Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection

Abstract

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.

Fig 1. Association of HBV occurrence with the modes of transmission, patients’ age groups and HBV genotypes among samples included in the study.

(A) Sexual promiscuity is the major mode by which HBV is transmitted in the study population. (B) Occurrence of HBV is highest in the age bracket between 15 to 30 years. (C) HBV/D2 is the dominant sub-genotype and is associated majorly with sexual promiscuity. (D) Occurrence of HBV/D2 is highest in the age bracket between 15 to 30 years.

Fig 2. Analysis of the association of HBV sub-genotypes with biochemical parameters.

Median (A) ALT (IU/liter) and (B) bilirubin (IU/liter) levels in patients vary with circulating HBV sub-genotypes. Only significant differences (p<0.05) have been marked in the figure.

Fig 3. Phylogenetic analysis of HBV isolates from Eastern India with acute infection created using the neighbour-joining method.

Phylogenetic tree was constructed from surface gene sequences of this study (denoted by AEI) along with reference sequences derived from GenBank (denoted by accession numbers).

Fig 4. Distribution of HBV genotypes and sub-genotypes among acute and chronic patients of eastern India.

(A) Difference in HBV genotype distribution among acute and chronic patients. (B) Difference in the distribution of HBV sub-genotypes under genotype D among acute and chronic patients. Only significant results (*p<0.05, **P<0.01) have been marked in the figure.

Fig 5. Frequencies of some principal mutations in the HBV genome compared in acute and chronic Hepatitis B patients.

Figure depicts some signature mutations in the surface gene (S), basal core promoter/pre-core regions (BCP/PC) and X gene.

Fig 6. Comparison of nucleotide diversity in acute Vs chronic infection.

Nucleotide diversity in the (A) surface gene, (B) BCP/PC region and (C) X gene has been compared. The nucleotide sequences of the chronic sequences were compared to that of the acute sequences using Shannon’s Entropy. The blue lines indicated the specific positions where changes were noted. The red lines indicate the positions where the frequency of substitution between acute and chronic sequences was significantly different. (D) The average ΔH (average difference in entropy between chronic and acute infection) was highest in the BCP region of the HBV genome. (E) Calculation of genetic diversity at the genotype level showing that the BCP/PC region of genotype C isolates exhibit highest average ΔH.