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 ~  Abstract
 ~  Materials and Me...
 ~  Reverse Transcri...
 ~  PCR profile
 ~  Radioimmunopreci...
 ~  Indirect immunof...
 ~  DNA immunization...
 ~  Assay for anti-H...
 ~  Results
 ~  Elisa
 ~  Discussion
 ~  Acknowledgements
 ~  References

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ORIGINAL ARTICLE
Year : 2003  |  Volume : 21  |  Issue : 2  |  Page : 93-97
 

Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct


Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 110 029, India

Correspondence Address:
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 110 029, India

 ~ Abstract 

PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA) and immunofluorescence (IFA). The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

How to cite this article:
Ray S, Broor S I, Vaishnav Y, Dar L, Seth P, Broor S. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct. Indian J Med Microbiol 2003;21:93-7


How to cite this URL:
Ray S, Broor S I, Vaishnav Y, Dar L, Seth P, Broor S. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct. Indian J Med Microbiol [serial online] 2003 [cited 2019 Aug 25];21:93-7. Available from: http://www.ijmm.org/text.asp?2003/21/2/93/7982


Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis.[1] Infection with HCV proceeds to chronicity in more than 70% of the cases with progression to cirrhosis and hepatocellular carcinoma.[2],[3] Interferon alfa (INF-a) treatment alone, benefits only 15-25% of patients with chronic HCV infection whereas the use of the combination of interferon with the nucleoside analogue like ribavarin results in a sustained virological response in 30-40% of patients, but may be associated with side effects.[4],[5] Thus the need for an effective vaccine in HCV to prevent infection is obvious. In this regard, direct injection of HCV cDNA into animals is a novel and promising method for delivering specific antigens for immunization. This approach has been successfully shown to elicit protective immune response against several viral infections like influenza virus, herpes simplex virus type 2, hepatitis B virus and other viral models.[6]
HCV is a single, positive stranded RNA virus belonging to the genus hepacivirus in family flaviviridae. The linear genome of 9.5 kb contains a large open reading frame that encodes a polyprotein precursor, 3010 to 3033 amino acids long.[7] The viral structural and non-structural proteins are produced as a result of cleavage of a large polyprotein precursor by host cell signal peptidases and viral proteinases respectively.[8] Among the structural proteins the nucleotide and the predicted amino acid sequence of the core protein is well conserved in different HCV isolates,[9] whereas the envelope proteins E1 (gp32-35) and E2 (gp68-72) show genetic heterogeneity. Due to high rate of mutations in the envelope region, quasispecies are generated which may provide an immune escape mechanism allowing viral persistence in the host.
Both humoral and cellular immune responses are likely to be important in immunity to HCV. Many recent reports have focused on DNA based immunization using HCV core and envelope antigens.[6],[10],[11],[12],[13] However, in most of these studies a good humoral response was not elicited.[11],[13] In this study, we have explored the efficacy of humoral immune responses in mice inoculated with a plasmid DNA containing subgenomic fragments of HCV DNA to lay a foundation for HCV nucleic acid vaccine development in the future.

