The Development Of The Reverse-genetics System And Gene-function Research For Infectious Bursal Disease Virus

Infectious bursal disease(IBD), mediatied by infectious bursal disease virus(IBDV), is the causative agent of an acute and highly contagious disease in 3-6 week-old chickens. The first outbreak of IBD was reported at Gumboro in American in 1957. During the past 50 years, IBD has been threatenned the poultry industry constantly. The immunosuppression. antigenic variation, especially the emergence of very virulent IBDV(vvIBDV), represents a new challenge for effective prevention and control of IBD. Recently, IBD was estimated having considerable socio-economic importance at the international level during the 63rd General Session of the Office Internationnal des Epizooties(OIE, Paris). The researches about the molecular basis of the antigenic variation, virulence and cell tropism of IBDV have been constantly emphasized.In one of our previous researches, the vvIBDV Gx strain was attenuated to Gt strain through continue passage in specific-pathogen free(SPF) chicken embryos(5 generation) and in chicken embryo fibroblast(CEF) cell cultures(20 generation). The Gx strain has the motality of above 60% and can not adapt to CEF culture. The 9th generation in CEF(named F9) has lower motality and adapt to CEF culture.In this research, the long-accurate PCR(LA-PCR) was developed and the full-length genome of Gt strain was cloned and sequenced. Also, the VP2 gene of F9 was cloned and sequenced. Compared with Gx, there are two amino acids mutation(Q253H and A284T) in VP2 of F9, and another ten amino acids mutation in VP2 of Gt. Besides, there are four and six amino acids mutation in VP3 and VP4 between strain Gx and Gt, respectively. The aim of this research is to develop an efficient reverse genetic system of IBDV and to research the influence of these amino acids mutation on the cell tropism, replication and virulence of IBDV.To date, three kinds of reverse genetics systems, in vitro transcription system, system induced by endogeny T7 RNA polymerase and system induced by RNA polymeraseâ…¡, were used to research IBDV. But these systems have some shortcomings in the efficiency and convenience of virus rescue. In this research, it was the first time that an efficient RNA polymeraseâ…¡-based reverse genetic system of IBDV was developed using an intelligent ideas. The full-length genome cDNA containing genetic tags and flanked by Hammerhead ribozyme(HamRz) and Hepatitis delta ribozyme(HdvRz) were cloned downstream of the CMV enhancer and the chicken beta actin promoter of the vector pCAGGS. After purified by Plasmid Midi Kit(QIAGEN), the recombinant infectious clones named pCAGGmGtAHRT and pCAGGmGtBHRT were directly co-transfected DFI cells indued by Lipofectamine^TM 2000(Invitrogen). 72h post transfection. the supernatant of cell cuture was harvested and continue passaged in CEF cell. The results of indirect immunofluorescence. RT-PCR and electron microscope all verified that IBDV was rescued successfully. And. the genetic tags was also existed. The rescued IBDV could cause special CPE in CEF and has consistent replication kinetics curves as it's parent virus Gt. The RNA polymeraseâ…¡-based reverse genetics system developed in this research is more efficient. stable. convenient, fit to various cells(at least DFI. Vero/E6, Vero/P12 cells). This system not only provides the basis for the gene function research of IBDV, but also can be applied to the reverse genetics research of other Birnaviridae viruses.Using fusion PCR, the VP2 of Gx strain(vvIBDV) was exchanged with the corresponding gene of F9 and Gt strain, respectively. Also, the VP2 of Gt strain was exchanged with the corresponding gene of F9 strain. By the reverse genetic system developed in this research, the corresponding gene-mosaic viruses were rescued. The results of replication kinetics and animal experiment showed that the mutagenesis of amino acids residues Q253H and A284T of VP2 allowed adaptaion of vvIBDV to CEF cuture and acutely influenced the virulence of IBDV; the amino acids residues 222, 242, 256, 279, 294, 299, 330 of VP2 influence not only the replication(in CEF) but also the virulence of IBDV.Additionally, the VP3/3'NCR, VP4/VP3/3'NCR, VP4 of Gt was exchanged with the corresponding domain of Gx, respectively. By the reverse genetic system mentioned above, the corresponding gene-mosaic viruses were rescued. The results of replication kinetics and animal experiment showed that VP3 influenced the replicaton of IBDV in CEF and the mutagenesis of amino acids residues P981L could promote the replication; VP3 couldn't influence the virulent of IBDV; VP4 couldn't influence replication in CEF and virulent of IBDV, and had no synergistic effect to VP3.It was the first time to develop an efficient RNA polymeraseâ…¡-based reverse genetics system of IBDV using an intelligent ideas. Then the function of VP2, VP3, VP4 was reseached deeply. This research is benefit to further understand the relationship between gene structure and function, to identify the molecular basis for gene variation, to reveal the molecular mechanism for virulence and immunity, to explore the molecular attenuation method, and to develop the advanced vaccine of IBDV which is fit to the prevalent strain.