Mobuck virus genome sequence and phylogenetic analysis: identification of a novel Orbivirus isolated from a white-tailed deer in Missouri, USA.

The genus Orbivirus includes a diverse group of segmented dsRNA viruses that are transmitted via arthropods, have a global distribution and affect a wide range of hosts. A novel orbivirus was co-isolated with epizootic haemorrhagic disease virus (EHDV) from a white-tailed deer (Odocoileus virginianus) exhibiting clinical signs characteristic of EHDV. Using antiserum generated against EHDV, a pure isolate of the novel non-cytopathic orbivirus was obtained in Aedes albopictus cell culture. Genomic sequencing and phylogenetic analysis of predicted ORFs showed that eight of the ten ORFs were most homologous to Peruvian horse sickness virus (PHSV), with amino acid identities of 44.3-73.7 %. The remaining two ORFs, VP3 and VP5, were most similar to Middle Point orbivirus (35.9 %) and Yunnan orbivirus (59.8 %), respectively. Taxonomic classification of orbiviruses is largely based on homology of the major subcore structural protein VP2(T2), encoded by segment 2 for mobuck virus. With only 69.1 % amino acid identity to PHSV, we propose mobuck virus as the prototype of a new species of Orbivirus.

Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.

The effects of differential extraction conditions on the premature lysis of spermatozoa.

The purpose of this study was to determine the effect Proteinase K, sodium dodecyl sulfate (SDS), incubation times, and temperatures had on differential extraction efficiencies and the premature lysis of spermatozoa. The effect was measured using Quantifiler® Duo and Identifiler™ PCR Amplification kits, where the resultant male and female DNA concentrations and their ratios within the nonsperm- and sperm fractions (SFs) were determined. Comparisons between expected and observed ratios illustrate the quantity of female DNA in the SF increased when Proteinase K was absent during the initial incubation. Additionally, there is no indication of simultaneous sperm and epithelial cell lysis in the absence of DTT at Proteinase K concentrations ranging from 10 to 300 µg/mL. All other conditions exhibited minimal variation in DNA concentration. Therefore, despite the various protocols used for the differential lysis of cell mixtures encountered in casework, the method is robust and successful at most conditions.