| Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease (IBD). IBD causes significant losses in the poultry industries due to its high mortality and immunodepression in young birds by the destruction of the developing B lymphocytes in the bursa of Fabricus (BF). There are two distinct serotypes of IBDV. Viruses of the serotype I group are pathogenic to chickens, sub-divided into the classical virus, the antigenic variant virus, the very virulent virus and attenuated virus according to their virulence. Whereas the serotype II, mostly isolated from turkey, are avirulent to chickens. VP5 was first discovered only in the IBDV-infected cells but not packed into virion particles. VP5 was not essential for the viral replication, but played a key role in the viral infection of the disease. Although a few progresses of VP5 had been made because of its importance, concrete functions of VP5 and molecular pathogenic mechanisms in viral infection need be further elucidated.To study the function of VP5 in vivo and in vitro, two mutant viruses deficient in expressing VP5 protein were generated using attenuated virus (rGt strain) and cell-cultured virulent virus (rGx-F9VP2 strain) as parental viruses by RNA polymerase II system. Site-directed mutagenesis technique was used to alter start codon of VP5 gene. The 5'NCR and 3'NCR of A segments were fused by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) cDNA sequences respectively, which were cloned into eukaryotic expression vector, pCAGGs, to get infectious clones of A segment, pCGtA△VP5HRT and pCGx-F9VP2A△VP5HRT. The mutant viruses were rescued by co-transfection pCGtA△VP5HRT and pCmGtBHRT or pCGx-F9VP2A△VP5HRT and pCmGxBHRT into DF I cells and identified by IFA and RT-PCR. In comparison to the characteristics of parental viruses in vitro, the mutant viruses demonstrated lower viral titers and lower cytopathogenicity. To understand the role of VP5 in the pathogenesis in vivo, animal experiments were conducted. Results suggested that chickens inoculated with rGx-F9VP2△VP5 showed minimal bursal lesion with more normal bursal weight to body weight index (BBIX) and lower histopathologic bursal lesion scores (HBLS) than those inoculated with rGx-F9VP2. The titers of AIV hemagglutination inhibition (HI) antibodies of the rGx-F9VP2△VP5 inoculated group were higher than those of the rGx-F9VP2 inoculated group, indicating that deficiency of VP5 decreased the immunosupression of rGx-F9VP2△VP5 in chickens. All data indicates that VP5 plays an important role in viral replication and pathogenesis both in vitro and in vivo.To study the functional differences between VP5 of vvIBDV and that of attenuanted virus, two mosaic viruses, rGtGxVP5 and rGxGtVP5, were constructed and rescued by exchanging the VP5 gene between virulent virus (rGx-F9VP2) and attenuated virus (rGt). In comparison to the characteristics of rGt in vitro, rGtGxVP5 demonstrated the same viral replication efficiency and cytopathogenicity. The results of animal experiments showed that both mosaic virus rGxGtVP5 and parental virus rGx-F9VP2 had the same BBIX and HBLS and induced the identical antibody level. All results indicate that there is no difference between the functions of VP5 of vvIBDV and that of attenuanted virus in replication and pathogenicity. VP5 is highly basic, cyteine-rich and it is a class II transmembrance protein with an N-terminal cytoplasmic tail and a C-terminal extracellular domain. Three mutant infectious clones sectorial lacking expression of VP5 according to its transmembrance structure by site-directed mutagenesis technique. Mutant viruses, rGt△VP5C51, rGt△VP5N70 and rGt△VP5N90, were rescued by co-transfecting with pCmGtBHRT into DF I cells, respectively, and identified by RT-PCR and IFA. In comparison to the characteristics of parental virus rGt in vitro, the rGt△VP5C51 virus demonstrated the same replication efficiency and final viral titer, however, rGt△VP5N70 and rGt△VP5N90 showed lower replication efficiency and lower final viral titers. Results indicate that 1-94 aa of N terminal of VP5 affects the replication efficiency in IBDV infection in vitro.It was found that more interferon (IFN) was produced by the cell infected with rGt△VP5 than cells infected with rGt did. This result implied that VP5 might inhibit the production of IFN by host cells. The recombinant eukaryotic expression vector, pCAGGVP5, was constructed by inserting the ORF of VP5 gene into pCAGGs vector. The expression levels of firefly luciferases were analyzed by co-transfecting pCAGGVP5 with the reporter gene plasmids, pISRE-luc, pNF-κB-luc and pMx-luc, which were regulated by ISRE promoter, NF-κB promoter and Mx promoter, respectively. The results showed that the stimulations of ISRE promoter, NF-κB promoter and Mx promoter were all inhibited by the expression of VP5 in vitro. The results indicated that VP5 inhibited the production of IFN in host cells, which might be one of molecular mechanisms of viral replication and pathogenicity affected by VP5.VP5 gene was considered as an potential marker for developing marker vaccine, due to its speciality in viral infecton. The characteristicses of rGt2382VP5 and rGx-F9VP2△VP5 was analyzed in vitro and in vivo. Fortunately, an McAb (5D3) was got which only reacted with VP5 of serotype II IBDV strains. The mosaic virus, rGt2382VP5, were generated by exchanging the VP5 of rGt with that of serotype II IBDV (23/82 strain), which was discriminated by the anti-IBDV-VP5 McAb (5D3). The analysis of mosaic virus characteristics showed that exchangement of VP5 gene decreased the mosaic virus replication efficiency and cytopathogenicity. This indicates that VP5 might contribute to the distinct pathogenesis of serotype I and serotype II strains. The VP5-deficient mutant virus rGx-F9VP2△VP5 and its parental virus rGx-F9VP2 were used to inoculate 3-week-old SPF chickens. The IBDV antibody was examined and the inoculated SPF chickens were challenged with vvIBDV (Gx strain) 4 weeks post-infection. All chichens inoculated with rGx-F9VP2△VP5 or rGx-F9VP2 were protected.In this study, a series of deficient and mosaic viruses were rescued and analyzed in vitro and in vivo. The results indicate that VP5 plays an important role in viral infection, which affects IBDV replication and pathogenicity. The function of VP5 between virulent virus and attenuated virus show no difference. The 1-94 aa of VP5 protein N-terminal affects replication efficiency of IBDV in vitro. VP5 influences the viral replication and pathogenicity by inhibiting the IFN producd by host cells. VP5 is also one of the determinants of distinct pathogenesis between two serotypes IBDV. VP5-dificient mutant virus, rGx-F9VP2△VP5, induces protection of chickens against challenge with vvIBDV, which is a potential marker vaccine candidate. This study further elucidates the functions of VP5 and the molecular pathogenesis mechanisms for virulence and immunity of IBDV, which provides good rationales and materials to research and develop marker vaccine base on VP5 gene. |
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