RESUMO
OBJECTIVE: To assess genomic sequence conservation and variation in the proviral promoter of enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) in tissue samples from 3 sheep with nasal adenocarcinoma associated with ENTV and 3 sheep with pulmonary adenocarcinoma associated with JSRV and to identify a cell culture system that supports transcriptional activity of the ENTV and JSRV viral promoters. ANIMALS: 6 adult sheep. PROCEDURES: Standard PCR procedures for detection of the ENTV and JSRV long terminal repeat (LTR) promoter region were performed on samples from the 3 nasal adenocarcinomas and 3 pulmonary adenocarcinomas, respectively. The LTRs were cloned into shuttle vectors, amplified, sequenced, and analyzed. The cloned LTR regions were transferred into reporter plasmids and multiple human and ruminant cell lines, and primary cells were transfected with the promoter-reporter plasmids. The viral promoter activity was evaluated by use of an in vitro ß-galactosidase reporter assay. RESULTS: Each isolate had a unique nucleotide sequence. Single nucleotide polymorphisms were the most common LTR mutation and rarely occurred at transcription factor binding sites. Relative to ENTV, the JSRV promoter isolates had a conserved 66-bp U3 insertion, including the lung-specific transcription factor HNF-3ß binding site. Among the cell lines used, human embryonic kidney (293T) and goat synovial membrane cells supported promoter transcription. CONCLUSIONS AND CLINICAL RELEVANCE: The LTRs of ENTV and JSRV have extensive blocks of sequence conservation. Human 293T and goat synovial membrane cell lines may be suitable in vitro cell culture systems for further research of viral promoter functions.
Assuntos
Betaretrovirus/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Provírus/genética , Infecções por Retroviridae/veterinária , Doenças dos Ovinos/virologia , Infecções Tumorais por Vírus/veterinária , Adenocarcinoma/veterinária , Adenocarcinoma/virologia , Animais , Sequência de Bases , Betaretrovirus/metabolismo , Técnicas de Cultura de Células/veterinária , Linhagem Celular , Sequência Conservada , DNA Viral/metabolismo , Feminino , Vetores Genéticos/genética , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/metabolismo , Masculino , Dados de Sequência Molecular , Doenças Nasais/veterinária , Doenças Nasais/virologia , Filogenia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Adenomatose Pulmonar Ovina/virologia , Infecções por Retroviridae/virologia , Ovinos , Sequências Repetidas Terminais , Infecções Tumorais por Vírus/virologiaRESUMO
BACKGROUND: Feline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment. RESULTS: In an approximately 2 year-long experimental infection study, cats infected with a biological isolate of FIV clade C demonstrated undetectable plasma viral loads from 10 months post-infection onward. Viral DNA was detected in CD4+CD25+ and CD4+CD25- T cells isolated from infected cats whereas viral RNA was not detected at multiple time points during the early chronic phase of infection. Viral transcription could be reactivated in latently infected CD4+ T cells ex vivo as demonstrated by detectable FIV gag RNA and 2-long terminal repeat (LTR) circle junctions. Viral LTR and gag sequences amplified from peripheral blood mononuclear cells during early and chronic stages of infection demonstrated minimal to no viral sequence variation. CONCLUSIONS: Collectively, these findings are consistent with FIV latency in peripheral blood CD4+ T cells isolated from chronically infected cats. The ability to isolate latently FIV-infected CD4+ T lymphocytes from FIV-infected cats provides a platform for the study of in vivo mechanisms of lentiviral latency.
