|Year : 2013 | Volume
| Issue : 4 | Page : 349-353
Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India
J Christdas, J Sivakumar, J David, HDJ Daniel, S Raghuraman, P Abraham
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Submission||06-Mar-2013|
|Date of Acceptance||23-Jul-2013|
|Date of Web Publication||25-Sep-2013|
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Hepatitis C virus (HCV) is a leading cause of chronic liver disease (CLD) that can progress to cirrhosis and hepatocellular carcinoma. Genotypes of HCV can vary in pathogenicity and can impact on treatment outcome. Objectives: To study the different genotypes among patients with HCV related CLD attending a tertiary care hospital in south India during 2002-2012. Study Design: Study subjects were those referred to clinical virology from the liver clinic. Genotyping was performed using the genotype specific core primers in nested polymerase chain reaction (PCR), 5′ non-coding regions based PCR- restriction fragment length polymorphism and NS5B sequencing methods. With the latter method, obtained sequences were compared with published GenBank sequences to determine the genotype. Results: Of the 451 samples tested, HCV genotype 3 was found to be the most predominant (63.85%). Other genotypes detected were genotype 1 (25.72%), genotype 2 (0.002%), genotype 4 (7.5%) and genotype 6 (2.7%). Genotype 3 was the common genotype in patients from Eastern India while genotype 1 and 4 were mainly seen in South Indian patients. Genotype 6 was seen exclusively in patients from North-Eastern India. Two other patients were infected with recombinants of genotype 1 and 2. Conclusions: In this study spanning a decade, HCV genotype 3 and genotype 1 were found to be the predominant genotypes in the Indian sub-continent. Genotype 4 and genotype 6 appeared to show some geographic restriction. A continued monitoring of HCV genotypes is essential for the optimum management of these chronically infected patients. In addition, knowledge of circulating genotypes could impact on future vaccine formulations.
Keywords: Hepatitis C virus genotypes, Indian sub-continent, NS5B sequencing, treatment outcome
|How to cite this article:|
Christdas J, Sivakumar J, David J, Daniel H, Raghuraman S, Abraham P. Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India. Indian J Med Microbiol 2013;31:349-53
|How to cite this URL:|
Christdas J, Sivakumar J, David J, Daniel H, Raghuraman S, Abraham P. Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India. Indian J Med Microbiol [serial online] 2013 [cited 2020 Aug 10];31:349-53. Available from: http://www.ijmm.org/text.asp?2013/31/4/349/118875
| ~ Introduction|| |
Chronic infection with hepatitis C virus (HCV) is one of the major causes of liver cirrhosis and hepatocellular carcinoma. According to the World Health Organization, there are 180 million people affected with HCV world-wide  and about 12.5 million carriers in India.  The high rate of chronicity combined with the lack of a successful vaccine makes HCV infection a serious public health challenge.
HCV is an enveloped positive stranded ribonucleic acid (RNA) virus belonging to genus Hepacivirus in the family Flaviviridae. It was discovered in 1989 and was the first virus to be detected by employing molecular techniques.  The RNA genome comprises of about 9500 nucleotides with a single open reading frame that encodes a polypeptide precursor of 3000 amino-acids and is flanked by the non-coding regions (NCR) at both the 5′ and the 3′ termini.  The polyprotein precursor is cotranslationally processed by host signal peptidases to yield the structural (core[c] and envelope [E1 and E2]) and the non-structural proteins (NS1, NS2, NS3, NS4A, NS4B, NS5A and NS5B). 
HCV has been classified into six major genotypes and into more than 90 subtypes distributed across the world.  This hypervariable region 1 region codes for a 34 amino-acid domain, located at the amino terminal end of the envelope protein. It is a major target for the neutralizing antibodies,  but due to the error prone RNA dependent RNA lacks proof reading mechanism.  HCV is also described to have quasispecies in chronically infected individuals. 
