Isolation And Identification Of CIAV And Establishment Of Indirect ELISA For Antibodies Detection

Chicken infectious anemia virus(CIAV)is classified as the only recognized species of the Gyrovirus genus of the Anelloviridae.It is one of the common external pollutants of poultry attenuated vaccine,causing chicken anemia and immune suppression,and widely exists in chicken farms in the world.Because there is no effective surveillance antibody technology in China,it has caused huge economic losses to our chicken industry.The virus consists of three proteins,namely VP1,VP2,and VP3.VP1 is a capsid protein,VP2 is a chaperone protein that helps VP1 form the correct conformation and also has bi-functional phosphorylase activity,and VP3 is an apopsin protein that promotes cell apoptosis.Based on the above,on the basis of isolating and identifying a strain of chicken infectious anemia virus,this study established an indirect ELISA method for detecting antibodies to CIAV,and carried out preliminary application on clinical samples.At the same time,three strains of specific monoclonal antibodies against CIAV VP1 protein were developed by hybridoma technology.The results of this study lay a foundation for further CIAV research.1.Isolation,identification and genetic evolution analysis of chicken infectious anemia virusAfter treatment of the samples suspected to be infected with CIAV,the genome was extracted for PCR amplification,and specific bands with molecular weight of about 582bp were amplified.The samples were sterilized and inoculated into MSB-1 cells for subculture,and then the MSB1 cell suspension virus genome was extracted for PCR,The results showed that there were also specific bands of the above size.A CIAV strain was successfully isolated.The whole genome sequencing of this strain showed that the total genome size of this strain was 2298bp,which was named CIAV-SX-2104.The nucleotide homology analysis with representative strains showed that the nucleotide homology between this strain and domestic and foreign strains was 96.5%～99.4%.It had the highest homology with Ahhui1998 strain(99.4%),and the lowest homology with CIAV F10 strain(96.5%).This strain was closely related to Ahhui1998 and CIA-1 strains,but was far related to 26P4 and Cux-1 strains.Amino acid sequence analysis of CIAV-SX-2104 showed that amino acid 394 of VP1 protein was Q,indicating that this strain may be highly pathogenic.2.Establishment and preliminary application of an indirect ELISA method for detecting antibodies of chicken infectious anemia virusIn order to reduce the non-specific reaction caused by antigen impurity,DNAstar software was used in this study to analyze the antigenicity of CIAV VP1,and a peptide with strong antigenicity and high surface accessibility was selected for artificial synthesis.The results showed that peptide 1 had obvious broad spectrum and strong reactivity through ELISA detection of known anti-CIAV positive sera.Then,peptide 1 was used as an antigen-coated ELISA plate to establish an ELISA method for the detection of CIAV antibody by optimizing relevant conditions.The method had no cross-reactivity with ALV,AIV,MDV,NDV,FAdV,etc.and had strong specificity to CIAV.The effect variability of the same batch was small,and the detection result was stable.This method can detect CIAV antibody in the same clinical sera as imported commercial kit.The successful development of the kit provides conditions for clinical monitoring of CIAV.3.Preparation of monoclonal antibody against VP1 protein of CIAVBALB/c mice were immunized peritoneally by mixing the synthetic peptides with complete adjuvant,and immunized again with incomplete adjuvant 2 weeks later.After boost immunization,spleen cells of mice were fused with myeloma cells SP2/0,and 6 hybridoma cells secreting anti-CIAV VP1 protein were obtained by ELISA screening.The 6 monoclonal antibodies showed good reaction in ELISA and Western blot.These 6 monoclonal antibodies could recognize VP1 protein from MSB-1 cells infected with CIAV,and could react with VP1 protein expressed by prokaryotic expression system.One of the monoclonal antibodies,3D11,was able to recognize VP1 protein expressed in Sf9 cells and DF-1 cells transfected by eukaryotic plasmid in IFA.The monoclonal antibody provides a foundation for the subsequent study of CIAV protein interaction and the establishment of ELISA methods.