Indian Journal of Experimental Biology Yol. 41, June 2003, pp. 549-562

Review Article

Hepatitis C virus (HCV) - A review Molecular biology of the virus, immunodiagnostics, genomic heterogeneity

and the role of virus in hepatocellular carcinoma

CD Poduri*

Department of Biotechnology, Indian Institute of Technology, Guwahati

North Guwahati, Guwahati 781039, Assam, India

Tel: +913612690321 to 2690328 ext. 2229

Fax : +913612690762; Email: chetan@iitg.ernet.in; chetan_poduri@hotmail.com

Hepatitis C virus (HCY), an RNA and a hepatotropic virus, is the leading cause of viral hepatitis worldwide. Infection with this virus causes a repertoire of liver diseases that include acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), in addition to a number of extra-hepatic manifestations such as lichen planus, oral cancer, etc. At present, patients infected with this virus are treated with interferon either alone or in combination with ribavirin, a guanosine-like nucleoside analog. However, response to this treatment has been rather disappointing. For about a decade, lack of an alternative animal model other than chimpanzee, and an efficient cell culture system that could support long-term replication of the virus, hampered research on HCY. Despite this, a significant amount of information with regard to the molecular biology of the virus is available using bacterial cloning-expression sys tems, and based on computer predictions and analys is. Recent discovery of a cellular receptor to which the virus binds, identification of efficient cell culture/cell-free systems, HCY replicons and the development of a chimeric mouse model, provide a platform to verify the existi ng knowledge about this virus in the coming years. Additionally these developments aid the researchers in identifying novel therapeutic agents, apart from allowing us to reassess the efficiency of the currently available therapeutics. Presented in this article are a review of existing information with regard to the molecular biology of the virus, immunodiagnostic assays, genomic heterogeneity and the role of the virus in hepatocellular carcinoma. Likely therapeutic strategies other than those currently available are also introduced.

Keywords: Genomic heterogeneity , Hepatitis C virus, Hepatocellular carcinoma, Immunodiagnostics, Molecular biology, Therapeutic strategies

As early as in 1974, it was apparent that a third virus other than hepatitis A virus (HAY) and hepatitis B ViruS (HBY) is responsible for a significant percentage of post-transfusion hepatitis (PTH) cases l

,2. However, it was only in 1989 that researchers at the Chiron Corporation, USA3

, could discover the virus responsible for the PTH cases. This virus was subsequently designated Hepatitis C virus (HCY). Discovery of HCY and its establishment as the leading cause of non-A, non-B hepatitis4 represents a major breakthrough in the fields of bio-medical biotechnology, in general, and viral hepatitis, in particular. Current estimates put the prevalence of HCY infection at 1-2% of the World population. While illicit intravenous drug use and parenteral transmission remain the key routes of acquiring HCY,

*Formerly with Rajiv Gandhi Centre for Biotechnology, Thiru­vananthapuram 695 014, India Center for Cellular and Molecular Biology , Hyderabad 500 007 , India

the mode of acquisition in almost 50% of the cases is still uncertain; such cases are labeled as "community acquired" and most likely result from cultural practices such as tattooing, body piercing, sharing razors, etc .

In a vast majority of the infected population, infec­tion with this virus typically results in mild acute dis­ease with apparent complete recovery in about 20% of cases, and about 80% of cases progress to asympto­matic chronic infection. Approximately 20% of such chronically infected patients develop cirrhosis after 22 years of infection on average. A number of such cases eventually progress to hepatocellular carcinoma (HCQ5.9. Nevertheless, documentary evidence shows that chronically infected cases can also progress di­rectly to HCC without the intervening cirrhotic stage, and only a fraction progress to HCC via cirrhosis 10. 11.

In addition to this, HCY is now associated with a number of extra-hepatic manifestations such as lichen planus, oral cancer, meml1ranoproliferative glomeru-I h . . 12·14 onep ntts, etc. .

550 INDIAN J EXP BIOL, JUNE 2003

Presently, diagnosis of the viral infection is done by a commercially available third generation ELISA, RT-PCR and strip-based diagnostics that include RIBA, LIA, etc., which are reviewed here. However, it might be worth mentioning at this stage that although the procedures mentioned here are sensitive and specific for detecting HCY infection, lack of an universally accepted standard procedure resulted in the development of a number of in-house protocols and assays, review of which is beyond the scope of this article.

