Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành y học dành cho các bạn tham khảo đề tài: Genetic modification of Bluetongue virus by uptake of “synthetic” genome segments | van Gennip et al. Virology Journal 2010 7 261 http content 7 1 261 VIROLOGY JOURNAL SHORT REPORT Open Access Genetic modification of Bluetongue virus by uptake of synthetic genome segments René GP van Gennip Daniel Veldman Sandra GP van de Water Piet A van Rijn Abstract 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 .
