Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi.

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

Study of VIPER and TATE in kinetoplastids and the evolution of tyrosine recombinase retrotransposons.

BACKGROUND: Kinetoplastids are a flagellated group of protists, including some parasites, such as Trypanosoma and Leishmania species, that can cause diseases in humans and other animals. The genomes of these species enclose a fraction of retrotransposons including VIPER and TATE, two poorly studied transposable elements that encode a tyrosine recombinase (YR) and were previously classified as DIRS elements. This study investigated the distribution and evolution of VIPER and TATE in kinetoplastids to understand the relationships of these elements with other retrotransposons. RESULTS: We observed that VIPER and TATE have a discontinuous distribution among Trypanosomatidae, with several events of loss and degeneration occurring during a vertical transfer evolution. We were able to identify the terminal repeats of these elements for the first time, and we showed that these elements are potentially active in some species, including T. cruzi copies of VIPER. We found that VIPER and TATE are strictly related elements, which were named in this study as VIPER-like. The reverse transcriptase (RT) tree presented a low resolution, and the origin and relationships among YR groups remain uncertain. Conversely, for RH, VIPER-like grouped with Hepadnavirus, whereas for YR, VIPER-like sequences constituted two different clades that are closely allied to Crypton. Distinct topologies among RT, RH and YR trees suggest ancient rearrangements/exchanges in domains and a modular pattern of evolution with putative independent origins for each ORF. CONCLUSIONS: Due to the presence of both elements in Bodo saltans, a nontrypanosomatid species, we suggested that VIPER and TATE have survived and remained active for more than 400 million years or were reactivated during the evolution of the host species. We did not find clear evidence of independent origins of VIPER-like from the other YR retroelements, supporting the maintenance of the DIRS group of retrotransposons. Nevertheless, according to phylogenetic findings and sequence structure obtained by this study and other works, we proposed separating DIRS elements into four subgroups: DIRS-like, PAT-like, Ngaro-like, and VIPER-like.

Selection and characterization of brivudin resistant bovine herpesvirus type 5.

Bovine herpesvirus type 5 (BoHV-5) is the agent of meningoencephalitis, an important disease of cattle in South America. The neuropathogenesis of BoHV-5 infection is poorly understood and most previous research focused on the role of envelope glicoproteins in neurovirulence. Thymidine kinase (TK) is a viral enzyme necessary for virus replication in neurons and, therefore, represents a potential target for virus attenuation. The selection and characterization of BoHV-5 variants resistant to the nucleoside analog brivudin (BVDU), which selects TK-defective viruses is here described. Several BVDU-resistant clones were obtained after multiple passages in tissue culture in the presence of BVDU and one clone (BoHV-5/R-27) was further characterized. The selected clone replicated to similar titers and produced plaques with similar size and morphology to those of wild-type virus (SV507/99). The genetic stability of the resistant virus was demonstrated after ten passages in cell culture in the absence of the drug. Moreover, the drug-resistant virus showed reduced virulence in a rabbit model: virus inoculation in four rabbits did not result in disease, in contrast with 75% morbidity (3/4) and 50% mortality (2/2) among rabbits inoculated with the parental virus. These results demonstrate that BoHV-5 is sensitive to BVDU and that drug-resistant mutants can be readily selected upon BVDU treatment. BVDU-resistant mutants, likely defective in TK, retained their ability to replicate in tissue culture yet were attenuated for rabbits. This strategy to obtain TK-defective BoHV-5 may be useful to study the role of TK in BoHV-5 neuropathogenesis and for vaccine development.