Initial euphoria about the discovery of HCY died down when it was realized that this virus is a fastidi­ous organism and no cell-culture system could sup­port the long-term replication of the viru$. Moreover, chimpanzee being the only animal model restricts studies on this virus to a very few labs the world over. Despite this, a significant amount of data are available with regard to the molecular biology of the virus using bacterial cloning and expression systems. A

5'UTR C

mFBl ~ AUG (start codon)

Structural proteins

El

~ E2 p7 NS2 NS3

~ ~ i

summary of this information is presented as Tables I, 2, Figs 1, 2 and is reviewed here.

Molecular biology of the virus A schematic representation of the HCY genome

organization is shown as Fig. 1. Based on the comparative analysis of the genomes and the protein profiles of HCY isolates, the virus is now recognized as a separate genus-Hepaciviruses-under the family Flaviviridae 1S

-19

• Other genera of this family include the Flaviviruses and the Pestiviruses.

Hepatitis C virus (HCY) is a single-stranded positive sense RNA virus. The nucleic acid is approximately 9.4 kb in length and possesses a unique open reading frame (ORF) that encodes for a polyprotein precursor of approximately 3010 amino acids in length20

-23

. Comprehensive analysis of HCY isolates revealed differences in length of the nucleic acid24

, and diversities of around 30% in their whole genome, leading to the definition of at least six genotypes and over 80 subtypes25

, 26 .

Non­structural proteins

NS4a NS4b

ii i NS5a NS5b

3'UTR

i

~ t t

Cleaved by host Signal peptidase

Cleaved by Cpro-l )

Cleaved by Cpro-2

Virus encoded proteinases

Fig. I - Schematic representation of genomic organization of Hey, polyprotein and cleavage sites on polyprotein. Note: Recently an alternate reading frame (ARF) and an alternative reading frame protein (ARFP) concept has been put forward and par­tial evidence for the same documented 37.38. Depicted in this schematic representation is the original ORF concept and the polyprotein encoded by the same. [For more details, see Table 2]

PODURT: HEPATITIS C YTRUS-A REYIEW 551

Flanking the ORF on either side are the 5' untrans­lated region (UTR) and the 3' UTR. The 5' UTR is about 341 nucleotides in length, well conserved in almost all the isolates and forms extensive secondary structures guiding the ribosomes In translation27-29 . This secondary structure is termed the internal ribo­some entry site (IRES) and allows viral genome ex­pression using a cap-independent mechanism. Despite being relatively well conserved, the 5' UTR shows type-specific variations that can be successfully ex­ploited for genotyping of the virus isolates. On the other hand, the 3 'UTR is highly divergent and varies in length depending on the isolate. This region is composed of three characteristic elements: the con­ventional 3' UTR, subsequent poly U stretch and the newly identified 3' terminal sequence named the 3' X tail. It is believed that the 3' UTR might function in initiation and regulation of genomic replication of HCy30-32

The nascent viral polyprotein is cleaved, by a com­bination of host cellular and viral proteinases, into at least ten functional proteins - Core, El and E2 glyco­proteins (gp), p7, NS2, NS3, NS4a, NS4b, NS5a,

Table I - Characteristics of hepatitis C virus (HCY)

Family Flaviviridae (enveloped viruses)

Genus Hepaciviruses or hepatitis C-like viruses

Nucleic acid SsRNA, (+) sense; -9.5kb; single ORF; IRES on the 5'UTR; 3' poly UI A tract (?); 6 genotypes, -80 sUbtypes

Yiral proteins Ten - see Table II; 6 serotypes

Natural host Humans

Experimental host Chimpanzee

Clinical/disease profile Hepatotropic virus causing typical acute disease with apparent complete recovery progressing to symptom-less chronic liver disease and subsequently to cirrhosis and/or hepatocellular carcinoma.

Transmission

Treatment

Mostly parenterally; sexual and vertical transmission - rare but documented; approx. 50% of the community acquired cases-mode of transmission unknown.

Interferon either alone or in combination with ribavirin

ssRNA: single - stranded RNA; (+) sense: positive sense; ORF: Open reading frame; UTR: Un translated region; IRES: internal ribosome entry site; Other abbreviations carry their usual significance.

