In this work we analyzed genetic diversity in HBV isolates from asymptomatic (blood donors) and symptomatic chronic carriers in Mexico. Genotypes were determined with a commercially available reverse-hybridization technique and compared with the sequencing technique. Reverse hybridization allows the detection of infection with mixed genotypes, suggesting that this technique is more reliable than sequencing to detect quasispecies present in lower proportion in the sample; although in some cases it failed to establish the genotype. On the other hand, besides genotyping, nucleic acid sequencing provides information on nucleotide and amino acid sequences of the region investigated, while other molecular methods detect only the point mutations specified by the probes or primers used.
Of interest, a higher diversity in the infecting genotypes was found among asymptomatic carriers (H, C, F, C/H, C/F/H), than in symptomatic patients, where genotype H was found in all cases and in only one case a C/H infection was detected. These results would suggest that in our population, genotype H strains are more prevalent than C and F genotypes in symptomatic cases. This observation is relevant since studies indicate certain association of the genotype with the clinical outcome of the infection; thus, in hepatocellular carcinoma, genotype C was more prevalent in patients >50 years and genotype B more prevalent in patients [37]. Of interest, in our study, genotype C was identified in four of the 21 asymptomatic cases. Alternatively, LMV treatment in the symptomatic cases might be selecting for the H genotype. Diversity of HBV genotypes may also affect the accuracy of diagnostic tests and therapeutic decisions [32].
HBV has been classified in eight genotypes, A through H [18], which show a geographic distribution. In this study, genotype H was found as the predominant genotype in Mexican strains, followed by genotypes C and F. Genotype H has a close phylogenetic relationship with genotype F [17,18]. The genotype C is reported as prevalent in Asian populations [20,33], and patients observed in our population might correspond to imported cases, due to the increased mobility among Asian and Latin-American countries; in fact, recent reports have documented the presence of HBV genotype H strains among Japanese blood donors [34], as an evidence of the global mobility. The genotype prevalent in a population may determine the type of mutations prevalent in the infecting strains [35]; for example, the immune-escape mutant G145R is closely associated with genotype D [36]. HBsAg has also been classified in subtypes based on the sequence of the S gene and in identification of the amino acids encoded at specific positions [33]; in our population, subtypes, like genotypes, were more diverse among strains from asymptomatic carriers than in symptomatic cases.
The hepatic function tests and HBV viral load showed that in our symptomatic patients there was hepatic damage and high viral replication, which suggests that the antiviral treatment with LMV was not working, even thought the drug was administered for at least one year in all cases. It is likely that after a long failed therapy, resistance to LMV has been developed. The main mutations associated with LMV resistance are located at the RT gene in the 204 position (rtM204I/V), which is in the catalytic YMDD motif. The P gene overlaps the S gene and thus mutations selected during antiviral treatment may cause concomitant changes to the overlapping reading frame; in particular altering the C-terminal region of HBsAg. Thus for mutations associated with LMV resistance, the rtM204V change is associated with a I195M change in the S gene (sI195M), while the rtM204I change is associated with three possible changes in the S gene, sW196S, sW196L, or a stop codon. In addition, mutation rtA181T is associated with the stop mutation sW172stop in the S gene [37]. However, in our study, none of these mutations were found in the S gene of the strains from symptomatic cases.
There has been several reports on HBV S gene mutants affecting amino acid position 120, 123, 124 126, 129, 131, 141, 144 and 145 of the "a" determinant and preS region. The most relevant mutations seem to be the substitutions of amino acid G145R, K141E and T131I and insertion of 3 amino acids between residues 123 and 124, since they markedly affect the antigenic structure of HBsAg [38]. Mexican strains of the present study showed mutations at the first and second loop of the "a" determinant; although it should be noted that these mutations differed between asymptomatic and symptomatic strains. Thus, in the asymptomatic cases mutations were found at positions 100, 126, 129, and 146, whereas in symptomatic patients mutation were in positions 93 and128; none of the above mutations are reported to affect significantly the antigenic structure of the protein. It should be emphasized that all cases included in the study were selected because they were HBsAg positive and so those patients with mutations rendering the S protein undetectable with the antibodies tested, were excluded.
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