Development In Reverse Genetics System For Recovery Of Animal RNA Viruses

The reverse genetics of RNA virus allows precise study of the mechanisms as antigenicity,virulence, pathogenesis, maturation and replication of the virus at the molecular level. In this study, invitro and in vivo transcription systems were developed.1 Testing of biological properties, analyzing of bioinformatics of SVDV HK/70 strain anddevelopment in in vitro transcription system based on T7 RNA polymerase for the virusTo develop in vitro transcription system based on T7 RNA polymerase (T7 RNAP) for RNA virus,the swine vesicular disease virus HK/70 strain was selected as a model virus of cytoplasmicpositive-strand RNA virus in the present study. Firstly, the virus was isolated and cultured in mouse andIBRS-2 cells. Then, the biological properties such as 50ï¼…tissue culture infecting dose (TCID₅₀) andmouse virulence, and so on, were tested. The bioinformatics of the virus genome was also analyzedbased complete genome sequence by some software.At last, the full-length cDNA clone of swine vesicular disease virus HK/70 strain named pSVOK₁₂was constructed in order to study the antigenicity, replication, maturation and pathogenicity of swinevesicular disease virus. In vitro transcription RNA from pSVOK₁₂ transfected IBRS-2 cells and therecovered virus RNA was isolated and sequenced, then indirect hemagglutination test, indirectimmunofluorescence assays, eleectron microscope test, 50ï¼…tissue culture infecting dose (TCID₅₀)assays and mouse virulence studies were used to study the antigenicity and virulence of the recoveredvirus. The result showed that the infectious clones has obtained and the virus derived from pSVOK₁₂had the same biological properties as the parental strain HK/70; The full-length infectious cDNA clone,pSVOK₁₂, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation andreplication of SVDV.2 Development in in vivo transcription system based on T7 RNA polymeraseEstablishment of cell lines stably expressing T7 RNA polymerase by using retroviral gene transfertechnique for rescue of infectious RNA virusReverse genetics based on transfection of in vitro transcribed RNA to target cells has lowefficiency to recover RNA viruses. To develop an efficient and vaccinia virus-free recovery system(reverse genetics), three stable cell lines (designated as BHKT7, IBRST7 and SK6T7 respectively)constitutively expressing cytoplasmic bacteriophage T7 RNA polymerase (T7 RNAP) were developed.By using retroviral gene transfer technology, the T7 RNAP gene was integrated into the chromosome of these cells (BHK-21, IBRS-2 and SK6) and then these cell lines stably expressing T7 RNAP (BHKT7)were established under selection pressure. The T7 RNAP produced in the BHKT7 cell line was able toefficiently driving in vivo transcription of enhanced green fluorescent protein (EGFP) reporter genecontrolled by the T7 promoter. When the IBRST7/ BHKT7 cell line was directly transfected withlinearized full-length cDNA of swine vesicular disease viruse (SVDV) HK/70, infectious SVDV wassuccessfully recovered. Two-day-old mice inoculated intra-peritoneally with the recovered virus died at72 hours after inoculation. These data showed the T7 RNAP in BHKT7 cells has transcriptional activityand can be used for recovery of infectious RNA virus directly from full-length cDNAs. InfectiousCSFV was also successfully recovered from the SK6T7 cell line, and rabbit virulence studies were usedto study the virulence of the recovered virus.3 Development in the in vivo rescue system based on RNA polymerase I system.For some viruses refuse to grow in laboratory cell cultures, which cannot be recovered in cellcultures, or a limited number of mammalian cell lines are available for culture of virus which cannot betransfected with high efficiencies, which sometimes limits their use in reverse genetics systems forvaccine production and studying basic principles of molecular biology of the virus. To address thislimitation, we established a reverse genetics system that is entirely from a RNA polymeraseâ… -drivenplasmid required for virus generation. Here, we report the recovery of infectious SVDV entirely fromcDNA, which is a polymerase (pol)â… and polâ…¡-driven plasmid constructed which permits intracellulartranscription of the accurate viral RNA (vRNA) and caped mRNA of the viral protein to be derivedfrom the same template resulted in the efficient formation of infectious virus with genetic tags in thegenome from IBRS-2 cell transfected and suckling mice directly inoculated with a RNA polymeraseâ… /â…¡-driven vector. The cDNA-derived viruses behaved identically to wild-type virus in both cell cultureand infected mice. Importantly, the virus can be recovered from the mice directly injected with theplasmid, which also would develop a method to rescue the viruses refuse to grow in laboratory cellcultures.This technology provides an important basis for investigating various fields of virological research.The reverse genetic procedures are simplified to a faster, one step protocol to recover virus andovercome the obstacle to rescue the viruses which have no adaptive cell line. In addition, our findingsdemonstrate that polâ… /â…¡-based vector systems may represent an efficient alternative strategy for therecovery of cytoplasmic positive-strand RNA viruses from cDNA. Then, infectious FMDV was alsosuccessfully recovered from BHK-21 cell line by the in vivo rescue system, it is the first time that theresults shown that the RNA polymeraseâ… could drive transcripts of about 8.2 kb in length,4 Research on recombinant viruses with marker as vaccines against viral diseases by reversegenetics system To develop a marker vaccination: designing unique antigens to be added to vaccines todifferentiate between natural infection and vaccination. The full-length cDNA clone of SVDVreplication-competent recombinant virus with bio-mark 5B19 was constructed. Then the recombinantvirus was recovered by reverse genetics system.