Identification Of Biological Marker And Cell SAGE Libraries Of Infectious Bursal Disease Virus Infection

Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus in the family Birnaviridae, has two serotype, serotype 1 and serotype 2. Serotype 1 viruses are pathogenic for chickens, whereas serotype 2 viruses are avirulent. Genome of IBDV consists of two segments (A and B) of double-stranded RNA (dsRNA) and codes 5 proteins. The large segment A, contains two open reading frames (ORFs). A small ORF encodes the nonstructural protein VP5, which overlaps the N terminal region of a precursor polyprotein from the large ORF. The precursor polyprotein is autoproteolytically cleaved to form mature viral proteins VP2-VP4. Segment B encodes VP1, which has RNA-dependent RNA polymerase (RDRP) activity. Infectious bursal disease (IBD) is an immunosuppression disease; IBD cause the death of infected chicken or vaccination failure of other vaccines for immunosuppression. Today, this disease still threatens poultry industry. The pathogenesis is still unclear. In the present study, the interactions of IBDV and hosts were deeply researched at transcriptomic and humoral levels.Firstly, at transcriptome level, this study analyzed the impacts of IBDV infection or IBDV coding genes transformation on host transcriptome. Based on 5 Long-SAGE libraries:normal Vero cell, IBDV infected Vero cell, monoclonal Vero cell line expressing A-segment coding proteins, monoclonal Vero cell line expressing VP243 protein, monoclonal Vero cell line expressing VP5 protein, differentially expressed genes were analyzed (p<0.05),217 differentially expressed genes were identified. the influences of different viral genes on Vero cell transcriptome and metabolism were summarized. By classing the differential expression genes, we discovered that the protein metabolism, energy metabolism and cytoskeleton related genes were influenced. Real-time PCR analysis was performed to verify 57 genes expression which were differentially expressed in Long-SAGE library. The coincidence of the real-time PCR data and Long-SAGE results ranges from 60% to 70%, this verified the correctness of Long-SAGE data. These data provides much information to research the pathogenesis of IBDV.Total RNA was isolated from IBDV infected/uninfected bursa of Fabricius and chicken embryo fibroblast, and 15 differentially expressed genes in Long-SAGE and 17 pro-inflammatory cytokines were selected to perform real-time PCR to analyze the transcript changes. In chicken embryo fibroblast, HMGN2 was down-regulated significantly, it is possible that this gene involved in apoptosis of infected cells; pro-inflammatory cytokines were all up-regulated, IFN-P was up-regulated more than 500 times. Other pro-inflammatory cytokines such as IFN-y, BAFF, IL-6, IL-1B, IL-8 and INOS were up-reguated more than 20 times. In IBDV infected bursa of Fabricius, CLIC4 was up-regulated significantly, it is presumed that CLIC4 involved in virus release; pro-inflammatory cytokines were all up-regulated, IL-6 was up-regulated 1746 times. Other pro-inflammatory cytokines TNFSF8, IFN-β, IFN-y, TNFRSF1A, IL-8 and iNOS were up-reguated more than 20 times. These data indicates that the category of differential transcript genes in different host cells infected with IBDV is coincident, but, the tendency of gene transcript changes is not coincident, thus presents the infection variability of different type cells to IBDV.Coffilin was down-regulated significantly in IBDV and analyzed the relation of IBDV infection and cofilin expression using anti-cofilin polyclonal antibody, the result indicates that the cells expressing cofilin are susceptive to IBDV. Because that ADF and cofilin have 70% homology and similar function, for differentiating the function of this two genes in IBDV infection, we tried to prepare anti-ADF and anti-cofilin monoclonal antibody, only anti-ADF antibody was obtained. However, ADF is expressed in all cells.Based on the information of Long-SAGE libraries and real-time PCR, for understanding host feeding back to IBDV VP4 protein, using recombinant expressed VP3 and VP4 proteins, the humoral immunity to VP4 protein was analyzed in 21-day old SPF Leghorn chicken infected respectively with virulent IBDV (NB strain, BC6/85), intermediate plus virulent IBDV (MB43), intermediate virulent IBDV (B87) and attenuated IBDV (attenuated NB strain). The serum antibody was detected by ELISA:in virulent IBDV infected chickens, VP4 antibody seroconverted at 23 days post infection (dpi), seropositivity ratio of VP4 antibody is more than 70% at 40 dpi; VP3-antibody seroconverts at 20 dpi, seropositivity ratio of VP3 antibody is 100% at 23 dpi. In intermediate plus virulent IBDV infected chickens, VP4 antibody seroconverted at 23 dpi, seropositivity ratio of VP4 antibody is less than 55% at 40 dpi; seropositivity ratio of VP3 antibody is 100% at 27 dpi. In intermediate virulent IBDV infected chickens, VP4 antibody seroconverted at 23 dpi, seropositivity ratio of VP4 antibody is less than 37% at 40 dpi; seropositivity ratio of VP3 antibody is 100% at 27 dpi. In attenuated IBDV infected chickens, VP4 antibody was scarcely detectable after first vaccination, seropositivity ratio of VP4 antibody is less than 22% after second vaccination; seropositivity ratio of VP3 antibody was nearly 100% after first vaccination. Similar results were obtained by IFA using Vero cell expressing IBDV VP3 or VP4 protein. Furthermore, immunohistochemistry assay using aniti-VP3/VP4 monoclonal antibodies showed:VP3 and VP4 proteins can detected in bursa of Fabricius infected with virulent IBDV, however, in bursa of Fabricius infected with attenuated IBDV, only VP3 protein can be detected, the VP4 protein of IBDV can scarcely be detected. These data proves that VP4 protein antigen or specific antibodis are biomarker discriminating wild IBDV or vaccine virus infection.