Isolation And Identification Of Chicken Infectious Anaemia Virus And Development Of Subunit Vaccine And DNA Vaccine

Chicken infectious anemia（CIA）is an immunosuppressive disease caused by chicken infectious anemia virus（CIAV）,which is mainly characterized by aplastic anemia and lymphoid tissue atrophy.CIAV mainly infects chicks aged 10-14 days.The infected chickens are more susceptible to secondary infection of other pathogens,bringing huge economic loss to the poultry industry.In recent years,the positive rate of CIAV infection in chicken farms in China had been increasing.According to epidemiological survey data,CIAV has been widely found in various types of chicken flocks in China.Vaccine immunization is an important way to prevent and control this disease.However,the CIAV attenuated vaccine used abroad had a strong virulence.Although researches about inactivated vaccines and subunit vaccines had been reported,there were some disadvantages exsited such as high production cost and poor protection efficiency.In this study,the suspected CIAV tissues collected from broilers in Shandong Province were detected,and the whole genome of 33 strains were sequenced.Subsequently,CIAV subunit vaccine and DNA vaccine were prepared respectively,and the protective effect of the "prime-boost" immunization strategy was evaluated.1.Isolation,identification and whole genome sequence analysis of CIAV from broilers in some areas of Shandong ProvinceIn order to investigate the CIAV infection status of broilers and the molecular characteristics of CIAV strains in Shandong Province,the suspected broilers samples were collected from January 2020 to September 2021 and detected by PCR.The results showed that the positive rate of CIAV of those collected samples was 52.05%（38/73）.Then,positive samples were inoculated into MDCC-MSB1 cells,and 33 strains of CIAV were successfully isolated.Subsequently,the whole genome sequence of the 33 strains of virus was determined and analyzed.The results showed that the homology of the whole genome sequence of 33 strains of CIAV was ranged 95.7%-99.3%,and the homology with Gen Bank reference strain was ranged 95.3%-99.3%.The homology of VP1 nucleotide sequence of 33 strains of CIAV was ranged 95.1%-99.8%,and a total of 35 amino acid sites were found in VP1 amino acid sequence.The 394 th position of VP1 was Q,suggesting that these isolates had a strong pathogenicity.Through the prediction of possible genome recombination by RDP4 software, it is found that four strains SD2014,SD2009,SD2103 and SD2102 occuered genetic recombination.The above results were helpful to deeply understand the molecular epidemiological characteristics and evolution of CIAV,and awared us the importance to strengthen the monitoring and prevention and control of CIAV.2.Preparation and evaluation of protective efficiency of CIAV subunit vaccine and DNA vaccineIn order to prepare a safe and effective CIAV vaccine and explore an efficient vaccination strategy,the prokaryotic expression plasmids pET32-VP1 and pET28-VP2 were constructed.The expression of VP1 and VP2 recombinant protein were induced by E.coli expression system,and SDS-PAGE and Western blot showed that the recombinant protein was successfully expressed.The two proteins were mixed in 1:1 and then mixed with Freund’s complete adjuvant to prepare subunit vaccine.Then,CIAV VP1 gene and VP2 gene were connected into pBud CE 4.0 bidirectional expression vector to construct eukaryotic expression plasmid pBud-VP1-VP2,and DF-1 cells were transfected with pBud-VP1-VP2.Western blot analysis showed that the proteins could be expressed successfully.pBud-VP1-VP2 plasmid was mixed with liposome to prepare DNA vaccine.According to the research reports,subunit vaccine immunization could stimulate the production of antibodies,but was difficult to induce cellular immunity.The immune response induced by DNA vaccine was more comprehensive,while the protect efficiency was unsatisfactory.Therefore,we explored the "prime-boost" vaccination strategy in this study.Sixty 1-day-old SPF chickens were randomly divided into 4 groups.The first group was immunized with DNA vaccine at the age of 1 and 14 days old;Group 2 was immunized with subunit vaccine at the age of 1 and 14 days;Group 3 was immunized with DNA vaccine at 1day old and subunit vaccine at 14 days old;Group 4 was immunized empty vector pBud CE4.0 at the age of 1 and 14 days.After the first immunization,the serum was collected every week to detect the CIAV antibody titer.The results showed that both subunit vaccine and DNA vaccine could induce antibody immune response,and the antibody level induced by DNA subunit vaccine combined immunization was the highest.In order to investigate the effect of vaccination on cellular immune function,the lymphocyte proliferation ability in each group was measured by lymphocyte proliferation test.The results showed that the SI index of the three vaccine immunized groups was higher than that of the control group,and the SI index of the DNA-subunit vaccine combined immunization group was significantly higher than that of the single immunization group（P < 0.05）.Then,the lymphocytes were stimulated by VP1 recombinant protein,and IL-2,IL6 and IFN-γin the supernatant were detected by ELISA.The results showed that IL-2,IL-4 and IFN-γin the three vaccination groups were increased,and the expression level of cytokines in DNA-subunit vaccine combined with immunization group was the highest（P<0.05）.In order to further investigate the protective efficiency of “prime-boost” strategy,animal challenge experiment was carried out.75 1-day-old SPF chickens were randomly divided into5 groups: DNA vaccine group,subunit vaccine group,DNA-subunit vaccine combined group,CIAV group and control group.Group 1-4 were injected with CIAV SD15 strain at the age of21 days,and the protective effect was evaluated by hematocrit（HCT）,thymus index and viral load.The results showed that the HCT value and thymus index of CIAV group decreased significantly,while the HCT value and thymus index of immune group recovered,and the recovery of DNA-subunit combined group was the most significant.The results of viral load showed that viremia could be detected in all groups except the control group.The virus load of DNA-subunit combined group and the number of positive individuals was the lowest,indicating that combined immunization could provide a better protective efficiency.This study showed that the “prime-boost” strategy could induce both a higher level of antibody response and cellular immune response,which had a good application prospect in CIAV vaccine immunization.