The conventional antiviral therapy against HCV is either monotherapy with interferon (IFN) that may be pegylated or in combination with ribavirin. The penultimate response to therapy is genotype dependent and the duration of therapy is guided by the rate of response to therapy that is monitored at different time intervals during therapy. , The response to therapy differs across genotypes. While up to 80% of the genotypes 2 and 3 can be cured with standard of care treatment consisting of pegylated or standard IFN-alfa and ribavirin,  genotypes 1, 4, 5 and 6 have been reported to show poorer response. In the present study, we sought to retrospectively analyze the genotypes detected among HCV infected individuals attending our hospital, spanning a decade. Currently, there is a paucity of such data from various regions within India.
| ~ Materials and Methods|| |
A retrospective analysis was performed with patients who were genotyped during the time period 2002-2012. Patients attending the Liver Clinic of Christian Medical College from 2002 were referred to the Department of Clinical Virology of the Institution. Blood samples were collected by venipuncture and stored in the sterile tubes containing the anticoagulant ethylenediaminetetraacetic acid. Separated plasma was stored in aliquots at −60°C for prospective analysis. The RNA was extracted using Qiagen extraction protocol (QIAamp Viral RNA Mini Kit, Qiagen GmbH, Hilden, Germany) and reverse transcribed using random primers and moloney murine leukemia virus reverse transcriptase enzyme (Invitrogen, Corp., California, USA). A total number of 451 patient's samples were genotyped during the above mentioned time interval. Out of the total 451 patients studied, 27 were genotyped using primers targeting the core region of the genome, which was standardized in our laboratory. For the second round amplification, two reaction mixes were made containing primers specific for different genotypes. Sequencing of the core region was performed using the sense and antisense primers of the first round with cycling condition similar to that of the second round amplification.  A total of 99 samples were genotyped using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) employing primer specific for the 5′NCR region of the HCV genome that involves a nested PCR followed by restriction endonuclease digestion spanning about 174 bp product.  The remaining 325 samples were genotyped by sequencing 350 nucleotides of the NS5B region, which again involves the nested PCR.  The sequencing was carried out using ABI PRISM 310 Genetic Analyzer (PE Applied Bio systems. California, USA). The generated sequences are posted into the HCV database (Los Alamos National Laboratory; http://hcv.lanl.gov/content/sequence/Basic_Blast/basic_blast.html ) and this BLAST search yielded the genotype of HCV. Generated sequences were further compared with GenBank sequences corresponding to the various HCV strains earlier identified from this hospital. All these three techniques were previously compared and the level of agreement between core type specific PCR and NS5B sequencing and 5′NCR based PCR-RFLP and NS5B sequencing were good (κ = 0.913, P < 0.001, κ = 0.794, P < 0.001) respectively. 
| ~ Results|| |
The trend of HCV genotypes has not changed over the entire study period i.e. March 2002 to March 2012. Consistently, genotype 3 (63.85%) is more prevalent followed by genotype 1, 4 and 6 (25.72%, 7.5% and 2.7%). Genotype 2 was detected in only one patient from Northeast India since 2004. The distribution of the genotypes detected each year during the last decade is represented in [Table 1]. There were no reports of genotype 5 over a decade in our setting [Table 1]. Region-wise distribution of genotypes was shown in [Figure 1]. South India had a higher distribution of HCV genotype 1 followed by genotype 3 and 4, whereas Eastern India and Northeast India genotype 3 was predominantly seen. Genotype 4 was seen in higher numbers from South India and genotype 6 was in Eastern and North Eastern region of India. Two patients were found to be infected with a recombinant strain of HCV genotype 1b and 2k and one of them was represented in [Figure 2]. Apart from these regions, we also had patients from Delhi, Haryana, Rajasthan, Nepal, Muscat, Pakistan and Islands like Myanmar where the sample numbers are very less and the genotypes prevailing in these regions were 3 and 1.
