PCR-Based Reverse Genetics Strategy For Bluetongue Virus Recovery

Bluetongue virus(BTV)belongs to the genus Circovirus of the Reoviridae family.The genome of the virus consists of 10 linearized double-stranded RNA gene segments.BTV is spread mainly through blood-sucking insects,and many domestic and wild ruminants are susceptible to infection.The virus exists in most regions and countries of the world,and has an adverse effect on agricultural production and import and export trade.At present,the reverse genetics technique used to rescue the bluetongue virus mainly relies on the use of complementary DNA(cDNA)of viral genome segments to synthesize RNA transcripts in vitro to rescue the virus with the help of auxiliary plasmids.Among them,the process of RNA synthesis is a laborious and complicated task,requiring expensive reagents and careful operating procedures,and RNA is very easy to degrade.In addition,this technology requires the target gene to be cloned into a specific vector and used as a template for RNA transcription.This study established a reverse genetics technique that only requires two steps of transfection to rescue BTV based on PCR products.we first transfected 7 helper plasmids that can express VP1,VP3,VP4,VP6,VP7,NS1,and NS2 proteins of BTV into BSR cells,and then transfected the PCR products of 10 gene segments of BTV into BSR cells,And finally we can save BTV.Then we verified the reverse genetics technology,and successfully rescued one skeleton virus and three segmented recombinant BTV strains using this technology,and identified rescued viruses.We first draw the growth curves of the wild virus wtBTV1,the rescued skeleton virus rBTV1,and the recombinant virus rBTVls2,and compare their growth and reproduction characteristics.It was found that the recombinant virus containing the foreign gene segment S2 had a slower growth rate than the other two strains.This technology is expected to replace the traditional BTV rescue method based on in vitro transcribed RNA,or as a useful supplement to the classical method.In addition,the reverse genetic method can provide a reference for establishing or optimizing other reverse genetic techniques of circovirus.