NS5b, in that order7,20,22,33,34. The cleavage sites on the

polyprotein, their products and functions are summa­rized in Table 2 and Fig. 1. The core, El and E2 pro­teinsare the putative structural proteins of the virus; the non-structural ones include p7, NS2, NS3, NS4a, NS4b, NS5a and NS5b. Functions of these cleavage products have been speculated by sequence compari­sons with other known viruses, and evidence support­ing these speculations was demonstrated in bacterial systems expressing these genes 7,20,22,33,36. Particularly

Table 2-Summary of cleavage site on the polyprotein of HCY, their products and functions in the virus replication

Site of cleavage Cleaving (a. a., position enzyme

on HCY polyprotein)

1911192 HSP

383/384 HSP

746/747 HSP

809/810 HSP

102611027 Cpro - 1

165711658 Cpro - 2

171111712 Cpro - 2

1972/1973 Cpro - 2

2419/2420 Cpro - 2

2419/2420 Cpro - 2

Product Size Function

C

E1

E2

p7

NS2

NS3

NS4a

NS4b

NS5a

NS5b

(kDa)

21 Nucleocapsid; Onco­

Protein (?) 31 Envelope

Glycoprotein; heterodimer with

E2 70 Envelope

Glycoprotein; heterodimer with

E1 7 Unknown

function 23 Forms

autoprotease with NS3

70 Serine protease, NTPasel

Helicase (?); Oncogene-like

function (?) 8 NS3-protease

cofactor; Modulator for

Kinase; Stabilizer ofNS3

27 Unknown

56 function

Interferon sensitivity

determining region;

phosphoprotein (?)

68 RNA-dependent RNA-polymerase

Source - references 9, 137. HSP: Host signal peptidase; Cpro - 1 and Cpro - 2 are a HCY - coded metalloproteinase and serine­proteinase, respectively. Other abbreviations carry their usual significance.

552 INDIAN J EXP BIOL, JUNE 2003

noteworthy is the establishment of the function of the NS5b protein. The sequence of the NS5b protein, in general, and the amino acid motif G-D-D in particu­lar, is totally conserved not only in HCY but also in Flaviviruses, Pestiviruses, Polioviruses, Tobacco mo­saic virus and most of the RNA viruses35. This motif is characteristic of RNA-dependent RNA-polymerases, and hence the function of NS5b in HCY has been speculated to be the viral polymerase. Behrens et al. 36

,

demonstrated experimental evidence in support of this. Many viruses are reported to have overlapping

reading frames (OLRF) . This might also be true in the case of HCY. Partial scientific evidence in this direc­tion comes from the work of Walewski and col­leagues at Mount Sinai School of Medicine, New York 37, wherein they have shown the presence of a computer predicted alternate reading frame (ARF) . The authors demonstrated the presence of an alternate reading frame protein (ARFP) using synthetic pep­tides corresponding to the predicted ARFP sequence. Results from this and their subsequent study 38 are interesting. The possibility of more number of ARFs or OLRFs in the genome of HCY cannot be ruled out. If found true with regard to all the genotypes and the numerous reported isolates, and coupled with the pre­sent concept of a single ORF, the ARF concept might provide an explanation for many of the questions left unanswered with the ORF concept that include persis­tence of the virus in human host despite a robust hu­moral and cellular immune response. Although specu­lative and needs to be verified, the virus might be switching, during its replication and survival, between the ORF and ARF probably without a loss in function of the proteins encoded by either of the reading frames.

Evolution of immunodiagnostic assays Immunoscreening with the serum of a chronic

NANBH patient of a cDNA library, constructed using random primers, of the viral nucleic acid extracted from the plasma of a chimpanzee infected with the infectious agent eventually led to the discovery of the virus3. This clone designated 5-1-1, formed the basis of development of reagents and methodologies essential for diagnosis of the HCY infection. Furthermore, this clone also formed the starting point for characterization of HCy3

.4.16. Subsequently, an antigen designated c 100-3, a 363 amino acid (a. a.) fusion polypeptide representing part of the now designated NS4 protein of HCY, constructed from three overlapping c1one~ of 5-1-1 , and expressed in

yeast along with super oxide dismutase (SOD), formed the first immunodiagnostic procedure for detecting anti-HCY antibodies in patient sera thereby diagnosing the viral infection39. This was the so-called first generation immunoassay to be made available. Soon it became apparent that there are a number of false positives associated with this assa/0-42 that could probably be attributed to non-specific binding of IgG or immune complexes to the solid-phase40.42. It was also reported that false positives were common in patients with auto-immune-chronic active hepatitis, hypergammaglobulinaemia and in stored frozen

4344 L k f . . . f h' I sera '. ac 0 sensitivity 0 t IS assay was a so d45-47 reporte .

This situation eventually led to the development of the second-generation assay that included the c22-3 antigen along with the cI00-3, representing a. a, 2-120 of HCY polyprotein47.48, and the region amino acid 1192-1931 (includes NS3 and NS4 proteins), designated c200 or a smaller fragment of this termed c33c (only NS3)46-48. This assay was found to provide significant improvements in both sensitivity and specificity compared to the first generation assay. Presently a third generation assay is available in the market that includes an additional antigen, the NS5 protein product (a. a., 2054-2955). Together with this antigen, 60% of the total a. a., profile of HCY polyprotein is represented Il1 the present day commercial assays49.

