Pathogenicity And Genomic Sequence Comparison Of A Chicken Infectious Anemia Virus Field Isolate

Chicken anemia virus (CAV) causes transient severe aplastic anemia due to destruction of erythroblastoid cells and generalized lymphoid atrophy with a concomitant immunosuppression. CAV causes considerable economic losses in poultry industry as a result of a co-infection and a secondary affection after its infection.In this study, Specific-pathogen-free(SPF) chickens were inoculated by intramuscular of a chicken infectious anemia virus field isolate CAV-C14 with 104 mean tissue culture infective dose(TCID50) at 1 day of age to elucidate the pathogenicity of CAV-C14. Weight of body and the quantity of red blood cell were decreased,the marrow of femur and focile turned pale and lymphatic organ (thymus and bursa) was atrophied at 28, 42 days postinoculation(DPI). but a dose of 104TCID50 of CAV-C14 can't induce death to SPF chickens.CAV and reticuloendotheliosis virus (REV) were used to infect 1-day-old SPF chickens, the experiments indicated that HI antibody titers to avian influenza virus (AIV) H5 and H9 were significantly lower in SPF chickens inoculated with CAV or REV alone at the age of 1 day than the control after vaccination of inactivated vaccines of AIV, and HI titers in birds co-infected with both viruses were even more lower than the birds infected with CAV or REV alone. CAV infection tended to decrease the antibody responses to vaccines against Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), but differences were not significant statistically. However, co-infection of CAV with REV could significantly suppress the antibody titers to NDV and IBDV compared with chickens infected with REV alone. The results demonstrated the synergism in immunosuppression in chickens co-infected with CAV and REV.A field strain C14 of chicken infectious anemia virus (CAV) was isolated from a 14 day-old broiler flock with growth runting syndromes. Antibody reactions to inactivated vaccines to avian influenza viruses (AIV) were suppressed in SPF chickens inoculated with C14 strain CAV at 1 day-old. Also C14 strain CAV and reticuloendotheliosis Virus demonstrated a synergism in immunosuppression when chickens were infected with both virus. The viral genomic DNA was amplified by PCR in 3 overlapped fragments and PCR products were cloned into T-vecor plasmid for sequencing. The sequencing results indicated that the total genome of C14 strain CAV was 2298bp, it contained 3 overlapped ORF and 1 non-coding regulation fragment. Its whole genome had 97.2%-99.2% of homogeneity to other several published CAV reference strains. Sequence data indicated that there are many motifs in the non-coding area of about 400 bp as the binding sites for transcriptional factors. All these motifs were very conservative. There were some mutations in 3 genes VP1, VP2 and VP3. Relatively, VP1 was less conservative than VP2 and VP3. Among different strains, mutations in these 3 genes were not correlated.VP1 gene of CAV-C14 was amplified by PCR from a field strain CAV-C14 as the template with primers synthysized according to the published sequences. PCR product was cloned into pGEX-6p-1 down to its GST gene for expression of fusion protein. The E.coli BL21 was transformed with recombinant expressing plasmid, and selected BL21 was cultured at 37℃and induced with IPTG to express the fusion protein. fusion protein was collected from gel after electrophoresis and then used to immunize mice. In indirect fluorescence antibody test, the mouse sera to VP1 gave positive fluorescence in CAV infected MSB1 cells, the atibody titers reached at 26. The results indicated that protein expressed in E.coli still maitained their antigenicity partially, and the antibody titer to protein was high enough to be used as monovalent antisera for diagnosis purpose.