Hepatitis B virus genotypes and resistance mutations in patients under long term lamivudine therapy: characterization of genotype G in Brazil

Abstract

Background: Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results: Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 x 107 and 6.9 x 107 copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion: A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.

Figure 1

PCR-RFLP analysis of the pre-S/S genomic region. (A), EcoRI restriction endonuclease digestion. Lane 1, PCR products without EcoRI site (pattern predicted for genotype G). Lane 2, PCR products containing one EcoRI site (pattern deduced for most HBV genotype A isolates). Lane 3, PCR products derived from sample 09-hgg. (B), digestion with BamHI. Lane 1, PCR products without BamHI site (pattern predicted for genotype G). Lane 2, PCR products containing a BamHI restriction site at nt 491 (pattern predicted for genotypes B and C and some genotype A isolates). Lane 3, PCR products containing a BamHI site at nt 28 (subgenotype Aa and genotype G). Lane 4, PCR products derived from sample 09-hgg.

Figure 2

Phylogenetic tree based on full-length nucleotide sequences. Isolates from this work are named 05-hgg, 06-hac and 09-hgg. The other isolates are indicated by their GenBank accession number, followed by genotype/subgenotype designation. The horizontal bar shows a genetic distance scale.