One of the rather improperly documented aspects of HCY infection is the host humoral immune response, which in part could be attributed to the lack of a reliable assay system that can detect various antibodies elicited to different proteins of the virus. To overcome these impediments synthetic peptides or clones of the proteins of HCY are being used. Whereas the information thus generated does provide useful insights into the pathobiology of the virus, it needs to be confirmed and might not be accurate.

Initial reports on antibody responses involved the popular c100-3 antigen, which represents a portion of the NS4 protein. However, with the establishment of improper diagnosis using this antigen4o.44-48 and with the availability of full-length sequences of HCY, a more precise analysis of humoral response is being achieved with the aid of synthetic peptides. The humoral epitopes that are identified so far are shown schematically as Fig . 2. Also summarized in the figure are the different regions used in various different diagnostics , etc.

PODURI: HEPATITIS C VIR US - A REVIEW 553

The availability of full-length sequences of HCY isolates from different parts of the world also established the presence of variability in the sequences of the envelope gp and the NS5 protein 50

-52

.

In enveloped vi ruses, antibody responses to the envelope proteins play an important role in the prevention of infection, parti cularly re-infection or reactivation of the virus53

-58

. Especially in f1 av iviruses and pestiviruses, anti-envelope gp antibodies are a measu re of recovery or immunity against the respective viral infection. Should a similar situation exist in HCY infection, detectio n of anti-HCY envelope gp antibodies would be of immense value. However, there is contradic tory evidence avail able with regard to the protective ab ility of the envelope antibodies of HCY. Whereas Farci et at. 59 reported a lack of protective immunity against re infection with HCY, neutrali zing ability of the envelope antibodies was reported by Rosa et at. 60,6 1. Nevertheless, significant majority of the infec ted patients (60%­> 90%) develop antibodies against E2 gp of HCy62

-68

,

which is noteworthy. While the presence of anti-E2 gp antibodies in a majority of the infected individuals makes it an indi spensable diagnostic marker, the exact role of these antibodies in the viral infec tion , in vivo, needs a thorough investigation, At present, not all commercially available di agnostic kits incorporate representative sequences from the envelope proteins of HCY. It is believed that majority of the anti-E2 gp antibody response is directed against assembled/ discontinuous epitopes on the envelope gp that can be detected using E2 gp purified from mammalian cells, which was not availab le until recently. To overcome this imped iment, Poduri el at. 65-67 have used linear epitopes, from the immunodominant regions of the virus, without a compromise in either the sensitivity or the specificity of detection of the viral infection, as compared to the commercially ava il ab le assays. A noteworthy finding from these studies65

-67 is that over­

a-period of time, almost all the patients infected with the virus were found to harbor antibodies to all the immunodominan t epitopes shown in Fig. 2. This finding suggests that in lo ng-term disease statuses, such as cirrhosis and HCC, the anti -HCY antibody response is no longer neutrali zi ng the infec ti o n. In addition to thi s, it is no w well es tab li shed that antibodies to the hyper variable regio n (HYR 1) of E2 gp (immunodom inant epitope 383-409; ep itope 0 in F ig. 2) are isolate specifi c and might not be ab le to protect the indi viduals fro m reinfection and in chronic . f . 5'1 68 6'1 T k h h I f In ec tlons . '. a en toget er t ese resu ts by ar

suggest that the antibody response aga in st the immunodominant epitopes in HCY might be non­neutrali zing . One probable explanation for thi s could li e in the observation that HCY is associated with low-density lipoproteins (LDL) that might be masking the epitopes thereby making them inaccessible for anti body binding and neutrali zation7o

-72

. This being the situation with regard to the anti-HCY antibodies and their protective ability, cellul ar immunity has b d b .. h h 7374 een reporte to e protective In t e ost · . Particularly note worthy is the observation that there seems to be a vigorous response towards the core and the e2 proteins in patients who recover from the acute infection73

. Nonetheless, it appears that such an effec tive CTL response is only for a short period and persistence of the viral infection probably leads to a decline in the antiviral control74

