| Chicken infectious anemia virus(CIAV)is a capsule-free,single-stranded circular DNA virus,which belongs to the family Circoviridae and genus Gyrovirus.CIAV is an immunosuppressive virus characterized by atrophy of bone marrow hematopoietic tissue in chickens,resulting in anemia.In 1979,CIAV was first isolated and identified in Japan,and the first case of CIAV infection in China was reported in Heilongjiang Province in 1992,and CIAV has been detected in various types of chickens in China.CIAV infection causes aplastic anemia and systemic lymphoid tissue atrophy,poor growth and development,and secondary infection after immunosuppression in the body,which has posed a significant threat to the global poultry industry.However,there is no effective treatment for CIAV infection,and breeder vaccination with attenuated virus is the main immunization strategy for CIAV prevention and control.Therefore,it is increasingly important to conduct epidemiological research on CIAV and establish effective detection methods in China to prevent and control the epidemic and infection of CIAV.In this study,the identification of CIAV were carried out in chickens with typical anemia symptoms,and a recombinant protein-based indirect ELISA that was established by using the baculovirus system to express simultaneously VP1/VP2/VP3 of CIAV,which was applied to the detection of clinical samples.These results provide a basis for the analysis of CIAV epidemic patterns and genetic variations,and detection techniques for clinical surveillance program.1.PCR identification of CIAV and sequence analysisIn this study,specific primers for VP3 were utilized to detect tissue samples of sick chickens with symptoms of CIAV,which were sent from Nantong,Tai’an,Yancheng and other breeder farms through PCR.The identification results suggested that CIAV infection was identified in these chickens.Based on another pair of specific primers designed and synthesized with reference to the sequence of the strain Cux-1,the whole genome cloning and sequence analysis of CIAV were carried out,8 CIAV strains were newly discovered from the tissue samples,named JS201020,JS210325,JS2104141,JS2104142,SD210421,JS210525,JS2107211 and JS2107212,respectively.And the results showed that the whole genome sequence length of all strains was 2298 bp.In comparison with 22 CIAV reference strains in different regions of China and other countries in GenBank,the nucleotide sequences of 8 CIAV strains had 95.90%～99.50%identity.According to different branches,the CIAV genetic evolutionary tree was divided into Group A,Group B and Group C.5 CIAV strains(JS210325\JS2104141\JS2104142\SD210421\JS2107211)with most domestic CIAV strains in China were distributed in Group A,and the remaining 3 strains(JS201020\JS210525\JS2107212)were located in Group B and Group C.Furthermore,the amino acid sequences of VP1,VP2 and VP3 of the CIAV strains were analyzed,8 CIAV strains were all glutamine at the amino acid site at 394 positions of VP1,indicating that these newly discovered CIAV strains were highly pathogenic.The VP1 protein of JS201020 and JS2107212 also had several mutations that probably were related to viral proliferation,whereas all the eight strains have less amino acid variability in VP2 and VP3.The whole genome sequencing cloning and analysis of the eight CIAV strains identified in this experiment would replenish the epidemiological information for further study of CIAV molecular evolution.2.Expression and purification of CIAV proteins in baculovirus expression systemPrimers were designed according to the sequence of eukaryotic expression vector pcDNA3.1 and the sequence of CIAV whole open reading frames(including VP1,VP2 and VP3),and the eukaryotic expression vector pcDNA3.1-CIAV was constructed.The pcDNA3.1-CIAV plasmid was used as template,and the whole open reading frames of the CIAV were cloned into the baculovirus vector pFastBac-HT A,then the recombinant bacmid rB-CIAV was obtained by transposing,which was used to transfect the insect Sf9 cells to generate the recombinant baculovirus that could express proteins encoded by all the open reading frames of CIAV,namely rBv-CIAV.Indirect immunofluorescence assay(IFA)revealed that the recombinant proteins expressed in the baculovirus system could be recognized by anti His-tag mAb.Western blot further demonstrated that the VP1,VP2 and VP3 proteins of CIAV were all expressed solubilly and showed biological reactivity,and the molecular weight of the expressed proteins were consistent with the predicted size of target proteins.The Sf9 infected with recombinant baculovirus was collected and the expressed proteins in the cell lysate supernatant were purified at a concentration of 1.02 mg/mL using Ni column.The purified CIAV proteins were detected by SDS-PAGE and Western blot,and the results indicated that the purified CIAV proteins were highly pure and reactive.The purified products of VP1/VP2/VP3 of CIAV obtained simultaneously in the baculovirus expression system could provide biomaterials for the further establishment of approaches for the detection of antibody against CIAV.3.Development of the indirect ELISA for detection of antibody against CIAVWith purified CIAV recombinant proteins obtained from above as specific coated antigens,an indirect ELISA(CIAV_ELISA)for detection of antibody against CIAV was established.In order to determine the suitable conditions for CIAV_ELISA,the working conditions including antigen coating concentration,serum dilution of the test samples,secondary antibody dilution concentration and other conditions were selected with square matrix titration.After optimization,the CIAV_ELISA conditions were as follows:antigen coating concentration was 2.50 μg/mL;serum dilution of the test samples was 1:400;The dilution of secondary antibody was 1:10000.The specificity,sensitivity and reproducibility of the CIAV_ELISA were detected,which indicated that the ELISA only reacted with sera against CIAV,but did not cross-react with positive sera of other avian viral infectious diseases such as Avian influenza virus(AIV),Avian leukosis virus(ALV),Infectious bronchitis virus(IBV),Fowl adenovirus(FAdV),Newcastle disease virus(NDV),etc..Compared with IFA,the CIAV_ELISA has higher sensitivity.Moreover,the CV values of the repeatability coefficient of variation between batches and within batches of the ELISA were less than 15%.In addition,for detecting the serum samples of clinical chickens,CIAV_ELISA had a 96.67%consistency rate with IDEXX kits.The above results revealed that the CIAV_ELISA based on the recombinant proteins expressed in the baculovirus system could provide a convenient and effective detection approach for monitoring the immunological statement of CIAV-immunized chickens and detecting the clinical infection of CIAV. |
