Similar evolutionary rates of hepatitis B virus in monoinfected and HIV-coinfected patients.

CASSINO L., MBAYED V., LAUFER N., CAMPOS R., QUARLERI J.

Lisboa, Portugal

Workshop; 5th International HIV and Hepatitis Co-infection workshop; 2009

Background: The study of the molecular evolution of HBV within patients is difficult due to HBV genomic complexity and need to study paired samples collected over long periods of time. The course of chronic HBV infection is modified by HIV coexistence. We aimed to investigate whether HBV variants characterized from monoinfected patients exhibit evolutionary differences with those from individuals coinfected with HIV by comparing at both inter- and intra-host levels. Methods: Thirty-five HBV infected patients (20 monoinfected and 15 HIV-coinfected) were included. Clinical records obtained include data on CD4+ cell counts, HBe serological status defined by ELISA (Abbott), HBV-VL, HIV-VL and 3TC therapy. Among isolates from HIV coinfected patients 11 exhibited 3TC resistance mutations, and 9 among monoinfected ones. HBV DNA was extracted from serum samples obtained during a 3-year follow up.  The basal core promoter (BCP), Precore (Pc) and S genomic regions were amplified, cloned and sequenced.  Phylogenetic tree was constructed using maximum-likelihood method and the best-fitted model of evolution and the topology was evaluated by bootstrap analysis. This allowed the classification of the HBV isolates according the genotype (Gt). Viral quasispecies diversity and the intra-host quasispecies complexity (by determining the Shannon entropy) were evaluated. The inter-patient genetic distance was also calculated. Statistical analysis was performed by using independent samples T-test (p<0.05 was considered statistically significant). Results: HBV phylogenetic analysis based on Pc-core alone or with S-gene concatenated sequences showed that all sequences from same genotype clustered together despite 3TC pressure or presence of resistance mutations.  No different ancestral origin according to HIV presence was observed.  The isolates from coinfected patients were ascribed to GtA (n=12), GtD (n=1) and Gt F (n=2). Those from monoinfected patients were classified as GtA (n=12) and GtF (n=8). Analysis of inter-patient distance based on BCP-Pc and S concatenated sequences showed a higher diversity among GtA isolates from HBeAg positive (+) and HBeAg negative (-) monoinfected patients (p<0.0002 and p<0.0001 respectively). Statistically significant differences were also obtained after BCP-Pc and S gene analysis of distances was performed for HBeAg+ versus HBeAg- patients (p<0.001 in both cases).  These results are consistent with a tendency of longer mean branch length of isolates from monoinfected patients in the phylogenetic tree. A slight tendency of higher complexity in the quasispecies distribution was observed among monoinfected hosts (p=0.13). However the distances values are consistently low (range: 0.001-0.02) and sustained during the 3-yr follow up both in monoinfected and coinfected patients.    Conclusions: In the present study, we demonstrated that there is not different phylogenetic clustering among HBV isolates from mono- and HIV co-infected patients in the same genotype, independently of the HBe status or 3TC therapy. HBV strains appear to evolve from a common ancestor independently of the HIV coexistence, but the HBV isolates from co-infected patients exhibited less diversity. At intrahost level, no significant changes at quasispecies distribution were found during the 3-yr follow-up of these patients, suggesting that HIV virus by itself appear no exert an influence on HBV evolution.