. The vigorous response mounted during acute infection apparently has a negative impact on the liver in the sense that CTLs are documented to have a dual role in infections involving hepatotropic viruses such as HBY and HCY. Based on these observat io ns it is tempting to speculate that a few of the epitopes on Hepatiti s C vira l proteins particularly the Core and E2 gp mi gh t be mimicking some of the cell surface proteins of hepatocytes and this makes sense given the fact that molecular mimicry by viruses is no w a well documented aspect of virology . Moreover, Hijikata et at.,71 have shown the presence of defective naked caps id particles in chronically infec ted pat ients, which might be a viral decoy for subverting the anti vira l response. Collectively, these observations suggest that immune response against the capsid protein of HCY at least is not protecti ve. Nevertheless, in addition to what has been stated earli er, it is essential to clarify the exact protective ability of anti-envel ope gp antibodies, as immune serum globulins (ISG) or polyclonal immune globulin preparations offer a safe, low-cost and effec ti ve _!}leans for preventing HCY infection parti cularl y in post-operative/pre- transfusion settings.

A vailability of full-l ength sequence informat ion of HCY from various parts of the world allowed re­searchers to design peptide assays that in most of the cases are far superior to the above three generations of immunoassays for HCY. Hi story of the development of synthetic peptide-based im munoassays was para ll e l to the development of the above-described three­generation immul1oassays. Of the earl iest immunoas­says developed, the first syn thetic peptide-based en­zyme immul10assays (PBEIA) used peptides from the

554 INDIAN J EXP BIOL, JUNE 2003

capsid/core protein of HCy75.76

. Subsequently, nu­merous reports us ing either oligopeptides or overlap­ping peptides appeared in various journals. Apart from permitting the formulation of immunosorbents with high signal/cut-off ratio for maximum sens itivity and specificity, peptide assays are cost-effective.

In addition to ELiSNantibody based diagnostics , ci rcu lating antigen in the patient sera is detected by scoring for the presence of HCY -RNA by RT-PCR. It should be borne in the mind of the reader that the discovery of the virus was via RT-PCR. A number of labs all over the world developed their own in-house primers for RT-PCR based diagnosis of HCY

infect ion. While 5' UTR of HCY-RNA is used for d · . d RFLP . f h . 677778 h lagnosls an typmg 0 t e virus . . , ot er regions such as the Core80 and NS5 81 are amplified using ei ther type specific primers or universal primers, essenti ally with an aim at genotyping of the virus once differential di agnosis has been done. RT­PCR based di agnostics, in addition to being sensitive and accurate, offers quantification of the pathogen that is essential once a patient is put on treatment. Yariants of PCR based diagnosis such as bONA

c

5'UTR

Amplified by RT-PCR

El E2 p7 NS2 NS3

Viral Antigens

assays, etc ., are ava ilable in the market. Put simply , to get a complete pi cture of the viral infection before a patient is given treatment and for monitoring the viral dynamics during and after the treatment, it is prudent to take into account the results of both antibody and RT-PCR tests. In spite of this, RT-PCR diagnosis is not routinely implemented in third world countries owing to either economic reasons o r lack of necessary facilities or qualified personnel.

Presently opinion is divided as to whether there is any need for further improvement of the currently ava ilab le 3'd generation immunoassays. While in countries such as USA, there might not be any necessity to improve the test, for reasons already mentioned, stored tropical samples continue to give fa lse positive results. If a patient is also infected with malaria, a tropical disease, widespread in this part of the globe, the person' s serum sample tends to give false positive results42

. Adding to this is the variations in the genome of the virus observed in a given region, makes the development of better-suited assays for detection of thi s viral infection, imperative. In thi s context RT-PCR based testing yields distinct results, which however as stated earlier is not a feasible

NS4aNS4b NS5a

Strip Test

NS5b 3'UTR

3rd Generation

2nd Generation

i " Generation

Fig. 2 - Schematic representation of major immunodominant epitopes iden ti fied on HCY polyprotcin RT-PCR: reverse transcription­polymerase chain reaction; UTR: Untranslated region; a - K: major immunodominant epitopes identified . Amino ac id positi on of epi­topes (a - K) on HCY polyprotein - a: 7_2565.67.75.76.149; ~: 35-6364 ; X: 222_24864 -67; 8: HYR 1,383-40962 ; £: 413_43662.65.68; <1': 440_46465-6H; y: 857_88065-67 ; 11 : 1188_146564-67 ; t : 1696_173964-67. 15°; <p : 1916-194464 ; K: 2263-23 1864-67

PODURI: HEPATITIS C VIRUS-A REVIEW 555

alternative in third world countries wherein the facilities, particularly in the rural areas, many a time are not available.

