Occult Hepatitis B Virus Infection in Chronic Liver Disease: Full-Length Genome and Analysis of Mutant Surface Promoter VAISHALI CHAUDHURI,* RUCHI TAYAL,* BAIBASWATA NAYAK,* SUBRAT KUMAR ACHARYA, ‡ and SUBRAT KUMAR PANDA* *Department of Pathology and ‡ Department of Gastroenterology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India Background & Aims: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty- six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1–S9) and 5 controls positive for HBsAg (SWT.1–SWT.5) were analyzed. Methods: Over- lapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fu- sion construct. Results: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1–S5, SWT.1–2, SWT.5) and 6 had infection with genotype D (S6 –S9, SWT.3– 4). S4 and S5.1 of genotype A had the characteristic nucleo- tide deletions in core and pre-S1 region seen in geno- type D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the sur- face promoters (nt 3025–54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Poly- merase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. Conclusions: These changes in the regulatory region with possible alter- ations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative. H epatitis B virus (HBV) infection has significant morbidity and mortality worldwide and its preva- lence varies from region to region. India lies in the intermediate prevalence zone for HBV endemicity with an estimated infection load of 40 million people. 1 The diagnosis of chronic infection by HBV is characterized by the persistent presence of viral envelope protein (hepati- tis B surface antigen [HBsAg]) in the blood. HBsAg- negative HBV infection or occult HBV infection is a recently recognized entity, whose exact magnitude, pathogenesis, and clinical relevance in various popula- tions is unclear. 2 Occult HBV infection is defined as “HBV DNA de- tectable by PCR [polymerase chain reaction] among patients negative for HBsAg” 3 and has been classified into seropositive and seronegative infection depending on positivity for anti– core antibody (HBc) and anti- HBs. 4 Usually individuals with occult HBV infection have low viremia. 5 However, the diagnostic approach and mechanism of anti–HBc-/anti–HBs-negative serology in individuals with occult HBV infection have not been elucidated clearly. A progressive decrease in HBV load as well as replication and various relevant mutations have been implicated in the explanation of HBsAg negativity in such infections. 6 The cause in 47% (216 of 458) of acute liver failure patients in India 7 is unidentifiable. Forty-one percent (13 of 32) of these patients were positive for HBV DNA by polymerase chain reaction (PCR) (unpublished results, 2002). Considering that chronically infected individuals are the major reservoirs of HBV infection, de novo infection with naturally occurring mutants of HBV from this popu- lation might be causing such occult HBV infection. The seronegativity in these patients may be caused by muta- tions, which alters either the immunoreactivity of various HBV proteins 8 or the quantity of HBsAg in serum. 9 Earlier studies have focused on the analysis of limited genome region 10,11 in occult HBV infection. It is elusive as to which mutations and variations and this can affect HBsAg detection. Therefore, the current study was aimed at assessing the extent of occult HBV infection among patients with chronic liver disease, at analyzing sequence changes of these patients with mutant HBV with respect to those patients with wild-type HBV in the Abbreviations used in this paper: aa, amino acid; ATG, antithymo- cyte globulin; fLUC, firefly luciferase; nt, nucleotide; PCR, polymerase chain reaction; S-rLUC, S gene-renilla luciferase. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gast.2004.08.003 GASTROENTEROLOGY 2004;127:1356 –1371
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