 ~ Materials and Methods Top

Plasmid construction
The BK-146 clone,[14] kindly gifted by Chisato Mori of Biken Research Foundation, Japan, comprises of nucleotides 39-4839 of the viral genome and is incorporated at the EcoR1 site in pUC19 vector. This construct was digested by Stu1 (3 sites of Stu I at 267, 3798 and 4555 nt positions) and the ~3.5 kb Stu I fragment (267-3798 nt position in the HCV genome) was gel purified. This segment was then ligated with Sma I digested, CIAP (calf intestinal alkaline phosphatase) treated and gel purified pMT3 vector,[15] an eukaryotic expression system with an adenoviral major late promoter [Figure - 1], to make pMT3-BK146 construct.
The orientation of the construct was checked by Cla I digestion (for which the restriction sites were present both in the cloning plasmid and the insert) that gave a 3.1 kb small and a 5.5 kb large fragment in the sense orientation. The fragments obtained on antisense orientation were of 432 bp and 8.2 kb (data not shown). This construct comprising of the Stu I fragment of 267-3798 nucleotides (~3.5 kb), has the potential to code for the entire core, E1, E2 / NS1, NS2 and a small portion of NS3. The presence of the insert in the pMT3- BK146 construct was checked by polymerase chain reaction (PCR) using core primers. Large-scale preparation of plasmid DNA was made by cesium chloride density gradient centrifugation and stored at -20C till further use.
Transfection
COS cells obtained from American type culture collection (ATCC, Rocksville, MD, USA) were maintained in Dulbecco 's Minimum Essential Medium (DMEM) (Life Technologies, USA) with 10 % FCS (fetal calf serum), while HepG2 (human hepatoblastoma) cells obtained from National Center for Cell Sciences, Pune, India, were maintained in Eagle's Minimum Essential Medium (EMEM), (HiMedia Laboratories, Mumbai, India) with 10 % FCS (fetal calf serum), and 0.01 % sodium pyruvate. Both the cells were grown in 60mm tissue culture dishes (Costar, Corning, NY, USA) and incubated at 37C in 5 % CO2 atmosphere. When the cells were 30 to 50 % confluent, they were transiently transfected with pMT3- BK146 HCV clone using lipofectin (Life Technologies, MD, USA), according to the manufacturer's instructions. The transfection efficiency was checked by b-galactosidase assay using pCMV-gal as the reporter plasmid. The protocol used was a standard colorimetric assay using ortho-nitrophenyl b-D galactopyranoside (ONPG).[16] Cells were harvested 72 hours post transfection and checked for expression of the desired mRNA and proteins by (a) RT-PCR (reverse transcription-polymerase chain reaction), (b) radio-immunoprecipitation and (c) indirect immunofluorescence. In each experiment mock transfected cells (cells transfected with the pMT3 vector alone) were used as control.
Expression in mammalian cells: RT- PCR, Radio-immunoprecipitation, and Indirect-immunofluorescence

 ~ Reverse Transcription-Polymerase chain reaction (RT-PCR) Top

RNA was extracted from the transfected COS and HepG2 cells by Chomczynski and Sacchis single step RNA extraction method.[17] First strand cDNA (complementary DNA) was synthesized using 7 U Avian Myeloblastoma virus (AMV) reverse transcriptase (Promega Corp. Madison, USA), and 100 picomolar (pM) of antisense primer from the HCV core region. Reverse transcription was carried out at 42C for one hour followed by enzyme inactivation at 65C for 5 minutes. PCR was carried out for HCV using primers from the core region.
Primer sequences[18]
Sense primer: 5'- CGC GCG ACT AGG AAG ACT TC-3' (nt 139-158).
Antisense primer: 5' - ATG TAC CCA TGA GGG TCG GC-3' (nt 391-410).

 ~ PCR profile Top

The cDNA was subjected to amplification by denaturing at 95C for 1 minute, annealing at 55C for 1 minute and extension at 72C for 2 minutes for 30 cycles using 0.2 mM of dNTPs, 20 pM of sense and antisense primers each and 2 U of Taq DNA Polymerase (Genei, Bangalore, India). Final extension was done for 7 minutes at 72C. The amplified product was run on a 1.5% agarose gel in Tris- borate EDTA (ethylenediamine tetra acetic acid) buffer containing ethidium bromide and visualized under an UV (ultra violet) transilluminator and photographed.

 ~ Radioimmunoprecipitation (RIPA) Top

After 72 hours post transfection the COS cells were washed with PBS (pH 7.2) and radiolabelled with 400 mCi of [35] S methionine (BARC, India) and incubated for 3 hours with constant shaking at 37C. Cell lysates were prepared in 500 mL of RIPA (radioimmunoprecipitation assay) buffer.[16] The cell lysate was treated with 20 mL (1:25 dil) of anti-HCV positive serum from a patient, which showed a strong band with the core antigens, no band with the envelope antigens and only a faint band with NS-3 antigen, by LIATEK (Line Immunoassay, Organon Technika, Netherlands). For control, anti HCV negative serum was used. The mixture was incubated for 1 hour at 4C with 40 mL of protein-A sepharose CL-4B (Pharmacia Biotech, Upsalla, Sweden). The final antigen - antibody complex was then centrifuged and the pellet washed and run on a 7.5 % polyacrylamide gel. The gel was then dried and developed by exposing it to an X-ray film (Kodak, Rochester, NY, USA).