Despite the fact that only -70-94% of repeatedly HCV antibody positive patient samples score positive for HCY-RNA by RT_PCR82. 83, HCV-RNA detection is many-a-time used to confirm HCV-antibody screening tests . This is where strip-based tests such as INNO-LIA, RIBA, etc., offer a reliable alternative64

.

Apart from the antigens used in the 3rd generation ELISA, representative sequences from the E2 gp (a. a. 383-410, HVR 1; epitope 8, Fig. 2) are also included in these strip-based tests, thereby, in certain situations, making them a feasible alternative to RT­PCR, even in the third world countries.

Prevalence and genetic heterogeneity of Hev (with specific reference to Indian Subcontinent)

Once a diagnostic assay is available, it is imperative to estimate the extent and magnitude of the viral infection in the population . While current estimates put the number of affected individuals at 170 million people worldwide, prevalence statistics for the Indian subcontinent range from as low as 0.34%84 to 2.5 %65.67.85 (Table 3). In addition to this, a

vast number of cases go unreported and the insufficiency of the currently available diagnostic immunoassays in the Indian context has been clearly demonstrated65-67. 86.87 .

Hepatitis C Virus is an RNA virus and still evolving with an estimated frequency of 1.4-1.9 x 10-3

mutations per nucleotide per year88.89. Because of this, the virus displays significant sequence diversities in the entire nucleic acid, in general, and in the amino terminus of the E2 gene, termed the Hyper variable region (HVR), in particular. This is due to the lack of

proof reading activity of RNA-dependent RNA polymerase of RNA viruses leading to a high frequency of nucleotide substitutions during replication . This genetic heterogeneity of HCV has been classified under two headings - Quasispecies and Genotypes. While the genetic heterogeneity of HCV population observed within a single individual is referred to as Quasispeciei6.34.88.90, the term Genotype is applied to the genome heterogeneity observed among different HCV isolates. Owing to accumulation of mutations during evolution of these viruses, HCV isolates can be divided into distinct groups25.26.34.50.5 1. To date, at least six genotypes and

close to 80 SUbtypes have been identified and more are in the process of being characterized. Hence the present classification of HCY is incomplete and more types and SUbtypes are likely to be added. HCV types are now designated by Arabic numerical in their order of di scovery (e.g., 1,2,3, etc.), while the SUbtypes are designated in lower case letters, also in their order of discovery (e.g., a, b, c, etc.)91. Overall there is no preponderance of genotypes geographically and more than one genotype is prevalent in almost all the countries from where data is available. However, the current pandemic of HCV is dominated by type 1, which is arguably the most severe form of the virus.

Prevalence of genotypes 165-67.85.92-96, 293, 365-67.85.92-95,

4 and 595 and their variants have been documented from the Indian sub-continent. A note-worthy obser­vation in these genotyping studies is that almost all the reported variants of genotype 3 (viz. a to g, except e) have been reported from India, while only three sub­types of 1 (i.e., la, 1b and lc) are found65.92-94. This pattern is suggestive of a long-standing endemic in­fection with ,genotype 3 of HCV in India, and the presence of fewer variants of type 1 is indicative of recent acquisition of this type.

Table 3 - Prevalence statistics for hepatitis C virus (HCV) infection in different categories in India

Category Prevalence range Cumulative total of individuals Actual anal yzed in different studies@ prevalence*

General popul alion# 0.34-2.5% 82228 0.70%

Patients with different liver disorderss 2.35-42.10% 4530 3.42%

Hospital staff 0.0-9 .09% 155 6.45% Patients visiting STD clinics 0.64-21.10% 616 9.60%

Total 87529 0.90%

# Apparently healthy voluntary blood donors taken as general popul ation sIncludes primarily patients with acute hepatiti s, chronic hepatiti s, cirrhosis, and hepatocellular carcinoma @Sum of all the individuals examined in the references cited § Assuming homogeneity in the samples In addition to the above, a prevalence of 7.89% was reported from Lisu community of Arunachal Pradesh l47

Reference

66,67,84,85, 140, 143, 146,147,149, 150, 151

66,67,138-141,144-146, 152

144,146,151 142,148

556 INDIAN J EXP BIOL, JUNE 2003

Presence of numerous genotypes and their variants pose problems both in effective diagnosis and management of the viral infection. This situation necessitates development of reagents and methodologies taking into account all the prevalent types in a given country. Consequently, the diagnostics developed by Panigrahi et al. 87 and Pod uri et al. 65

.67 attain significance, while specific therapeutic

regimen taking into account the prevalent types in the country as well as the variants of the virus as Quasispecies in a given individual is yet to evolve.

