Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V⁺) and uninfected (V⁻) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V⁺ compared with V⁻ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V⁺ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V⁺ and V⁻ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Development of monoclonal antibodies against the gp90 protein of reticuloendotheliosis virus and mapping of their recognition regions / 生物工程学报

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.

Human T-Lymphotropic virus (HTLV) type I in vivo integration in oral keratinocytes

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14 percent (8/14) HAM/TSP patients and 27.28 percent (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

Improved quality control protocol for fowl pox virus vaccines based on the use of PCR and sequence analysis to detect REV and ALV as contaminants

In the present study, twenty one Fowl pox virus [FPV] vaccines were collected in the period between 2003-2005 and tested using the currently used and an improved quality control protocols. Results of currently used quality control protocol revealed negativity of all tested vaccines for the presence of contaminants and the results were satisfactory. Using PCR to detect Reticuloendotheliosis virus [REV] as contaminant in such vaccines revealed negative result except one suspected contaminated vaccine. Inoculation of this vaccine in egg, chicken embryo fibroblasts [CEFs], Specific pathogen free [SPF] chicks for three passages and testing of samples collected from inoculated host revealed positive amplification using REV specific primers. Sequence analysis of the obtained amplification fragment for REV revealed its negativity and confirmed the non specific amplification of such primers which were previously published in several PCR studies for REV. Using avian leucosis virus [ALV] sets of primers to detect groups A, B, C, D and J in a PCR reaction revealed positive amplification of ALV fragment and confirming the contamination of tested vaccine with ALV. The study proposes the importance of using PCR followed by sequence analysis of the amplified product to confirm the contaminations fonnd in the FPV vaccines