Title:                     RFLP Typing of Hepatitis C Virus  (HCV) isolates from infected Indian patients.

 

 

 

 

Authors:                           C. D. Poduri and M. R. Das

and Affiliations                          Rajiv Gandhi Center for Biotechnology

Jagathy

Trivandrum – 695 014

Kerala, India.

 

 

Correspondence:                    Dr. M. R. Das

                                                Rajiv Gandhi Center for Biotechnology

                                                Jagathy

                                                TRIVANDRUM – 695 014

                                                Kerala, INDIA

                                                E-mail: rgcbt{at}md2.vsnl.net.in

                                                Fax: 0471-329472

 

 

Running Head:                            Typing of HCV isolates from India.

 

Paper Category:       Research Letter.

Summary.

 

RFLP typing of hepatitis C virus (HCV) isolates from seven of ten infected Indian patients was carried out and all were found to be infected with genotype 3b of HCV.  Earlier studies have suggested that type 1 could be the prominent form present in India.  Our results clearly suggest that there are two concurrent on-going infections of HCV in India caused by types 1 and 3.

 

 

Key Words:                    Hepatitis C Virus, RFLP Typing, Indian Isolates,

 

Abbreviations:            HCV:  Hepatitis C Virus; RFLP:  Restriction Fragment length Polymorphism; CH: chronic hepatitis; CIR: Cirrhosis; VBD: Voluntary blood donor; SHC: cases suspected for hepatitis C viral infection.

 

 

Hepatitis C virus (HCV), a major causative agent of viral hepatitis transmitted parenterally, belongs to the genus hepaciviruses, under the family Flaviviridae ¹, and is now well established to be a heterogeneous group.  Infection with this virus causes a repertoire of liver diseases; most significant ones include chronic hepatitis, cirrhosis and hepatocellular carcinoma.  Currently, the only available treatment for this viral infection is Interferon, either alone or in combination with Ribavirin, and only 11 – 30% of the infected patients respond to this treatment ².  With the identification of six genotypes and more than 40 subtypes of HCV, typing of the virus is essential taking into account problems associated with diagnosis of the viral infection and design of therapeutic strategies.  There are very few reports available on the different strains of HCV from India ^{3 - 5}. From these reports, it is inferred that although genotype 1 might be the predominant type in India, variants of type 3 do exist.  Consequently, owing to differences in strains from which the reagents are manufactured and genetic variations of the Indian isolates, dependability of diagnosis of this viral infection in Indian patients is somewhat questionable.

                Routine diagnosis of HCV infection in patients suspected for the viral infection was tested by both RT-PCR ³ and Immunoassays.  Patients were included in the present study based on the presence of sero-markers for HCV infection (positive either for anti-HCV antibodies by an in-house enzyme immunoassay or for HCV-RNA by RT-PCR ³).  Only those patients (total analyzed ten; mean age 46.30+10.71) who are not on treatment at the time of analysis were included in this study.  The study panel included patients with chronic hepatitis, CH (3/10), cirrhosis, CIR (2/10), voluntary blood donor, VBD (1/10), and patients suspected for hepatitis C virus infection, SHC (4/10).   Nine of the patients (2 CH, 2 CIR, 1 VBD, 4 SHC) were positive for anti-HCV antibodies, while one patient with CH was negative for the same although positive for HCV-RNA by RT-PCR. 

                Seven patients (1, CH; 2, CIR; 3, SHC; 1, VBD) scored positive for HCV-RNA by RT-PCR.   For identifying the infecting genotype of the virus in these patients, RFLP was carried out with Hae III and Mva I enzymes essentially as described ³.  However, as genotypes 2, 3 and JKO49 yield similar RFLP patterns with these enzymes, the PCR amplicons were further digested with Hinf I enzyme followed by a double digestion with Hinf I + Mva I enzymes.  The restricted amplicons were checked on a 3% agarose-TBE gel and the electrophoretic patterns thus obtained were compared with the predicted patterns of the corresponding region in full-length sequences (Table I).  All the seven isolates showed similar electrophoretic pattern (Figure 1) comparable to the type 3b.  This is in contrast to the earlier reports ^{3 - 5}  wherein type 1b was reported to be the predominant form in Southern India.

