Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments

Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in the Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus Orbivirus for which reverse genetics has not been developed yet

Does reservoir host mortality enhance transmission of West Nile virus?

<p>Abstract</p> <p>Background</p> <p>Since its 1999 emergence in New York City, West Nile virus (WNV) has become the most important and widespread cause of mosquito-transmitted disease in North America. Its sweeping spread from the Atlantic to the Pacific coast was accompanied by widespread mortality among wild birds, especially corvids. Only sporadic avian mortality had previously been associated with this infection in the Old World. Here, we examine the possibility that reservoir host mortality may intensify transmission, both by concentrating vector mosquitoes on remaining hosts and by preventing the accumulation of "herd immunity".</p> <p>Results</p> <p>Inspection of the Ross-Macdonald expression of the basic reproductive number (<it>R</it>₀) suggests that this quantity may increase with reservoir host mortality. Computer simulation confirms this finding and indicates that the level of virulence is positively associated with the numbers of infectious mosquitoes by the end of the epizootic. The presence of reservoir incompetent hosts in even moderate numbers largely eliminated the transmission-enhancing effect of host mortality. Local host die-off may prevent mosquitoes to "waste" infectious blood meals on immune host and may thus facilitate perpetuation and spread of transmission.</p> <p>Conclusion</p> <p>Under certain conditions, host mortality may enhance transmission of WNV and similarly maintained arboviruses and thus facilitate their emergence and spread. The validity of the assumptions upon which this argument is built need to be empirically examined.</p

First detection of Schmallenberg virus RNA in bovine semen, Germany, 2012

In analogy to the related Akabane virus, transmission of Schmallenberg virus (SBV) by contaminated semen has primarily been considered negligible. However, the potential economic consequences for stock-bull breeders prompted the investigation of reliable diagnostic methods for SBV-RNA detection in bovine semen. Twelve extraction methods were compared using a dilution series of SBV-spiked semen as well as serum and medium samples for control. The most promising methods were subsequently used with semen samples obtained in an intensive field study. In total, frozen semen from 95 SBV-seroconverted bulls collected in the field between May and November 2012 were tested for SBV-RNA with an optimised standard operating procedure. The highest diagnostic and analytical sensitivity for the extraction of SBV in semen was found for the Trizol® LS Reagent lysis with or without combined purification of the viral RNA with magnetic beads. A total of 29 of 766 semen batches from 11 of 95 SBV-infected bulls were PCR-positive (Cq-values 26−37). Intermittent virus excretion was observed in 2 of the bulls. SBV-RNA-positive semen was coincidentally detected with early SBV-antibodies in 4 bulls. In bulls that showed seroconversion together with consecutive positive semen batches, SBV-RNA was predominantly found in the seminal cell fraction, while in bulls with single positive results only, SBV-RNA was detected exclusively in the seminal plasma