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Indian Journal of Medical Microbiology, Vol. 26, No. 1, January-March, 2008, pp. 96-97 Correspondence Novel digestion patterns with hepatitis B virus strains from the Indian subcontinent detected using restriction fragment length polymorphism Vivekanandan P, Daniel HDJ, Raghuraman S, Daniel D, Shaji RV, Sridharan G, Chandy G, Abraham P Department of Clinical Virology, Christian Medical College, Vellore Date of Submission: 29-Jun-2007 Code Number: mb08030 Dear editor, Hepatitis B virus (HBV) genotypes differ in the rate of seroconversion to anti-HBe, [1] selection of core promoter, [1] disease progression [2] and response to treatment. [1] Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [3] is one of the most widely used HBV genotyping techniques. We studied 122 HBV DNA positive, chronic hepatitis B patients referred to the Clinical Virology Department from the Gastroenterology Department, Christian Medical College, Vellore. One hundred and five HBV DNA positive ′healthy′ blood donors from John Scudder Memorial Blood Bank, Christian Medical College, were also studied. We used the earlier described PCR-RFLP for determining HBV genotypes. [3] This technique involves the amplification of surface gene using primers P7 and P8, which flanked nucleotide positions 256 to 796. The PCR product was digested with Hinf I and 5 U of Tsp509 I, and digestion products were analysed in a 3% agarose gel by electrophoresis. Among the 227 subjects studied, HBV genotypes could be assigned for 215 subjects as previously described. [4] However, in 12 (5.3%) samples, the digestion pattern obtained did not match with any described pattern. Various investigators who used PCR-RFLP found a small proportion of their strains ′untypeable′. [1],[3],[4] To the best of our knowledge, none of the investigators studied this phenomenon further. We, therefore, studied the nucleotide sequences of these ′untypeable′ strains with a view to identify altered/novel restriction sites. Nucleotide sequencing was performed after amplification using primers P7 and P8. [3] Sequencing was carried out using the ABI PRISM 310 genetic analyzer (PE Applied Biosystems, CA, USA). The nucleotide sequences of these 12 ′untypeable′ samples were assigned HBV genotypes using the BLAST (basic local alignment search tool) (http://www.ncbi.nlm.nih.gov/BLAST/) programme. Eight samples were identified as genotype D, while the other four samples belonged to genotype C. The nucleotide positions of restriction sites and the digestion patterns of ′untypeable′ samples are shown in the [Table - 1]. We have described five novel digestion patterns, three for genotype D and two for genotype C. We have analysed the putative digestion patterns of 54 complete genome/surface gene sequences from India available with the NCBI database using NEB Cutter V2.0 (http://tools.neb.com/NEBcutter2/index.php). None of the Indian sequences we analysed matched with any of the five novel patterns of digestion described in this study. It is, therefore, possible that the availability of fewer Indian HBV sequences in the NCBI database contributed to a poorer representation of the sequence variability from this geographical region, leading to the inadequate choice of restriction enzymes by investigators who developed this technique. Genotype C was detected in 12 subjects by PCR-RFLP, and four ′untypeables′ were further identified as genotype C after nucleotide sequencing. Therefore, it is possible that one in every four genotype C samples from the subcontinent may yield an ′untypeable′ pattern, leading to an underestimation of infection with genotype C. The two novel patterns of digestion for genotype C described in this study could, therefore, be particularly useful for investigators using this technique for genotyping Indian strains. Studies from Southeast Asia show that genotype C is associated with more aggressive liver diseases [2],[4] and lower response rates to interferon alpha. [1] Underestimation or failure to detect infection with genotype C may, therefore, have direct implications on patient management and treatment algorithms. Genotype D is found universally. The three novel digestion patterns that we have described for genotype D could hence be encountered with strains from any part of the world. The users of this genotyping technique globally may encounter ′untypeable′ patterns. As clinical differences among HBV genotypes are becoming increasingly relevant, there is a need for genotyping methods with high throughput. PCR-RFLP is an excellent technique for genotyping HBV, although a small proportion of strains is ′untypeable′. The five novel digestion patterns described in our study may assist investigators using this technique resolve such ′untypeable′ samples, thereby saving on the need to use labour-intensive and expensive options, such as nucleotide sequencing. References
Copyright 2008 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb08030t1.jpg] |
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![[Table - 1]](/showimage?mb/photo/mb08030t1.jpg){kind=link}