As mentioned under 7.0 our contribution to the OrbiNet EMIDA project encompasses two main research areas. These are described in the EMIDA proposal. The respected part of the text is cited here:
" In order to characterize genetic and phenotypic properties of TOV, primarily being responsible for (i) the inability of the virus to replicate in cell culture, and (ii) its apathogenic phenotype in its caprine host, RG techniques will be used in attempts to create reassortants between TOV and BTV (e.g. BTV-1 or BTV-8), creating viruses containing different individual genome segments from TOV. For this purpose, strategies commonly used for RT-PCR amplification and cloning of dsRNA viral genomes from orbiviruses or other reoviruses propagated in cell culture will have to be optimized to allow cloning of complete genome segments starting from low amounts of viral RNA obtained directly from blood of TOV-infected goats. All 10 RNA segments of several TOV strains will be cloned into plasmids allowing the in vitro transcription of authentic ssRNA to be used for RG. MR will then be generated based on BTV-1 or BTV-8 as "backbone" where one of the 10 genome segments is replaced by the corresponding segment from TOV. This is done by transfecting in vitro transcribed ssRNA of all 10 genome segments into cells that support BTV replication. Such MR will serve to characterize the roles of the individual TOV genes, and ultimately also confirm the formal taxonomic status of TOV as BTV-25. MRs will also be used to identify the TOV gene(s) responsible for the apathogenic phenotype and the restricted host range. This will be done in collaboration with other partners of the Consortium who are performing animal experiments (e.g. partners 1, 6, 9). Furthermore, a complete set of TOV ssRNA transcripts will be used to identify TOV-susceptible cells for in vitro propagation/rescue of the virus upon RNA transfection, thereby complementing conventional inoculation studies.
No TOV-specific serological tools are available so far. Instead, TOV-specific antibodies are identified by a combination of (i) cross-reactivity in commonly used pan BTV ELISAs, (ii) negative results in BTV-8-specific ELISAS, (iii) negative results in serum neutralization tests against all 24 known BTV serotypes, and (iv) by focussing on samples collected from animals in areas without any prior epidemiolocical evidence of BTV infection nor vaccination. Therefore, TOV-specific tests are highly desirable. To this end, recombinant proteins derived from cloned TOV genes, primarily focussing on VP2 (genome segment L2), will be produced in a bacterial and/or baculovirus protein expression system and used as antigen in an ELISA. Truncated versions of VP2 could be used - either in a shotgun approach or upon comprehensive mapping of TOV-specific epitopes - if the full-length VP2 shows a (too) high degree of cross-reactivity with BTV antisera.
Once such a TOV antibody ELISA is established, it will be made available to other projects partners. Goat sera from several countries in Europe with a siginificant goat population, primarily those which are involved in the present EMIDA project, will then be examined for TOV-specific antibodies in order to obtain data about the Europe-wide distribution of TOV. Samples from goats yielding serological evidence of TOV infection will be tested for the presence of TOV-specific RNA by RT-qPCR. TOV RNA segments from positive samples will be cloned and sequenced, ultimately providing data about the molecular epidemiology of TOV.
Switzerland has experienced several BTV-8 outbreaks in 2007 and 2008. Blood samples from these cases will be made available to other partners (e.g. P1, P3) of OrbiNet EMIDA consortium who will be performing molecular epidemiological studies.
