| It has greatly promoted the study of RNA viruses at the molecular level and the development of new vaccines since the establishment of reverse genetics of RNA viruses. Double-stranded RNA(dsRNA) viruses have wide range of hosts. They have caused significant damage and economic losses to people or animals. However, reverse genetics of dsRNA viruses lag behind the positive-strand or minus-strand RNA viruses. The purpose of this study is to explore the reverse genetic of dsRNA viruses using infectious bursal disease virus (IBDV) as the model virus.Firstly, the full-length cDNA segment B of strain ZJ2000 was cloned and sequenced.The full-length cDNA segment B of strain ZJ2000 was 2827bp, and was deposited in the GenBank database (Accession No. DQ166818). Meanwhile, biological experiments of ZJ2000 and TL2004 strains in vivo and vitro were made. The results showed that ZJ2000 and TL2004 strains are able to directly adapted to CEF cells, and caused significant cytopathic effect. ZJ2000 strain exhibited delayed replication kinetics as compared to attenuated strains JD1 and HZ2. TL2004 strain was pathogenic to the 9-11 day-old SPF chicken embryoes and SPF chickenes. By the bioinformatics analysis of the sequences of Birnaviridae in GenBank, we found the reassortant phenomenon of IBDV and IPNV firstly at home and abroad. Reasortment of IBDV ZJ2000 strain indicate that VP2 is not the sole determinant of IBDV virulence, multiple factors may affect the virulence of IBDV, especially segment B playing an important role in IBDV virulence.Using IBDV as the model, the sequences of ribozyme HamRz and HDV with the function of self-cutting wer e introducted into the 5'ends and 3'ends, of segments A and B of IBDV genome using the method of over-lap PCR. Then, the segments containing ribozyme sequence were cloned into eukaryotic expression vector PCI and named as PCI-ma and PCI-mb. Parallel co-transfection of reverse-genetic system of IBDV was developed. The tandem vector, containing two independent transcription units(red and green reporter gene expression), was constructed. The results showed that expression of the reporter genes in the tandem vector was significantly better than that in the co-transfected of two reporter gene vectors in the same target cells. The similar tandem vector, containing two independent transcription units(IBDV genome segments A and B), was constructed. The vector was named as PCI-mab. PCI-mab was transfected to cells for virus rescue, and the rescue efficiency of this method was higher than that of the parallel transfection rescue by 100 times.Using IBDV as the model, the sequences of T7 promoter and ribozyme HDV were introduced into the 5'ends and 3'ends of segments A and B of IBDV genome using PCR. Then, the infectious vectors of PT-mA and PT-mB were constructed respectively. Both PT-mA and PT-mB were co-transfected into vero cells which were infected by recombinant vaccinia virus vTF7-3 (vTF7-3 can express the T7 RNA polymerase in vero cell) for 1 hour and the reverse genetics of IBDV that was based on vaccinia virus vTF7-3 was developed.Using IBDV as the model, A helper-virus-free method for rescue of dsRNA viruses was successfully established firstly. T7 RNA polymerase genes were cloned from the genome of prokaryotic expression bacteria strain BL21 and vaccinia virus vTF7-3 respectively. Sequence analysis showed that the gene cloned from the genome of prokaryotic expression bacteria strain BL21 shared 100% homology with that from vaccinia virus VTF7-3. T7 RNA polymerase gene was cloned into retroviral vector pLPCX. The retroviral vector was packaged to the infectiou particles JVT7 by the packaging cell PT67. JVT7 was infectious but witnot the function of self-replication. Vero cells were infected with JVT7 virus particles and selected with antibiotic puromycin, then the stable T7 RNA polymerase producing cell line(named as V-T7) was obtained. Using the cell line of V-T7, reverse genetics of the dsRNA viruses(using IBDV as the model) was successfully established firstly.
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