RESUMEN
We isolated East/Central/South African genotype chikungunya virus during the 2016 epidemic in Rio de Janeiro, Brazil. Genome sequencing revealed unique mutations in the nonstructural protein 4 (NSP4-A481D) and envelope protein 1 (E1-K211T). Moreover, all Brazil East/Central/South isolates shared the exclusive mutations E1-M407L and E2-A103T.
Asunto(s)
Aedes/virología , Fiebre Chikungunya/epidemiología , Fiebre Chikungunya/transmisión , Virus Chikungunya/genética , Insectos Vectores/virología , ARN Viral/genética , Adolescente , Adulto , África/epidemiología , Animales , Brasil/epidemiología , Fiebre Chikungunya/virología , Virus Chikungunya/clasificación , Virus Chikungunya/aislamiento & purificación , Chlorocebus aethiops , Femenino , Genotipo , Humanos , Masculino , Filogenia , Células VeroRESUMEN
Cantagalo virus (CTGV) is the etiologic agent of a pustular disease in dairy cows and dairy workers in Brazil with important economical and occupational impacts. Nevertheless, no antiviral therapy is currently available. ST-246 is a potent inhibitor of orthopoxvirus egress from cells and has proved its efficacy in cell culture and in animal models. In this work, we evaluated the effect of ST-246 on CTGV replication. Plaque reduction assays indicated that CTGV is 6-38 times more susceptible to the drug than VACV-WR and cowpox virus, respectively, with an EC50 of 0.0086µM and a selective index of >11,600. The analysis of ß-gal activity expressed by recombinant viruses in the presence of ST-246 confirmed these results. In addition, ST-246 had a greater effect on the reduction of CTGV spread in comet tail assays and on the production of extracellular virus relative to VACV-WR. Infection of mice with CTGV by tail scarification generated primary lesions at the site of scarification that appeared less severe than those induced by VACV-WR. Animals infected with CTGV and treated with ST-246 at 100mg/kg for 5days did not develop primary lesions and virus yields were inhibited by nearly 98%. In contrast, primary lesions induced by VACV-WR were not affected by ST-246. The analysis of F13 (p37) protein from CTGV revealed a unique substitution in residue 217 (D217N) not found in other orthopoxviruses. Construction of recombinant VACV-WR containing the D217N polymorphism did not lead to an increase in the susceptibility to ST-246. Therefore, it is still unknown why CTGV is more susceptible to the antiviral effects of ST-246 compared to VACV-WR. Nonetheless, our data demonstrates that ST-246 is a potent inhibitor of CTGV replication that should be further evaluated as a promising anti-CTGV therapy.
Asunto(s)
Antivirales/farmacología , Benzamidas/farmacología , Enfermedades de los Bovinos/virología , Isoindoles/farmacología , Orthopoxvirus/efectos de los fármacos , Infecciones por Poxviridae/veterinaria , Secuencia de Aminoácidos , Animales , Bovinos , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Orthopoxvirus/química , Orthopoxvirus/genética , Orthopoxvirus/fisiología , Infecciones por Poxviridae/tratamiento farmacológico , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Cotia virus (COTV) SPAn232 was isolated in 1961 from sentinel mice at Cotia field station, São Paulo, Brazil. Attempts to classify COTV within a recognized genus of the Poxviridae have generated contradictory findings. Studies by different researchers suggested some similarity to myxoma virus and swinepox virus, whereas another investigation characterized COTV SPAn232 as a vaccinia virus strain. Because of the lack of consensus, we have conducted an independent biological and molecular characterization of COTV. Virus growth curves reached maximum yields at approximately 24 to 48 h and were accompanied by virus DNA replication and a characteristic early/late pattern of viral protein synthesis. Interestingly, COTV did not induce detectable cytopathic effects in BSC-40 cells until 4 days postinfection and generated viral plaques only after 8 days. We determined the complete genomic sequence of COTV by using a combination of the next-generation DNA sequencing technologies 454 and Illumina. A unique contiguous sequence of 185,139 bp containing 185 genes, including the 90 genes conserved in all chordopoxviruses, was obtained. COTV has an interesting panel of open reading frames (ORFs) related to the evasion of host defense, including two novel genes encoding C-C chemokine-like proteins, each present in duplicate copies. Phylogenetic analysis revealed the highest amino acid identity scores with Cervidpoxvirus, Capripoxvirus, Suipoxvirus, Leporipoxvirus, and Yatapoxvirus. However, COTV grouped as an independent branch within this clade, which clearly excluded its classification as an Orthopoxvirus. Therefore, our data suggest that COTV could represent a new poxvirus genus.
Asunto(s)
Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Poxviridae/clasificación , Poxviridae/genética , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Chlorocebus aethiops , Reacciones Cruzadas/inmunología , Efecto Citopatogénico Viral , Genes Virales , Humanos , Macaca mulatta , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Filogenia , Poxviridae/fisiología , Conejos , Ratas , Alineación de Secuencia , Porcinos , Tropismo Viral , Replicación Viral/fisiologíaRESUMEN
We report a 1-step assay to screen antiviral substances combining the simultaneous application of cells, virus sample, and drug to 96-well plates. The results were obtained within 26 h when vaccinia virus plaques were counted or virus-induced cytopathic effect was measured. This fast cost-effective procedure may be used for other cytopathic viruses and is suitable to 1st screening tests.
