|Year : 2015 | Volume
| 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
YPS Malik1, N Kumar2, V Rawat3, K Sharma2, A Kumar4, M Singhai3, M Kumar3
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 Submission||21-Aug-2013|
|Date of Acceptance||30-Jul-2014|
|Date of Web Publication||6-Feb-2015|
Department of Microbiology, Government Medical College, Haldwani, Uttarakhand
Source of Support: None, Conflict of Interest: None
|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 2020 Apr 5];33, Suppl S1:161-3. Available from: http://www.ijmm.org/text.asp?2015/33/5/161/148838
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.  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.  HCV genotyping was performed by the RFLP method as described by Pour et al. 
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 , 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).
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.  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.  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|| |
The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI) for providing funds to carry out the research work.
| ~ References|| |
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