Isolation And Identification Of Chicken Infectious Anemia Virus Strains,Sequence Analysis Of Whole Genes And Pathogenicity

Chicken infectious anemia(CIA)is an infection characterized by avian infectious anemia virus(CIAV),characterized by aplastic anemia,atrophy of lymphatic tissues throughout the body,subcutaneous and muscle bleeding disease.CIAV mainly infects chicks,but chickens of all ages are susceptible.The CIAV serological survey was conducted on 14 farms in seven cities in Guangdong Province to understand the epidemic situation of CIAV in Guangdong Province.The suspected CIAV materials submitted for clinical examination were identified by PCR,sequence analysis,virus isolation,and virus identification.The results showed that the positive rate of CIAV antibodies in the collected serum samples was 62.15%(202/325);four samples of CIAV were detected by PCR;gene sequence comparisons revealed that the open reading frames of the four CIAV isolates did not have insertion or deletion of amino acid,and the 394th amino acid of VP1protein is Glutamine(Q).The nucleotide similarity of VP1,VP2 and VP3 protein genes with reference strains in China and abroad is 94.1%?99.6%,98.5%?100%,98.4%?100%;Genetic evolution analysis shows that the prevalence of CIAV in my country is mainly group C.The four strains of CIAV belong to group C2 and have a genetic distance from the vaccine strain,and it can be determined that they are wild virus strains;four isolates cannot grow in SPF chicken embryos were isolated by continuously passaging 5?6 days on MSB1 cells.Obvious cell lesions were observed under the microscope,including cell swelling,fragmentation,and increased debris.Indirect immunofluorescence test using CIAV-positive serum showed obvious green fluorescence,which proved that the 4 isolates of the virus were all CIAV.Specific primers for CIAV were designed and a real-time quantitative PCR detection method for clinical detection of CIAV was established.The standard equation is Y=-3.55lg X+37.98 with acorrelation coefficient R²=0.997,and the amplification efficiency E=91.3%.This method has showed a high specificity,and has no specific amplification for NDV,ARV,and FAd V-4,and non-specific amplification curves for AIV,but the melting curve of AIV is significantly different from the melting curve of CIAV.Combine the amplification curve and melting curve for negative determination,the detection limit was 27 copies/?L for positive plasmid,which is 10 times that of ordinary PCR;good repeatability,3 batches of 3 different batches of positive plasmids Intra-and inter-assay duplication,the coefficients of variation were less than 2.4%.The growth characteristics of CIAV-GDJM strain on MSB1 cells was further investigated.It was found that the virus proliferated very slowly and hardly proliferated from 0?24 h after infection.The virus began to proliferate at a faster rate 24?96 h after infection,and 96 after infection h virus titer reached the highest.To investigate the pathogenicity of the CIAV-GDJM strain in Guangdong Province to SPF chicks,the leg muscles of one-day-old SPF chicks were injected with0.2 m L virus solution(100 TCID₅₀),and the control leg muscles were injected with0.2 m L of PBS.Chicks were random grouped with a number of fifteen in each group.At 7 days,14 days,18 days,and 21 days after infection,three SPF chicks from each group were autopsied for testing.The results showed that the experimental group of SPF chickens showed clinical symptoms such as lying prone,pale crowns,and depressed spirits.Post-mortem examination showed that the heart,liver,spleen,lung,kidney,bursa,and intestine of SPF chicks in the experimental group had no obvious pathological changes,but the thymus was severely atrophied,bone marrow was yellow stained,fat deposits,no death and muscle were seen.The erythrocyte sedimentation rate test showed that the hematocrit value of SPF chickens in the test group was significantly lower than that in the control group(P<0.05).The pathological section showed that the pathological changes were more serious 14 days after infection;the thymus cortical area was severely degraded;the liver hepatic cord arrangement was disordered,the hepatic sinusoids were widened and there was a small amount of lymphocyte infiltration;Decrease;the nodules of the bursa of the bursa of the bursa atrophy and the gap widens slightly.Detection of antibodies and cytokines found that the production of CIAV-specific antibodies requires at least 7days;the concentrations of IL-2,IL-4,IL-10,and IFN-?in serum are all increased or decreased,of which IL-2 changes the most Obviously,followed by IL-4;IL-10 and IFN-?were not greatly affected.The viral load of thymus is greater than other organs,and the viral load of liver,spleen,bursa and bone marrow are larger than other time points 14 days after infection.In addition,most of the SPF chicks detected detoxification at four time points after infection(10/12),but the virus titer is lower(Ct>28).In this study,through serological investigation,virus isolation and identification,complete gene sequence analysis,rapid detection method establishment,virus growth characteristics research and pathogenicity test,the prevalence of CIAV in Guangdong Province and low dose CIAV can also cause SPF Chicks have clinical symptoms and pathological changes,which provide reference for the prevention and control of CIAV in Guangdong Province and the development of vaccines.