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  Table of Contents  
CORRESPONDENCE
Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 161-163
 

Detection and distribution pattern of prevalent genotypes of Hepatitis-C virus among chronic hepatitis patients from Kumaon region of Uttarakhand, India


1 Division of Biological Standardization; Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India
2 Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India
3 Department of Microbiology, Government Medical College, Haldwani, Uttarakhand, India
4 Department of Medicine, Government Medical College, Haldwani, Uttarakhand, India

Date of Submission21-Aug-2013
Date of Acceptance30-Jul-2014
Date of Web Publication6-Feb-2015

Correspondence Address:
V Rawat
Department of Microbiology, Government Medical College, Haldwani, Uttarakhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.148838

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How to cite this article:
Malik Y, Kumar N, Rawat V, Sharma K, Kumar A, Singhai M, Kumar M. Detection and distribution pattern of prevalent genotypes of Hepatitis-C virus among chronic hepatitis patients from Kumaon region of Uttarakhand, India. Indian J Med Microbiol 2015;33, Suppl S1:161-3

How to cite this URL:
Malik Y, Kumar N, Rawat V, Sharma K, Kumar A, Singhai M, Kumar M. Detection and distribution pattern of prevalent genotypes of Hepatitis-C virus among chronic hepatitis patients from Kumaon region of Uttarakhand, India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Aug 23];33, Suppl S1:161-3. Available from: http://www.ijmm.org/text.asp?2015/33/5/161/148838


Dear Editor,

Hepatitis C virus (HCV) has population-specific genotypes and provides valuable epidemiological and therapeutic information. Hence, the importance of genotype knowledge is high for clinicians in devising therapeutic strategies. Till date, no scientific data is available on the confirmation of genotypic distribution of HCV in the Kumaon region of Uttarakhand.

In this study, 39 serum samples positive for anti-HCV antibodies using 4 th generation HCV TRI-DOT (J. Mitra and Co. Pvt. Ltd., New Delhi) were further processed using nested RT-PCR and restriction fragment length polymorphism (RFLP)-based genotyping. Eleven representative samples from 33 confirmed positives in PCR/RFLP were subjected for sequencing (SciGenom Labs Pvt. Ltd., Kerala). To simplify the genotype and subtype, a phylogenetic tree was constructed using MEGA 5.05 software. [1] The approval of ethical committee was obtained prior to the study, and informed consent was obtained from individual patient.

RNA was extracted from 150 μl of serum by using Nucleo-pore RNA Sure Virus Kit (Genetix Biotech Asia Pvt. Ltd., New Delhi). Reverse transcription polymerase chain reaction (RT-PCR) was performed following the method described previously. [2] HCV genotyping was performed by the RFLP method as described by Pour et al. [3]

In the present study, out of 39 serum samples positive for anti-HCV antibodies by 4 th Generation TRI-DOT Immunoassay, 33 were found positive by RT-PCR assay, showing a detection rate 84.6%. The RFLP patterns (genotype 3a: 129 bp, 145 bp, 99 bp; genotype 3b: 97 bp, 145 bp, 99 bp) reported earlier [2],[3] using the same sets of restriction enzymes were different compared to the present study [Table 1]. The cogency of restrictions patterns that we found in this study were further confirmed by in silico digestion of sequenced PCR amplicons with the same restriction enzymes using MB advanced DNA analysis v6.84. This difference may be due to mutation at the recognition site of Apa I due to which tube-A showed a 145-bp band instead of 129 bp (3a) or 97 bp (3b).
Table 1: Restriction pattern observed for HCV‑3a and HCV‑3b isolates

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The sequence-based HCV genotyping and subtyping revealed predominance of HCV 3a genotype (72.72%) followed by 3b (18.18%) in the State. Studies from India suggest north-south divide, with predominance of HCV genotype 3 in the Northern states while genotype 1 in the Southern states. [4] In the present study, genotype 3 was found in all 33 patients based on restriction patterns and/or sequencing.

On comparing the Uttarakhand HCV isolates with different countries HCV isolates, seven of the HCV-3a Indian isolates showed clustering with the Canadian HCV-3a isolates and sequence homology of more than 85% for all isolates [Figure 1]. Similarly, the remaining three HCV-3b isolates from Uttarakhand also clustered with Canadian HCV-3b isolates with more than 86% identity at nucleotide level for all isolates [Figure 1]. The Uttarakhand HCV isolates when compared with each other were found to have more sequence similarities except one isolate (HCV-3a/UKD-2/India) which showed lower sequence identity with other HCV 3a isolates.
Figure 1: Phylogenetic tree shown is based on retrieved sequences of 5'UTR of HCV from different countries. HCV isolates from Uttarakhand region are highlighted with solid black dots. The alignment was done with Clustal W method, and neighbour-joining statistical method was used for constructing the phylogenetic tree

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In our study, out of 33 patients who were HCV positive by PCR assay, 28 (84.8%) were from Udham Singh Nagar (USN) district and the remaining 5 (15.2%) were from the Nainital District. In our previous study from this region on blood donors, significantly higher HCV seropositivity was seen among the Sikh population from the USN district. [5] USN is largely inhabited by the Sikh community, where many people are found to be associated in some form of working or visiting Canada. It is hypothesised that this could be the reason for the close association of Indian and Canadian HCV isolates. However, a larger cohort from this geographical region is required to test the hypothesis that this genotype may have spread from Canada to USN place via population migration and admixture.


 ~ Acknowledgments Top


The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI) for providing funds to carry out the research work.

 
 ~ References Top

1.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.  Back to cited text no. 1
    
2.
Bukh J, Purcell RH, Miller RH. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc Natl Acad Sci U S A 1992;89:187-91.  Back to cited text no. 2
    
3.
Pour MH, Keivani H, Sabahi F, Alavian SM. Determination of HCV Genotypes, in Iran by PCR-RFLP. Iran J Public Health 2006;35:54-61.  Back to cited text no. 3
    
4.
Chakravarti A, Ashraf A, Malik S. A study of changing trends of prevalence and genotypic distribution of hepatitis C virus among high risk groups in North India. Indian J Med Microbiol 2013;31:354-9.  Back to cited text no. 4
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5.
Rawat V, Bhatt U, Singhai M, Kumar A, Malik YP. Prevalence of hepatitis C virus infection among blood donors of Kumaon region of Uttarakhand. Indian J Med Microbiol 2013;31:313-4.  Back to cited text no. 5
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