Hepatitis C virus as a causative agent of hepatocel­lular carcinoma (HCC)

Viruses represent a major ri sk factor in a significant portion of human cancers. Whereas most of the viruses that cause cancer have DNA as their genetic material or have a DNA intermediate in their replication , HCY is a typical RNA virus with no DNA intermediate3

. Hence the possibility of integration of the viral genome is probably an unlikely event. Regardless of this, it is now well established that HCV is stro ngly linked to the development of hepatocellular carcinoma (HCC) thereby representing a major complication in patients chroni cally infected with the virus8

.9

.97

.

Quite a significant number of reports have associated HCV with HCC8.IO.II ,98.I OO. Contrary to the

earlier report that HCY might not be an etiologic f I d · . 101 Pd' I 6667 actor among n Jan patients , 0 un et a. . demonstrated that one majority (75 %) of the HCC patients had active HCY replication , while in the rest the virus appears to be sequestered. Moreover, all the patients with active viral replication have detectable antibodies to all of the identifi ed immunodominant epitopes. Tn those patients where the virus appears to be sequestered, the antibody response seems to be directed mostly to the epitopes on the core, E2 gp and the NS3 proteins of the virus. While these results suggest a probable association of HCV with HCC in Indian patients, it is tempting to speculate that these three prote ins , viz. E2 gp, Core & NS3, have a role to play in hepatocarcinogenesis .

Although these studies associate HCY with HCC, the exact role of the virus or which viral proteins are in volved in this process remains to be clarified. There being controversial evidence with respect to the . I f h 53 . d 10) ·104 h lI1 VO vement 0 t e p gene or Its pro uct - , t e core and the NS3 proteins are now specul ated to play

I . h . . 105· 107 I a ro e In epatocarcll1ogenes ls . t was put forward that these viral proteins might exert their

influence in hepatocarcinogenesis in a manner similar to the E6 protein of Human papilloma viruses (HPV) or the El protein of adenoviruses, that degrade the p53 gene product leading to tumor formation . In addition to this, Kane et aI. I03 have demonstrated the presence of high levels of inducible nitric oxide synthetase (iNOS) and nitric oxide in patients who are chronically infected with HCY. Kane et al. hypothesized that genomic instability results from prolonged exposure of hepatocytes to nitric oxide, a well-known mutagen . Subsequently, subversion of cellular repair mechani sms and apoptotic strategies by the viral proteins, such as the core and the NS3 , leads to a malignant transformation eventually progress ing to HCC. These hypotheses are put together as one poss ible di sposition of HCY in inducing hepatocellular carcinoma in Fig. 3 . Although researchers all over the world might turn their attention towards the ARF concept to clarify the role of the virus in HCC eventually, it would be premature at present to contemplate on the possible role of ARF/ARFPs in hepatocarcinogenesis .

Future perspectives While a satisfactory prophylaxis is still not

availabl e, interferon either alone or in combination with ribavirin are the currently used drugs of choice. However, only about 11-30% of the patients put on the treatment respond l08

, and approximately 50-60%

Nccro- inl1:lI1llll alory s l:llc

C hronic Ilev illkc ti o n

ImPAIDCFiJ-,uiA I~ CAJ~CIN~@ .. I lie" CO l't·/.\SJ·J ~

Over (' .'pl'cssioli of ii\OS

I Incrcasc<lICl'cls of1\O I t

Gcnolllic Insta bilil.' ·

! ~ }~

Fig. 3 - Probable role of HCY in hepatocellul ar carc inoma (HCC). Figure shows one pl ausibl e role of HCY in hepatoca r­cinogenesis. Chronic infec tion with HCY leads to chronic necro­inn ammatory state. In this s itu at ion, over ex press ion of inducibl e nitri c ox ide (iNOS) leads to excess ive production of nitric ox ide (NO), thi s is produced essentiall y as a vasod il ator to ensu re con­stant blood pressure. Nitric oxide is a well-known mutagen and constant prolonged ex posure of ce lls to NO leads to genomic in­stab ility . Cellul ar repa ir mechani sms of which , as well as apop­tot ic s trategies, viral protein s such as the core and the NS 3 pro­te ins inhibit. T hi s leads to malignant transformatio n eventua lly manifes ting as hepatocellula r ca rc in oma

