viremic patients from non-infectious patients, detection of the virus genetic material by using polymerase chain reaction (PCR) has been attempted. The application of PCR techniques to amplify reverse transcribed cDNA permitted a very sensitive assay for viral RNA circulating in the blood stream and in tissue biopsy specimens. Using PCR assay, the data obtained show that viremia can be detected within only a few days of exposure to the virus and many weeks before elevation of transaminases and antibody titers ,. The assay provides valuable information regarding the viremic status in anti-HCV positive patients with normal liver function  and in patients with chronic NANBH hepatitis who may be seronegative  . Furthermore, PCR offers a possibility to monitor anti-viral treatment for chronic HCV infection since a decrease of viral RNA level is noticed in patients with positive response during interferon therapy , . Also, PCR may be useful for diagnosing vertical transmission of HCV from chronically infected mothers to their offsprings ,  .
Performance of general PCR assays for HCV RNA requires that primers specific for the 5'-terminus (so called 5' untranslated) region be used since this region is highly conserved  . Also, it is absolutely essential that rigorous negative control reactions be included to exclude cross-contamination and the assay should be performed at least in triplicate for the results to be more reliable.
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Department of Pathology, College of Medicine, King Saud University, P.O. Box 2925, Riyadh 11461
[Figure - 1], [Figure - 2]
[Table - 1]