title: Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India.
abstract: OBJECTIVE
  To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.
  
  
  METHODS
  Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05.
  
  
  RESULTS
  Irrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D.
  
  
  CONCLUSIONS
  Kerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Deepak
authors.initials: D
authors.lastname: Gopalakrishnan
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Mark
authors.initials: M
authors.lastname: Keyter
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Kotacherry Trivikrama
authors.initials: KT
authors.lastname: Shenoy
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Kondarappassery Balakumaran
authors.initials: KB
authors.lastname: Leena
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Lakshmikanthan
authors.initials: L
authors.lastname: Thayumanavan
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Varghese
authors.initials: V
authors.lastname: Thomas
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Kr
authors.initials: K
authors.lastname: Vinayakumar
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Charles
authors.initials: C
authors.lastname: Panackel
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Arun T
authors.initials: AT
authors.lastname: Korah
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Nair
authors.affiliation: Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa.
authors.forename: Anna
authors.initials: A
authors.lastname: Kramvis
mesh_terms: D000293:Adolescent; D000328:Adult; D017668:Age of Onset; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D016009:Chi-Square Distribution; D003430:Cross-Sectional Studies; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood; D018450:Disease Progression; D019143:Evolution, Molecular; D005260:Female; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology*; D006801:Humans; D007194:India / Q000453:epidemiology; D008113:Liver Neoplasms / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016017:Odds Ratio; D010641:Phenotype; D010802:Phylogeny; D011498:Protein Precursors / Q000235:genetics; D012307:Risk Factors; D055815:Young Adult
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen
keywords: Chronic hepatitis; Cirrhosis; Genotype; Hepatocellular carcinoma; India; Phylogenetic analysis