RESUMEN
Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Linfocitos T/virología , Línea Celular , Humanos , Virus Sincitial Respiratorio Humano/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Ensamble de Virus , Replicación ViralRESUMEN
BACKGROUND: Bovine leukemia virus (BLV) is a member of retroviridae family, together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) belonging to the genes deltaretrovirus, and infects cattle worldwide. Previous studies have classified the env sequences of BLV provirus from different geographic locations into eight genetic groups. To investigate the genetic variability of BLV in South America, we performed phylogenetic analyses of whole genome and partial env gp51 sequences of BLV strains isolated from Peru, Paraguay and Bolivia, for which no the molecular characteristics of BLV have previously been published, and discovered a novel BLV genotype, genotype-9, in Bolivia. RESULTS: In Peru and Paraguay, 42.3 % (139/328) and over 50 % (76/139) of samples, respectively, were BLV positive. In Bolivia, the BLV infection rate was up to 30 % (156/507) at the individual level. In Argentina, 325/420 samples were BLV positive, with a BLV prevalence of 77.4 % at the individual level and up to 90.9 % at herd level. By contrast, relatively few BLV positive samples were detected in Chile, with a maximum of 29.1 % BLV infection at the individual level. We performed phylogenetic analyses using two different approaches, maximum likelihood (ML) tree and Bayesian inference, using 35 distinct partial env gp51 sequences from BLV strains isolated from Peru, Paraguay, and Bolivia, and 74 known BLV strains, representing eight different BLV genotypes from various geographical locations worldwide. The results indicated that Peruvian and Paraguayan BLV strains were grouped into genotypes-1, -2, and -6, while those from Bolivia were clustered into genotypes-1, -2, and -6, and a new genotype, genotype-9. Interestingly, these results were confirmed using ML phylogenetic analysis of whole genome sequences obtained by next generation sequencing of 25 BLV strains, assigned to four different genotypes (genotypes-1, -2, -6, and -9) from Peru, Paraguay, and Bolivia. Comparative analyses of complete genome sequences clearly showed some specific substitutions, in both structural and non-structural BLV genes, distinguishing the novel genotype-9 from known genotypes. CONCLUSIONS: Our results demonstrate widespread BLV infection in South American cattle and the existence of a new BLV genotype-9 in Bolivia. We conclude that at least seven BLV genotypes (genotypes-1, -2, -4, -5, -6, -7, and -9) are circulating in South America.