Study On The Establishment Of Reverse Genetics System Of Vesicular Stomatitis Virus Indiana Serotype And The Construction Of Recombinant Viruses

Vesicular stomatitis virus ( VSV ) belongs to the Rhabdovirdae.It is an arthropod-borne virus that primarily affects rodents, cattle, swine and horses, and it can also infect humans and other species but commonly harmless. It is thought that VSV spreads between hoofed animals and rodents via insect vectors. The two prevalent serotypes of VSV are Indiana (VSV–Ind) and New Jersey (VSV–NJ).VSV causes vesicular stomatitis (VS) which is a highly contagious acute disease, and is defined as list A disease by OIE. In China VS is a foieign disease, and it is classified as list B disease in the import and export examinations.The genome of VSV is a non-segmented negative single RNA, it is contains five functional genes.The transcription of each gene is controlled under the VSV polymerase specificly recognized gene start (GS) and gene end (GE) respectively. The VSV genome has a broad tolarence of foieign gene insertion between the two indipendent transcription unit.VSV can grow in the most of primry cells and cell lines and can reach up to high titer in short time.VSV can induce high level expression of IFN, also it is extremly sesitive to IFN. VSV replicates only in the cytoplasma, it can not integrate into the host cell genome thus can not make the cell transformation.The unique characteristics let the VSV have great potential and advantage to be a live vaccine vector and oncolysis viral vector.In this study, the full-length genome cDNA clone of VSV Indiana strain was constructed, two unique restriction endonuclease site (Mlu I and Nhe I) was introduced into the genome before or after VSV G gene. The recombinant virus was generated by reverse genetics technique. The rescued VSV was characterized by indirect immune fluorescence and electron microscope, it kept the similar fast and high titer growth with parent Indiana strain.In the further research we inserted an enhanced green fluroncent protein (EGFP) gene into the Mlu I or Nhe I site of the mutated full-length VSV genome cDNA clone, then the recombinant virus—rVSV-EGFP(M/N) were generated by in vitro transfection. The GFP expression of rVSV-EGFP kept stable for at least ten passages on BHK21 cells. There is no any significant difference in replication kinetics between rVSV-EGFP(M/N) and...