PODURI: HEPATITIS C VIRUS - A REVIEW 557

of patients respond to combination therap/4,109 making search for alternative strategies imperative, A plausible direction in this regard is given by the observation that there is improper vertical transmission of the virus and the lack of an efficient cell culture system. It is now documented that there is an apparent suppression of liver damage during gestation 110. While this observation supports the concept that liver damage in hepatitis C infection is related to immune response III, elevated levels of lactoferrin, a broad-spectrum immunomodulator, in gestating and lactating women 11 2 might be the actual reason for the improper vertical transmission of the virus, Supporting this statement is the documentation that lactoferrin binds HCy I1 3 and markedly inhibits the viral replication in cultured cells 11 4,115. Ongoing trials for treating HCY infected patients with lactoferrin are showing promising resultsI1 6-11 8. The small amount of lactoferrin present in fetal bovine serum (FBS) used, might in fact be inhibiting the viral replication in cell cultures, In thi s context, HCY replicons 119 and the cell free systems 120, 121 are bound

to make an impact. It was recently demonstrated that the success achieved with regard to HCY replicons and identification of mutations that permit HCY replication 122 in cell cultures need not necessarily apply to the in vivo infectivity in chimpanzees123. A probable explanation for this could lie in the observation that the HCY replicons are exceedingly artificial. HCY replicons were designed to be bicistronic resulting in translation of the first cistron (neomycin phosphotransferase) being directed by HCY-IRES and translation of the second cistron by encephalomyocarditis virus (ECMY) IRES , Additionally, these molecules were derived from an HCY consensus genome, Nevertheless, this system is greatly suitable for drug screening as all the known HCY enzymes are encoded by this system.

An alternative approach in treating HCY induced HCC lies in the observation that mice immunized with viral peptides complexed to heat shock protein (HSP-70) resulted in protective immunity and specific cytotoxic T-Iymphocytes (CTL)1 24, 125. Consistent with these observations in mice, it is reported that cancer patients immunized with autologous cancer derived heat shock protein preparations develop anti-tumor immunity 126, Moreover, HSP-peptide complexes can be reconstituted in vitro to elicit peptide-specific CTL response as well as anti-tumor immuni ty 127. Taken together these findings clearly suggest that prepara­tions of HSP-peptide complexes is an alternative and

viable option for combating viral infections where patients do not respond to the conventional strategies that are being currently used.

While the above two strategies take advantage of the host defense mechanisms, active research target­ing viral proteins or stages in the viral replication is on-going around the world . Significant among these . I d'b I'd I J?8 'b 129· mc u e n onuc eosl e ana ogs - , fl ozymes , antI -sense RNA 130, protease inhibitors 131. Among those that are in the pipeline, include Glycyrrhizin, a triter­pene glycoside and extract of licorice root, in combi­nation with interferon 132, This herbal preparation is known to have antiviral activity . It is used routinely in the traditional medicine in the eastern parts of the world to treat diabetes and nephritis. In addition to these, research on the protective ability of immune serum globulin (ISG) or polyclonal immune globulin preparations offer a vi able substitute particularly in post-operative/pre-transfusion settings , Whereas the discovery of CD81 as a possible receptor for entry of the virus into cells '33 does definitely provide an ex­planation to the extra-hepatic manifestations ob­served, identification of the actual receptor responsi­ble for hepatotropism should expedite the drug di s­covery process with regard to this viral infection, apart from enabling researchers in reassessing the ef­ficiency of the currently available therapeutic regi­men. Identification of cell culture systems that sup­port HCY replication I1 9,120,121.122,134, 135 and develop-

ment of the chimeric mouse-model '36 should also aid in thi s process.

Conclusion With the current pandemic of HCY affecting about

170 million people worldwide, emphasis should be mainly on evaluating the actual role of anti-E2 gp antibodies, as these form an indispensable diagnostic marker, probably might also provide an insight to the disease progression, Additionally, it can be predicted that a significant proportion of those affected eventually develop HCe. Hence elucidating whether HCY exerts its influence directly or indirectly in the development of HCC becomes imperative.

Recent noteworthy developments such as identification of CD81 as the possible receptor for entry of the virus into the cells 133

, identification of cell culture systems I 19-122,1 34 , the ARF concept37,38 and the

development of a mouse model system l36 should aid in delineating a number of hypotheses that are in circulation in the literature. Coming years will witness development of novel and much better therapeutics that might include polyherbal preparations,

558 INDIAN J EXP BIOL, JUNE 2003

Acknowledgement The author wishes to acknowledgement the all

round support of Drs. M . R. Das and Ch. Mohan Rao, and my colleagues both at RGCB and CCMB.

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