First Isolation and Molecular Characterization of Umatilla Virus (Sedoreoviridae, Orbivirus) in Brazil.

In this study, we provide a genomic description of the first isolation of the Umattila virus (UMAV) in Brazil. The virus was obtained from the blood of a bird (Turdus fumigatus) and isolated in a C6/36 cell culture. The viral genome contains ten segments, and its organization is characteristic of viruses of the genus Orbivirus (family Sedoreoviridae). The coding region of each segment was sequenced, demonstrating the nucleotide identity with UMAV. The phylogenetic inference results were in line with these findings and demonstrated the formation of two distinct monophyletic clades containing strains isolated around the world, where our isolate, belonging to the same clade as the prototype strain, was allocated to a different subclade, highlighting the genetic divergence between them. This work reports the first isolation of UMAV in Brazil, and due to the scarcity of information on this viral agent in the scientific literature, it is essential to carry out further studies to better understand its epidemiology, dispersion, and, in particular, its interactions with vertebrate hosts, vectors, and the environment.

Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus.

Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species-specific, or EEV type-specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia.

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.

Full-length sequencing and genetic characterization of Breu Branco virus (Reoviridae, Orbivirus) and two related strains isolated from Anopheles mosquitoes.

Breu Branco virus (BE AR 492347) was isolated from Anopheles (Nyssorhynchus) triannulatus mosquitoes captured in Tucuruí, Pará State, northern Brazil, in 1988. No cross-reactivity by complement-fixation tests was observed between Breu Branco virus and other known arboviruses. Results of electron microscopy and physicochemical tests suggested that Breu Branco virus may be a member of the family Reoviridae. In order to elucidate its taxonomic status, a comprehensive genetic characterization was conducted for Breu Branco virus and related strains (BE AR 494475 and BE AR 486204) that were also isolated from Anopheles mosquitoes in the same area. This included full-length genome sequencing, determination of genetic traits and phylogenetic analysis. Breu Branco virus showed a similar genome organization to members of the genus Orbivirus, family Reoviridae. Genetically, Breu Branco virus was indistinguishable from strains BE AR 494475 and BE AR 486204. Phylogenetic analysis suggested that Breu Branco virus BE AR 492347 and its related strains constitute a novel species of the genus Orbivirus. Breu Branco virus is the first Brazilian orbivirus and the fifth orbivirus in the world to be sequenced fully.