|Table 1: Year-wise and cumulative prevalence of all the genotypes during each year from March 2002 to March 2012|
Click here to view
| ~ Discussion|| |
Currently India harbors an estimated 10-15 million chronic carriers of HCV, which is a major cause of liver related mortality and morbidity. Knowledge of genotype is crucial for management of HCV infection and prediction of prognosis. Patients infected with HCV genotype 1 and 4 will have to receive IFN and ribavirin for a period of 48 weeks. Persons with these genotypes show a poor sustained viral response when tested 24 weeks after completion of therapy.  On the contrary, patients infected with HCV genotype 2 and 3 are reported to have better response to therapy.  This remarkable heterogeneity of HCV must be studied in order to develop strategies for designing a successful vaccine.
This is the first and largest retrospective study in India reporting the results of HCV genotyping spanning a complete decade. The rate of prevalence of HCV genotypes 3 and 1 has already been reported.  This study reemphasizes that HCV genotype 3 is the predominant genotype (63.85%) followed by genotype 1 (25.72%) in India. Genotype 4 mainly seen in Egypt and Middle East and genotype 6 earlier restricted to the Southeast Asian countries , is being increasingly reported in India. Genotypes 4 (7.5%) and 6 (2.7%) appear to be somewhat geographically restricted in their distribution within India.
In a previously conducted the study from this centre three techniques were compared for identifying HCV genotypes namely core type specific PCR, 5′NCR based PCR-RFLP and NS5B sequencing. Core type specific PCR and 5'NCR based PCR-RFLP in our experience has been found to be comparable and had overall good agreement with the sequencing of the NS5B region. Sequencing of the NS5B region is proven to be the most efficient method to discriminate HCV genotypes and subtypes as described by Murphy et al. and Laperche et al. , The sensitivity for detecting genotype 6 when comparing core specific PCR and 5′NCR based PCR-RFLP with NS5B based genotyping methods was low for the 5′NCR based PCR-RFLP. This may be due to the low numbers of genotype 6 included in the study. However, sequencing of these genotype 6 strains using core type specific primers correctly identified genotype 6 strains. 
Despite the differences in the frequency of occurrence of different genotypes across the Indian sub-continent, there is also a pattern of distribution of HCV genotypes throughout the country. Besides this there appears a trend of increased occurrence of genotype 4 and genotype 6 within India, which is evident from our previous studies , and the current study. Genotype 6 was found to be prevalent exclusively in patients from North Eastern parts of the country, whereas the genotypes 1 and 4 were found mostly in South Indian patients from Andhra Pradesh and Tamil Nadu. There was only one genotype 2 reported from this centre in 2004. Genotype 5 is confined to South Africa and Central parts of France and has not yet been reported from this region.
Two patients were found to be infected with a strain that is a recombinant of HCV genotype 1b and 2k and one of them was represented in [Figure 2]. They were from Jharkhand and Tamil Nadu and had no known high risk behavior such as intravenous drug use, a risk factor that has been earlier reported among patients harboring such strains. 
The information provided by the present study and earlier studies from this region provides valuable information to physicians in clinical decision making. It is interesting to note that the trends published almost a decade ago have not changed despite the use of different genotyping techniques and movement of population. Such epidemiological studies and trend analysis are also important to document in the wake of licensure of the newer generation directly acting antivirals for HCV such as bocepravir and teleprevir. In addition, this epidemiological information will be invaluable to future vaccination development strategies.
