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Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the serum in absence of hepatitis B surface antigen (HBsAg). Studies were conducted to screen for occult HBV infection among family members of HBV carriers, incidentally detected positive for HBV infection with a view to assess the pattern of virus transmission among them. Nested PCR assay, employing independent sets of primers to surface and core genes, was used for detection of HBV DNA in serum samples from 28 index cases with asymptomatic HBV infection, and in serum samples from 72 HBsAg negative/anti-HBc positive family members. HBV DNA was detected in 15 HBsAg negative family members of 10 HBsAg positive index patients and was studied in detail. Direct sequencing of S gene region of 25 isolates (10 index cases and 15 contacts) and phylogenetic analysis with data base sequences revealed that genotypes A, C, and D and subtype adw2, adr, and ayw3 were present among them. Evidence of transmission from outside family sources was found in addition to intrafamilial transmission among individuals with occult infection. Mutations in the major hydrophilic loop (MHL) of the S gene region were also detected, including the 'vaccine escape' mutation G145R in three cases. Although majority of the occult infection was associated with low viral load, 3/15 (20%) cases were with higher viral load and potential infectivity. These cases are especially notable in diagnostic, blood banking, and transplantation services.

Mutations in the basal core promoter (BCP) and precore (PC) regions are associated with persistent and intermittently high hepatitis B virus (HBV) replication in several patients. The variability in the functional domains of BCP and PC region of HBV and their association with disease progression and clinical outcome were assessed in Eastern India, an unique region where three HBV genotypes, A, D, and C are prevalent among the same ethnic group. PCR amplification and direct sequencing of BCP and PC region was done on sera obtained from 130 HBsAg positive subjects with different clinical presentations. Associations of the apparent risk factors with clinical advancement were evaluated by statistical methods including multiple logistic regression analyses (MLR). HBV genotype A was present in 33.08%, C in 25.38%, and D in 41.54% cases. Genotypes A and C were associated with higher rate of T1762/A1764 mutations than the most predominant genotype D. HBeAg negative state was associated with considerably higher rate of C1753 mutation. T1762/A1764 along with C1753 was common among cirrhosis and T1762/A1764 without C1753 was frequent among chronic liver disease cases. No significant association was found between A1896 point mutation and clinical status. Multivariate analysis revealed that T1762/A1764 double mutation, HBV/A, age ≥25 years, C1753 and A1899 were critical factors for clinical advancement while age ≥25 years and C1753 as significant predictor for cirrhosis in comparison with chronic liver disease. In conclusion, the analysis of the BCP variability may help in monitoring the progression towards advanced liver disease in Eastern Indian patients. J. Med. Virol. 83:253–260, 2011. © 2010 Wiley-Liss, Inc.

Hepatitis B virus (HBV) has been classified into eight genotypes, and several subgenotypes, distinctly distributed geographically. The genotypes A and D were previously reported to be predominant in India. Recent studies indicated evidence of circulation of genotype C in Eastern part of India. With the aim to confirm the phylogenetic relation and molecular genetic characteristics of the HBV circulating in Kolkata, the most populous city in Eastern India, 11 strains were isolated and the complete genome sequences were analyzed. Phylogenetic analysis determined; three genotype C (adr-serotype) isolates closely related with C1 (Cs) subgenotype references from South East Asia, and three genotype A (adw2-serotype) isolates, related to Asia-variant references of subgenotype A1 (Aa). Whereas, five genotype D (ayw2, ayw3 serotype) isolates were highly divergent; one was related to subgenotype D1, two to subgenotype D3, and the remaining two clustered with a single genotype D isolate from Japan belonging to an unclassified subgenotype. Together, these two isolates differed from HBV D1-D4 subgenotypes by nucleotide differences ranging from 5.0 to 5.49%, probably indicating a new subgenotype, which we designate as D5. All serotype ayw3 of genotype D isolates had specific amino acid substitution Threonine at codon 118 and Methionine at codon 125 in antigenic determinant of surface gene that has not been reported previously in isolates from other parts of India. In conclusion; using the complete genome analyses this study has confirmed circulation of the genotype C in Eastern part of India and demonstrated considerable genotypic heterogeneity of the Indian genotype D.