 ~ Indirect immunofluorescence Top

HepG2 cells were grown in 24 well plates (Costar, Corning, NY, USA) containing 12mm coverslips. At 30 to 50 % confluency the cells were transiently transfected with pMT3- BK146 clone of HCV using lipofectin (Life Technologies, MD, USA). At 72 hours post transfection, the cells on coverslips were washed with PBS and fixed with chilled acetone. Indirect immunofluorescence was carried out with the same human sera (1:20 dilutions), which was used in RIPA. Serum samples that tested negative for HCV and mock-transfected cells were used as negative controls. The coverslips were incubated at 37C for one hour in a humid chamber after adding the primary antibody, followed by 3 washes with PBS (pH 7.2). Rabbit anti-human FITC (fluorescein isothiocyanate conjugate) (Sigma, St. Louis, MO, USA) was used as secondary antibody and the cover slips were incubated for one hour at 37C. At the end of incubation the coverslips were washed as before and counterstained with Evan's blue, mounted in glycerol buffer and viewed under the epifluorescence microscope (Carl Zeiss, West Germany), using 20x and 40x neofluar objectives.

 ~ DNA immunization of BALB/c mice Top

Twenty mg of DNA (pMT3-BK146) was injected into the footpads of three (X,Yand Z), 8 weeks old female BALB/c mice followed by a booster dose with the same amount of DNA after 6 weeks. Additional three mice (A,B, and C) were injected only with the vector DNA to serve as controls. The experiment was repeated in another set of 3 mice each (X1, Y1 and Z1) as test and A1, B1 and C1 as control animals.

 ~ Assay for anti-HCV antibodies in immune sera of mice Top

Serum from immunized mice was collected at 4 weeks after primary immunization and 6 weeks after the booster, by retro-orbital bleeding and the presence of anti- HCV antibodies was detected by ELISA (UBI Inc, USA) and indirect immunofluorescence. Pre immune sera collected from the same mice was used as control. ELISA was carried out at 1:20 dilution of mice sera according to the manufacturer's instructions in the kit except that an anti-mouse IgG peroxidase conjugate (Sigma, St.Louis, MO, USA) was used in place of the kit's anti-human peroxidase conjugate. Indirect imunofluorescence was done in HepG2 cells transiently transfected with the pMT3 BK146 clone using 1:10 dilution of mice sera as mentioned before except the conjugate used here was goat -anti mouse FITC (Genei, Bangalore, India).

 ~ Results Top

In-vitro expression of HCV m-RNA and proteins
Expression of HCV mRNA and protein was determined in COS and HepG2 cells transiently transfected with pMT3-BK146 clone of HCV. The transfection efficiency of these cells were determined using b-galactosidase assay by which the transfected cells showed an O.D of ~ 0.4 while the mock transfected cells showed <0.2 when assessed spectrophoto-metrically.
Expression of m-RNA for HCV core by RT-PCR
A 272 bp band was obtained by RT-PCR from the RNA extracted from COS and HepG2 cells transiently transfected with the pMT3-BK146 clone of HCV. No such band was observed in mock transfected cells or untransfected cells [Figure - 2]. These results prove the expression of HCV core specific mRNA in cells transfected with the HCV clone only. In the DNA control in which directly amplification was done without reverse transcription to check DNA contamination, no amplicon was detected. These results confirm the presence of RNA transcript and rules out any false positive amplification due to DNA contamination.
Lane M: Molecular weight marker (HaeIII digested phiX DNA), Lane 1: Negative control, Lane 2: Mock transfected HepG2 cells, Lane 3: 272bp band of HCV core: obtained after RT-PCR of mRNA, obtained from transfected HepG2 cells. Lane 4. Positive control: A band of 272 bp, PCR product of pUC19-BK146 clone
Radioimmunoprecipitation
A band of 46 kDa was obtained by RIPA in cell lysates treated with anti HCV positive serum whereas no such band was observed in cell lysates treated with anti-HCV negative serum [Figure:3].
Lane 1: Protein molecular weight marker, Lane 2: Empty well, Lane 3: HCV- transfected COS cell lysates treated with normal human serum (NHS) negative for anti-HCV antibody, Lane 4: A 46 kDa band of core protein (dimer) was seen in cell lysates, treated with anti-HCV antibody positive serum.
Expression of HCV proteins by indirect immunofluorescence
Specific apple green fluorescence was observed in HepG2 cells transiently transfected with the pMT3-BK146 clone of HCV and stained with human serum positive for anti-HCV antibodies . No fluorescence was observed either in transfected cells treated with human serum negative for anti- HCV antibody or mock transfected cells treated with human sera both positive and negative for anti - HCV antibody. This indicates the expression of the HCV proteins in the cells transfected with the clone of HCV.
Antibody response to HCV in BALB/c mice

