Pathogenicity Of Chicken Infectious Anemia Virus M Mutant And Susceptibility Of DF-1 To It

Chicken infectious anemia virus (CIAV) is the pathogen of chicken infectious anemia (CIA), belong to Circoviridae, and have a small covalently closed circular singal-stranded negative sense DNA genome. The nearly 5'end of CIAV genome transcription contains one AUG anticodon, while the internal contains two AUG anticodons. The transcription was divided to three mutual part repetitive Open Read Frames (ORFs). ORF3 is located in ORF2, and ORF2 is overlapped on ORF1. The three ORFs encode three virus proteins-VP3 (13kDa), VP2 (24kDa), VPl (52kDa), respectively. VP1 is the structural protein, forming the neutralizing epitope of CIAV. VP2 is chaperonin of VP1, acting as a scaffold protein. VP3 is the main pathogenic protein, inducing cell apoptosis. The recombinant plasmid pTCAVm was constructed successfully used PCR site-directed mutagenesis by Yongheng Xia, Beijing Academic of Agriculture and forestry sciences. The recombinant plasmid contains The AUG in transcription of CIAV VP3 genes was moved from 487nt to 526nt. Based on the previous research, this experiment is to study the pathogenicity of CIAV M mutant and the susceptibility of DF-1 to it, to lay the foundation of development methods of the attenuated CIAV vaccine.Positive cloning strains pTCAVm was reanimated and extracted using Xia'methods. The mutated fragment of CIAV genomic DNA was digested from recombinant plasmid pTCAVm, re-ligated in vitro, and transfected into the host cell by liposome-mediated method. Based on identification by PT-PCR, sequence and IFA assay, one infection mutant named CIAV M mutant was obtained. Based on the system study of 1 day-old SPF chickens poison attack experiment by intraperitoneal injection, the pathogenicity of CIAV M mutant to SPF Chicken and MDCC-MSB1 was studied. The IFA result showed that TCID₅₀ of CIAV M mutant was 10^4.5/mL. Postinfected 7d, 14d, 21d by CIAV M mutant, the SPF chicken manifested clinical and pathogenic symptoms showed that the body weight of SPF Chicken increased slowly, immune organs were atrophy, such as apoptosis of T lymphocyte cell in thymus, bone marrow color was pale yellow, and the number of red blood cells decreased significantly. The humoral antibody level was a little higher than the negative control, suggesting that CIAV M mutant have antigenicity. Detected by the flow cytometry, CIAV M mutant obtained from infected SPF Chicken cannot inhibit the cell cycle phase, but it can induce icell apoptosis. Compare with CUX-1, The pathogenicity of CIAV M mutant is lower.The viral titer is low in the MDCC-MSB1 host cell, which does not favor the CIAV vaccine production. Therefore, DF-1 cell line was selected in this experiment, to identify the susceptibility to CIAV M mutant. CIAV M mutant was incubated in DF-1 cell line. Undergoing 5d or7d incubation, the cells of this phase had not all showed the cell pathology in six repitition. Transferring generation once more, the virus from the parent cell incubated CIAV destructed by freeze-thaw method or from the directly takes. The result had not all showed the cell pathological effect of CIAV M mutant in six repitition. Many times transfering generations than fifteen, the result of CIAV detected by PCR showed positive, but showed negative when the virus detected by IFA assay and RT-PCR method. From these results, the conclusion was that the mutant was not adapt to propgate on DF-1 cell line, and CIAV M mutant may not have the DF-1 cell tropsm.