It must be stressed however, that the reported distribution of the various genotypes can be expected to change with increasing migration of population and changes in high risk behavior and life-style. There is a need for public education in this country about routes of transmission of viruses such as HCV. As tattooing and body piercing are still common practice in our country, it is important to educate and to create awareness in our population to prevent the acquisition of viruses such as HCV, especially in this era where we still await a suitable HCV vaccine.
| ~ References|| |
|1.||Hepatitis C. Initiative for Vaccine Research. World Health Organisation WHO; 2009. Available from: http://www.who.int/vaccine_research/diseases/viral_cancers/en/index2.html. [Accessed on 2011 Aug 06]. |
|2.||Chattopadhyay S, Hepatitis C. A major health problem of India. Curr Sci 2002;83:9. |
|3.||Choo QL, 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 U S A 1991;88:2451-5. |
|4.||Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-62. |
|5.||Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Shimotohno K. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A 1991;88:5547-51. |
|6.||Hoofnagle JH. Course and outcome of hepatitis C. Hepatology 2002;36:S21-9. |
|7.||Esumi M, Zhou YH, Tanoue T, Tomoguri T, Hayasaka I. In vivo and in vitro evidence that cross-reactive antibodies to C-terminus of hypervariable region 1 do not neutralize heterologous hepatitis C virus. Vaccine 2002;20:3095-103. |
|8.||Ray U, Ray PS, Das S. Ribosome-RNA interaction: A potential target for developing antiviral against hepatitis C virus. Curr Sci 2012;102:405-12. |
|9.||Kalinina O, Norder H, Mukomolov S, Magnius LO. A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol 2002;76:4034-43. |
|10.||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. Hepatitis interventional therapy group. N Engl J Med 1998;339:1485-92. |
|11.||Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: A randomised trial. Lancet 2001;358:958-65. |
|12.||Ohno O, Mizokami M, Wu RR, Saleh MG, Ohba K, Orito E, et al. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. J Clin Microbiol 1997;35:201-7. |
|13.||Pohjanpelto P, Lappalainen M, Widell A, Asikainen K, Paunio M. Hepatitis C genotypes in Finland determined by RFLP. Clin Diagn Virol 1996;7:7-16. |
|14.||Harris KA, Teo CG. Diversity of hepatitis C virus quasispecies evaluated by denaturing gradient gel electrophoresis. Clin Diagn Lab Immunol 2001;8:62-73. |
|15.||Laperche S, Lunel F, Izopet J, Alain S, Dény P, Duverlie G, et al. Comparison of hepatitis C virus NS5b and 5′ noncoding gene sequencing methods in a multicenter study. J Clin Microbiol 2005;43:733-9. |
|16.||Hadziyannis SJ, Sette H Jr, Morgan TR, Balan V, Diago M, Marcellin P, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: A randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;140:346-55. |
|17.||Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med 2000;132:296-305. |
|18.||Raghuraman S, Shaji RV, Sridharan G, Radhakrishnan S, Chandy G, Ramakrishna BS, et al. Distribution of the different genotypes of HCV among patients attending a tertiary care hospital in South India. J Clin Virol 2003;26:61-9. |
|19.||Raghuraman S, Abraham P, Sridharan G, Daniel HD, Ramakrishna BS, Shaji RV. HCV genotype 4 - An emerging threat as a cause of chronic liver disease in Indian (South) patients. J Clin Virol 2004;31:253-8. |
|20.||Raghuraman S, Abraham P, Sridharan G, Ramakrishna BS. Hepatitis C virus genotype 6 infection in India. Indian J Gastroenterol 2005;24:72-3. |
|21.||Nguyen MH, Keeffe EB. Prevalence and treatment of hepatitis C virus genotypes 4, 5, and 6. Clin Gastroenterol Hepatol 2005;3:S97-101. |
|22.||Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001;345:41-52. |
|23.||Daniel HD. Improved methods of direct detection of hepatitis C virus (HCV) in donated blood and high-risk patient groups and extension of this approach for the detection of other major blood borne viruses. PhD Dissertation. Chennai: The Tamil Nadu Dr. M.G.R. Medical University; 2007. |
|24.||Murphy DG, Willems B, Deschênes M, Hilzenrat N, Mousseau R, Sabbah S. Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5′ untranslated region sequences. J Clin Microbiol 2007;45:1102-12. |
[Figure 1], [Figure 2]