 ~ Elisa Top

The mean O.D. (optical density) values of the pre and post immune mice sera (1: 20 dilution) for anti HCV antibody by ELISA are shown in [Table - 1] (results of one set of experiment).
Any optical density value greater than twice that of the pre-immunized serum was taken as positive. Mice X did not show any antibody response after the first immunization but all the 3 mice (X, Y and Z) showed a >2 fold increase in O.D values as compared to the values in the serum obtained after primary immunization. In control mice the OD values before and after immunization with vector DNA were almost the same. In the second set of experiment all the 3 mice showed antibody response after first dose only (data not shown).
Indirect Immunofluorescence
Specific apple green fluorescence was observed in HepG2 cells transiently transfected with the pMT3-BK146 clone of HCV and stained with the above mentioned immunized mice serum at 1:10 dilution. No fluorescence was observed either in transfected cells treated with pre-immunized mice sera or mock-transfected cells treated with immunized mice sera.

 ~ Discussion Top

DNA- based immunization has been shown to be useful in inducing immune responses to proteins of several pathogens like influenza virus A, human immunodeficiency virus 1, bovine herpes virus, plasmodia, shigella, herpes simplex virus type 2 and hepatitis B virus as well as mutant p53, a tumor antigen.[6] Immunization of experimental animals with recombinant plasmid DNA encoding influenza virus proteins has been shown to elicit specific humoral and T-cell response and resulted in protection from subsequent challenge.[19] Neutralizing antibodies were elicited to homologous DNA vaccines encoding HIV1 envelope glycoprotein,[20] and bovine herpes virus 1,[21] in experimental animals. These studies indicate that DNA immunization can elicit protective immune responses and is a promising area for subunit vaccine development. In HCV infection, DNA based immunization appears to be a good approach for prevention of infection or as an adjunct to antiviral therapy.
In this study, we have prepared a construct derived from the structural fragment of the HCV genome using pMT3,[15] an eukaryotic expression vector with an adenoviral major late promoter. Earlier reports have used recombinant vaccinia and Sindbis virus vectors or eukaryotic expression vectors containing cytomegalovirus promoters.[6],[22] Although DNA immunization has been shown to induce strong antibody response to many viruses,[19],[20] the antibody response following DNA immunization with HCV core gene constructs was either weak or only IgM response was observed.[10],[13] The pMT3 vector has the advantage that adenovirus tripartite leader sequence and VAI (adenovirus associated RNA I), gene products of this vector enhance the translatability of the mRNA of interest.[23] The demonstration of core specific HCV mRNA in both COS and HepG2 cells by RT-PCR and the presence of HCV core protein in transiently transfected cells by indirect immunofluorescence showed that there is an effective transcription and expression of the target genes using this vector. The expression of HCV was further confirmed by RIPA in which a 46kDa band was obtained by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This band could be a dimer of the 23kDa core protein as the latter is highly basic and has the tendency to form homodimers and multimers and has multiple functions.[24] Alternatively it could be a combination of one or more HCV proteins. A strong HCV antibody response was observed in mice after injection of our c-DNA construct by intramuscular route. Further an enhanced immunologic response after the booster dose suggests that this vector may prove useful in eliciting a strong antibody response to HCV structural proteins.
Genotype II or 1b of HCV has been reported to be the most frequent HCV genotype prevalent in India[25],[26],[27] and our clone BK146 also is derived from genotype 1b. Thus our construct may be useful for DNA vaccine development studies in the Indian setting.
In conclusion, it may be said that although several plasmid based systems have been tried previously, the pMT3 vector with the BK 146 insert produced a good antibody response and thus may be an initial step towards vaccine development for HCV. The current study is only an experimental exercise to determine the immunogenicity of HCV cDNA in pMT3 vector. Further studies with a construct containing only the core gene of HCV, are underway in our laboratory, in order to specifically demonstrate the immunogenic potential of the core protein.

 ~ Acknowledgements Top

The authors thank Dr.Chisato Mori, Biken Research Foundation, Japan, for the gift of the BK-146 clone of HCV. This work was supported by a grant from the University Grants Commission, India. 

