Isolation,Pathogenicity And Bioinformatics Analysis Of A Virulent Strain Of Chicken Infectious Anemia Virus

Chicken anemia virus can cause aplastic anemia,which damages the bone marrow,generalized lymphoid atrophy and in growing chickens with immunodeficiency,eventually they are now the leading cause of death insecondary bacterial infection.Chicken infectious anemia virus has been widespread in many countries of the world,which brings greatest losses to poultry industry and become a global problem.Due to the virus is also one of the most common exogenous viral contaminants in the attenuated vaccine,therefore,we designed the simulated experiments to observe the effect of the immune and weight of chickens after use the attenuated vaccine with low dose of CAV.It has been reported that CAV is only pathogenic to chicks,in large age of chicken with infectious anemia syndromes are also reported.In this study,a CAV wild-strain was isolated from a large number of chickens with obvious anemia symptoms.The pathogenicity of different doses,and different ages of infection was conducted by experimental treatment,the biological information analysis based on the whole genome were systematically analyzed.1.Effect of low dose of CAV in attenuated vaccine on SPF chicken body weight and vaccine immune antibodyIn this study,the effects of low dose of CAV on the weight of SPF chickens and NDV antibody production were observed by simulated experiments.The results showed that 10EID50 and 5 EID50 CAV per plume attenuated NDV vaccines were used to cause the weight loss of SPF chickens.NDV antibody levels compared with the use of the non contaminated groups also decreased after use the vaccine with two doses of CAV contaminated.It has significant difference.A certain proportion of CAV antibody positive was detected at thebeginning of the second week after use the NDV vaccine with two doses of CAV contaminated.The detection of a high proportion of CAV nucleic acid was detected in the first week after the use of a contaminated vaccine.The results of the study demonstrate the effects of CAV pollution on the production and immune function of SPF chickens,and it is suggested that increasing the detection of viral nucleic acid can help save time and improve the detection rate in the detection of exogenous virus contamination by SPF chicken test method.2.Pathogenicity of CAV strain to one day old SPF chickensTwo months sick chickens were growing slowly,then individual difference increased gradually in breeding chicken farm in shandong,2015.The clinical symptoms and signs manifestations of comb and caruncle were pale appearance.The main characteristics of pathological necropsy were that focile marrow was pink or pale yellow,thymus atrophied.DNA was extracted from the original samples which were inoculated to chicken embryos and confirmed that CAV positive by dot blot.A CAV strain was isolated successfully and named CAV-SD15.Four dose of SD15 strain(150EID50,300EID50,700EID50,1500EID50)were injected via intramuscular to 1 day old SPF chickens.In order to study the pathogenicity of different doses CAV to 1 day old SPF chickens,the body weight,blood index,antibody level and viremia were detected every week.At the same time,the mortality rate was recorded,and the immune organ index was measured from all necropsy birds at three weeks.The results showed that different doses of infection group had significant growth retardation and immunosuppression compared with the control group(p<0.05),the main manifestation of immunosuppression showed that the thymus index and bursa index of the infected group were significantly decreased,and the decrease of the antibody level of NDV vaccine was significantly(p<0.05).However,there was no significant difference between the two groups of 150EID50 and 300EID50 groups.The mortality rate of chickens can be caused by different doses,the mortality rate of 1500EID50 group can reach 65%(7/20).3.Pathogenicity of CAV strain to 10 week old SPF chickensDue to CAV generally causes clinical symptoms of young chickens,but the strain was isolated from the 2 months old chicken.In this study,a total of 10 10 week old SPF chickenswere injected with SD15 strain(1500EID50/plume)via intramuscular,the pathogenicity of the isolated strain to SPF chickens was observed.The body weight,blood index,antibody level and viremia were measured every week last five weeks.All the chickens were necropsied and the immune organ index was measured at the end of 15 weeks.The results showed that the infection group showed significant growth retardation and immunosuppression compared with the control group.The main manifestation of immunosuppression was that the thymus index and bursa index of the infected group were significantly decreased,and the antibody level of AIV-H9 vaccine was significantly reduced(p<0.05).The infection group showed that the bone marrow was seriously yellow and the lowest number of red blood cells was only about 50% of the control group and showing significant anemia symptoms.The incidence of death was not found in the infected group,which showed that the isolate had obvious pathogenicity to 10 weeks old SPF chickens,but the mortality rate was 0%.4.Bioinformatics analysis of CAV strainAfter the whole genome sequence of CAV isolate was obtained by amplification and sequencing,MEGA,BioEdit and other bioinformatics software were used to analyze the genetic evolution relationship and amino acids in coding region of CAV-SD15 strain.The results showed that the SD15 strain had the highest homology(98.9%)with CAV18 strain isolated from Taiwan,and had the lowest homology(93.9%)with the NC001427 strain isolated from the United States.The phylogenetic tree can be found on the over 34 isolates were mainly divided into two branches,the SD15 strain is in the second branch,CAV18 strain is distributed in the same branch,shows a close genetic evolutionary relationship.There were16 amino acid sites were different from other reference strains,the mutation of the two amino acid sites was not found in the past.In the VP3(apoptosis protein region),SD15 has two amino acid mutations and the mutations located in the weak apoptosis-inducing domain,we speculated that the mutation enhances apoptosis induced by weak apoptosis-inducing domain and enhanced the virulence of strain SD15 eventually.