Two patients (1 SHC; 1 CIR), both infected with type 3b, in the present study, were positive for HBsAg.  However, they scored negative for HBV-DNA by PCR.  Suppression of HBV replication in HBV carriers (HBsAg positive, but negative for HBV-DNA by PCR), in patients co-infected with HCV has been documented.  HCV appears to be the causative agent of active hepatitis in such co-infected patients. 

Three patients (2 CH; 1 SHC), who scored negative for HCV-RNA by RT-PCR, were positive for anti-HCV antibodies by a third generation commercial ELISA as well as the in-house PBEIA.  Taken together these results would suggest sequestration of the virus in the liver of these patients.

                In the present RFLP typing, we could not successfully separate genotype 1 from genotype JKO46.  It may be noted that genotype JKO46 is more closely related to genotype 1 than to other types included in the present analysis.  In conclusion, the results from this study indicate that genotype 3b is the prevalent genotype in this part of the country, thereby suggesting that there are two concurrent on-going infections of HCV in India caused by genotypes 1 and 3.

 

ACKNOWLEDGEMENTS

 

C. D. Poduri acknowledges CSIR for the fellowship.  Help rendered by Dr. V. S. Sugunan and Mr. D. Sanjai is gratefully acknowledged.  We thank Dr. Satish Mundayoor for his critical comments.

References

 

1.      Choo Q-L, Richman KH, Han JH, Berger K, Lee C, Dong C, et al.  Genetic organization and diversity of the hepatitis C virus.  Proc Natl Acad Sci USA, 1991; 88: 2451 - 2455.

2.      Neumann AU, Nancy LP, Dahari H, Gretch DR, Wiley TE, Layden TJ, Perelson AS.  Hepatitis C viral Dynamics in vivo and the antiviral efficacy of Interferon-a Therapy.  Science, 1998; 282: 103 – 107.

3.      Das MR, Naushad Ali, Aruna B, Rama Durga, Nambiar A, Rehana Z, et al.  Indian Strains of Hepatitis C Virus: Prevalence and Detection.  Curr Sci, 1993: 65 (6), 477 – 483.

4.      Panigrahi AK, Roca J, Acharya SK, Jameel S, Panda SK.  Genotypic determination of HCV from Northern India: identification of a new subtype.  J Med Virol, 1996; 48 (2): 191 - 198.

5.      Valliammai T, Thyagarajan SP, Zuckerman AJ, Harrison TJ.  Diversity of genotypes of HCV in Southern India.  J Gen Virol, 1995; 76: 71 – 76.

 

Table 1: Combination of predicted electrophoretic patterns in the present RFLP typing

 

Legend: 

A: 213bp, 43bp; B: 160bp, 53bp, 43bp; C: 104bp, 63bp, 48bp, 41bp; D: 208bp, 48bp; E: 256bp (no digestion); F: 150bp, 106bp; G: 150bp, 58bp, 48bp, 48bp.

Size of the undigested PCR amplicon is 256bp.  The PCR amplicons were digested with all the above three mentioned restriction enzymes either alone or in combination as indicated, and the electrophoretypes were compared with the above predicted patterns to determine the genotype.  For generating the above patterns, representative full-length sequences of various genotypes [HCV-1 (1a); HCV-J (1b); HC-G9 (1c); JKO46 (11a?); HC-J6 (2a); HC-J8 (2b); Bebe1 (2c); NZL1 (3a); TR (3b); JKO49 (10a?); and ED43 (4a)] were selected and the electrophoretypes predicted using RESTRI and DIGEST programs of PCGENE software package.  Hae III and Mva I enzymes were originally used for typing of the HCV isolates from India by Das et al. ³ The combination of patterns showed by all the seven patients positive for HCV-RNA was B, D, F, and G as exemplified from figure 1.

 

List of Figures

 

Figure 1.  Title:                 Representative Electrophoretic pattern of Hepatitis C Virus observed in the present study.

 

 

 

Figure 1.  Legend:          The PCR amplicon was digested with the restriction enzymes Hae III (Lane 1), Hinf I (Lane 2), Mva I (Lane 3), followed by a double digestion with Hinf I and Mva I (Lane 4).  The restricted amplicons were run on a 3% agarose-TBE gel to give the above electrophoretype.  Lane 5: Unrestricted PCR amplicon (size 256bp); Lane 6.  DNA size standards – 100bp ladder (New England Biolabs, UK).