 ~ References Top

1.Alter HJ, Purcell RH, Shih JWK, Melpolder JC, Houghton M, Choo Q-L, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. New Engl J Med 1989;321:1494-500.  Back to cited text no. 1    
2.Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, et al. The natural history of community-acquired hepatitis C in the United States.The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. New Engl J Med 1992;327:1899-905.  Back to cited text no. 2    
3.Saito I, Miyamura T, Ohbayashi A, Harada H, Katayama T, Kikuchi S, et al. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci USA 1990; 87:6547-49.  Back to cited text no. 3    
4.McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998;339:1485-92.   Back to cited text no. 4    
5.Davis GL, Esteban - Mur R, Rustgi V, Hoefs J, Gordon SC, Trepo C, et al. Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. N Engl J Med 1998;339:1493-9.   Back to cited text no. 5    
6.Saito T, Sherman J, Kurokohchi K, Guo ZP, Donets M, Yu MY, et al. Plasmid DNA - based immunization for hepatitis C virus structural proteins: immune responses in mice. Gastroenterology 1997;112:1321-30.  Back to cited text no. 6    
7.Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, et al. Genetic organisation and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991; 88: 2451-55.  Back to cited text no. 7    
8.Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993; 67:1385-95.  Back to cited text no. 8    
9.Bukh J, Purcell RH, Miller RH. Sequence analysis of the core gene of 14 hepatitis C virus genotypes. Proc Natl Acad Sci USA 1994; 91: 8239-43.  Back to cited text no. 9    
10.Tokushige K, Wakita T, Pachuk C, Moradpour D, Weiner DB, Zurawskiv R Jr, et al. Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology 1996;24:14 -20.  Back to cited text no. 10    
11.Lagging LM, Meyer K, Hoft D, Houghton M, Belshe RB, Ray R. Immune responses to plasmid DNA encoding the hepatitis C virus core protein. J Virol 1995;69:5859-63.  Back to cited text no. 11    
12.Tedeschi V, Akatsuka T, Shih JWK, Battegay M, Feinstone SM. A specific antibody response to HCV E2 elicited in mice by intramuscular inoculation of plasmid DNA containing coding sequences for E2. Hepatology 1997;25:459-62.  Back to cited text no. 12    
13.Hu G-J, Wang RYH, Han DS, Alter HJ, Shih JWK. Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization. Vaccine 1999;17:3160 -70.  Back to cited text no. 13    
14.Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, et al. Structure and organisation of the hepatitis C virus genome isolated from human carriers. J Virol 1991;65:1105-13.  Back to cited text no. 14    
15.Swick AG, Janicot M, Cheneval-Kastelic T, McLenithan JC, Lane MD. Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes. Proc Natl Acad Sci USA 1992; 89:1812-16.  Back to cited text no. 15    
16.Sambrook J, Russel DW. Molecular Cloning- A Laboratory Manual. 3rd Ed. Cold Spring Harbour Laboratory Press, New York, USA; 2001,pp -17.48-17.51  Back to cited text no. 16    
17.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-59.  Back to cited text no. 17    
18.Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673-9.   Back to cited text no. 18    
19.Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993;259:1745 -49.  Back to cited text no. 19    
20.Wang B, Ugen KE, Srikatan V, Agadjanyan MG, Dang K, Refaeli Y, et al. Gene inoculation generates immune responses against human immuno deficiency virus type 1. Proc Natl Acad Sci USA 1993;90:4156 - 60.  Back to cited text no. 20    
21.Cox GJM, Zamb TJ, Babiuk LA. Bovine herpesvirus 1 immune responses in mice and cattle injected with plasmid DNA. J Virol 1993;67:5664 -67.  Back to cited text no. 21    
22.Dubuisson J, Hsu HH, Cheung RC, Greenberg HB, Russell DG, Rice CM. Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses. J Virol 1994; 68:6147-60.  Back to cited text no. 22    
23.Kaufman RJ, Davies MV, Pathak VK, Hershey JWB. The phosphorylation state of eukaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol 1989;9: 946-58.  Back to cited text no. 23    
24.Matsumoto M, Hwang SB, Jeng K-S, Zhu N, Lai M.MC. Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 1996;218: 43-51.  Back to cited text no. 24    
25.Nousbaum JB. Genomic subtypes of hepatitis C virus: epidemiology, diagnosis and clinical consequences. Bull Soc Pathol Exot 1998;91:29-33.  Back to cited text no. 25    
26.Panigrahi AK, Roca J, Acharya SK, Jameel S, Panda SK. Genotype determination of hepatitis C virus from northern India: identification of a new subtype. J Med Virol 1996;48:191-8.  Back to cited text no. 26    
27.Valliammai T, Thyagarajan SP, Zuckerman AJ, Harrison TJ. Diversity of genotypes of hepatitis C virus in southern India. J Gen Virol 1995;76:711-6.  Back to cited text no. 27    
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