| Chicken infectious anemia（Chicken infectious anemia,CIA）is a kind of poultry immunosuppressive disease caused by chicken infectious anemia virus（Chicken infection anemiavirus,CIAV）.It is mainly characterized by aplastic anemia and atrophy of immune organs such as thymus and bursa of Fabricius.CIAV gene encodes three proteins,namely VP1,VP2 and VP3.VP2 protein is not only an important protective antigen of virus,but also plays an important role in the pathogenesis of virus infection.In this study,based on the infectious clone of CIAV,the CIAV mutant was constructed by site-directed mutation of the amino acid site related to VP2 gene,and the pathogenicity of the mutant was compared,which laid the foundation for the subsequent study of CIAV virulence and mechanism.In this study,according to the whole gene sequence of infectious clone SD1520 strain of CIAV,site-directed mutagenesis of amino acid 101 of VP2 gene and 161 and 162 amino acid of VP2 gene was carried out by PCR mutation method.The whole genome sequence of the mutated CIAVSD1520 strain was sequenced,and the results showed that the amino acids at position 101（arginine→glycine）,161（aspartate→glycine）and 162（glutamate→glycine）of the VP2 gene of SD1520 strain were mutated successfully,which were named SD1520/101 and SD1520/16 respectively.The whole genomes of SD1520/101 and SD1520/16 were amplified by PCR,cyclized by T4 ligase and transfected into MDCC-MSB1 cells.The virus was rescued and the rescued CIAV mutants were identified by indirect immunofluorescence assay and sequencing.The results showed that the MDCC-MSB1 cells transfected with the mutant strain could emit a specific bright green by CIAVVP2 polyclonal antibody.The sequencing results further confirmed that the 101 st and 162 nd amino acids of CIAV VP2 gene and the 161 st and 162 nd amino acids of VP2 gene were mutated successfully,but no mutation occurred at other sites.In order to quantify the CIAV genome accurately,a fluorescence quantitative PCR detection method for VP2 gene was established,which could specifically amplify the typical "S" curve for CIAV,but had no response to Marek’s virus,infectious bursal virus and avian leukemia virus.The sensitivity was 100 times higher than that of conventional PCR.The coefficient of variation of intra-batch and inter-batch repeated experiments was less than 2%.A large number of clinical products could be detected quickly and accurately at the same time.To provide technical support for diagnosis and epidemiological analysis of CIAV.In order to verify whether the mutant is infectious to chicken embryo,the supernatant of mutant SD1520/101 and SD1520/16 virus was inoculated into 7-day-old chicken embryo through yolk sac,and the DNA was extracted from thymus,spleen,bone marrow and other tissues at 19 days old for PCR detection.The results showed that the genome of CIAV mutant was distributed in different tissues of infected chicken embryo,which further confirmed that the infectious clone of mutant was constructed successfully.Subsequently,the mutants SD1520/101 and SD1520/16 were inoculated into1-day-old SPF chicks for animal infection test,and the SD1520 strain challenge control group and blank control group were set up to detect the effects of mutants on growth performance,HCT level,immune organ index,antibody level and virus load in various tissues and organs of SPF chickens at different ages.Autopsy showed that SD1520 group,SD1520/101 group and SD1520/16 group could cause immune organ atrophy,bone marrow yellowing,chest and leg muscle bleeding and other clinical symptoms in infected chickens,but the symptoms in SD1520 group were the most serious,and those in SD1520/101 group and SD1520/16 group were significantly lower than those in SD1520 group.The body weight of SD1520 group,SD1520/101 group and SD1520/16 group was lower than that of blank control group,and on the 14 th day,the body weight of SD1520/101 group and SD1520/16 group was higher than that of SD1520 group（P < 0.05,05）.On the 14 th and 21 st day,the hematocrit of SD1520 group and SD1520/16 group was significantly lower than that of the blank control group（P <0.05）.On the 28 th day,the hematocrit of the SD1520 group was still lower than that of the blank control group.The immune organ index of SD1520 group,SD1520/101 group and SD1520/16 group was lower than that of blank control group（P < 0.05）.The immune organ index of SD1520 group was the most serious,and on the 14 th and 28 th day,the immune organ index of SD1520/101 group and SD1520/16 group was lower than that of SD1520 group.SD1520/101 group and SD1520/16 group could significantly inhibit the titer of HI antibody induced by NDV and AIV-H9 vaccine,but the inhibitory effect of SD1520/10 group and SD520/16 group was lower than that of SD1520 group,the viral load of SD1520/10 group and SD520/16 group was lower than that of SD1520 group,and the content of CIAV in thymus and bone marrow was the highest.To sum up,this study successfully constructed the mutant at the amino acid site related to VP2 gene of CIAVSD1520.It was confirmed that the 101 st amino acid of mutant VP2 gene and the 161 st and 162 nd amino acid of VP2 gene could slow down the replication ability of CIAV and weaken the pathogenicity of CIAV.The pathogenicity of mutant SD1520/10 was the most significant,which laid a foundation for further study of the pathogenic mechanism of CIAV and provided an important scientific reference for the study of new gene vaccine candidates. |
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