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Abstract: RNA viruses replicate their genomes with a very high error rate and constitute highly heterogeneous mutant distributions similar to the molecular quasispecies introduced to explain the evolution of prebiotic replicators. The genetic information included in a quasispecies can only be faithfully transmitted below a critical error rate. When the error threshold is crossed, the population structure disorganizes, and it is substituted by a randomly distributed mutant spectrum. For viral quasispecies, the increase in error rate is associated with a decrease in specific infectivity that can lead to the extinction of the population. In contrast, a strong resistance to extinction has been observed in populations subjected to bottleneck events despite the increased accumulation of mutations. In the present study, we show that the mutagenic nucleoside analogue 5-azacytidine (AZC) is a potent mutagen for bacteriophage Qβ. We have evaluated the effect of the increase in the replication error rate in populations of the bacteriophage Qβ evolving either in liquid medium or during development of clonal populations in semisolid agar. Populations evolving in liquid medium in the presence of AZC were extinguished, while during plaque development in the presence of AZC, the virus experienced a significant increase in the replicative ability. Individual viruses isolated from preextinction populations could withstand high error rates during a number of plaque-to-plaque transfers. The response to mutagenesis is interpreted in the light of features of plaque development versus infections by free-moving virus particles and the distance to a mutation–selection equilibrium. The results suggest that clonal bacteriophage populations away from equilibrium derive replicative benefits from increased mutation rates. This is relevant to the application of lethal mutagenesis in vivo, in the case of viruses that encounter changing environments and are transmitted from cell to cell under conditions of limited diffusion that mimic the events taking place during plaque development. [Copyright &y& Elsevier]

Abstract: Human immunodeficiency virus type 1 (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)-terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the β3–β4 hairpin loop, which plays a key role in the resistance mechanism. [Copyright &y& Elsevier]

Abstract: Susceptibility to Chagas disease infections and its clinical manifestations may be influenced by host genetic factors. Among cytokines, the multifunctional transforming growth factor beta 1 (TGFβ1), plays a major role in the establishment and pathogenesis of Trypanosoma cruzi infection, the etiological agent of Chagas disease. Several single-nucleotide polymorphisms (SNP) in the TGFβ1 gene that may affect cytokine production have been described. We investigated, by PCR methods, five SNP in the TGFβ1 gene of known or suggested functional significance (−988 C/A; −800 G/A; −509 C/T; 10 T/C and 263 C/T) in 347 seropositive (asymptomatic, n =175; cardiomyopathic, n =172) and 279 seronegative unrelated individuals from a Peruvian and a Colombian population where T. cruzi is highly endemic. We found a significant difference in the distribution of the TGFβ1 10T and 10C alleles between patients and healthy controls in both cohorts, analyzed independently and combined. The frequency of the high TGFβ1 producer genotype 10 C/C was increased in the patients groups of both populations. Our data suggests that TGFβ1 genetic polymorphisms at codon 10 may be involved in a differential susceptibility to T. cruzi infection in these South American samples. [Copyright &y& Elsevier]

Abstract: In this study we investigated the association of functional single nucleotide polymorphisms of tumor necrosis factor-alfa (TNFA) and TNF receptor 2 (TNFR2) genes in determining the susceptibility to Chagas’ disease. This study included 313 patients from Colombia serologically positive for Trypanosoma cruzi antigens (cardiomyopathic, N =159; asymptomatic, N =154). Genotypes were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism method. We found a significant difference in the distribution of the TNFA -1031C (p =0.0153, OR=1.69, CI=1.10–2.58) and -308A (p =0.0002, OR=2.60, CI=1.53–4.39) alleles between cardiomyopathic and asymptomatic subjects. In addition, we observed that the TNFR2 +676T allele was monomorphic in our population. Our results suggest that TNFA -1031C and -308A gene polymorphisms may influence the susceptibility to develop chagasic cardiomyopathy in the population under study. [Copyright &y& Elsevier]