The presence of resistance mutations to protease and polymerase inhibitors in Hepatitis C virus sequences from the Los Alamos databank.

Several new direct-acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase have been the major targets. HCV variants displaying drug-resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment-naïve HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a = 680, 1b = 498 and 3a = 205) and 806 NS5B polymerase sequences (genotypes 1a = 471, 1b = 329, 3a = 6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low-level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre-existence of HCV variants resistant to first-generation protease inhibitors and to non-nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre-existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes.

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-α and ribavirin therapy. Major histocompatibility complex class I restricted CD8(+) T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

Relation of pretreatment sequence diversity in NS5A region ofHCV genotype 1 with immune response between pegylated- INF/ribavirin therapy outcomes

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responsesand the combination of pegylated interferon (INF)-a and ribavirin therapy. Major histocompatibility complex class Irestricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggestprotection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated a-INF with ribavirin therapy and test evidence of natural.

Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region.

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among fulminant hepatitis cases from the western Amazon Basin of Brazil were characterized in this study. HBV and HDV isolates were obtained from liver samples from 14 patients who developed fulminant hepatitis and died during 1978-1989. HBV DNA and HDV RNA were detected in all samples. Phylogenetic analyses of HDV sequences showed that they all clustered with previously characterized sequences of HDV genotype 3 (HDV-3). HBV genotypes F, A and D were found in 50.0, 28.6 and 21.4 % of cases, respectively. These results confirm the predominance of HDV-3 in South America and its association with the severe form of hepatitis, and the finding of the co-infection of HDV-3 with different genotypes of HBV suggests that the association between HDV-3 and HBV-F is not necessarily causally related to a more severe clinical course of infection.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation.

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.