Isolation And Identification Of Goose Astrovirus And Preliminary Study On Subunit Vaccine

Since 2016,an infectious disease characterized by visceral and joint gout has broken out in goslings in many regions in China.Many studies have confirmed that the key causative pathogen is a novel avian astrovirus,named goose astrovirus(GAstV)due to its widespread in geese.GAstV mainly affects 3-15 daysold goslings,with the motality rate of about 30%.GAstV mainly transmits horizontally through digestive tract and respiratory tract,and can also be transmitted vertically through contaminated embryonated eggs.GAstV is a non-enveloped,single-stranded positive-strand RNA virus with stable structure,which is resistant to acid and heat,and could keep long-lasting infectivity in the external environment.Therefore,it quickly spreads to Jiangsu,Shandong,Liaoning,Henan,and other regions within a few years after the first discovery.Since lacking effective drugs and vaccines to prevent and curb the spread of GAstV,the specific prevention and control measures are relatively limited,seriously threatening the healthy development of goose breeding industry.1.Isolation and identification of goose astrovirusIn this study,the suspected clinical sample was ground with liquid nitrogen,and then inoculated into goose embryo fibroblasts(GEF)for pathogen identification and isolation.After PCR/RT-PCR detection,gene sequencing analysis,and IFA,the pathogen was confirmed to be GAstV and named CH-HNXX-2021.The virus titer was 106 TCID500.1mL-1,and it replicated well in GEF.The virus titer reached peak at 48 h post infection,and then decreased slowly.Goslings negative for GAstV by RT-PCR test were randomly divided into two groups.Each was inoculated through oral administration and leg intramuscular injection with 0.1mL GAstV,respectively.The animal regression test was carried out during the observation period of 14 days.One died on the 8th day in the infected group,and autopsy showed enlargement of kidney,spleen,and joint,with white urate deposition on the surface of ureter,liver,and heart.Body weight monitoring showed that there was no significant difference in weight between the animals in challenge group and its counterpart on the 1st,2nd,and 3rd day after infection.From the 4th day,the body weight of animals in challenge group slightly decreased,and then slowly increased.At the end of the experiment,the average body weight of animals in challenge group was 285.71 g,while that of the control group was 458.75 g,indicating that GAstV infection stunted the growth and development of goslings.Fluorescence quantitative PCR was used to detected virus shed from cloaca of infected animals,and results showed that thevirus titer reached the peak of 5.069 lg copies/μL on the 9th day,and then decreased slowly to 2.145 lg copies/μL on the 14th day.The above results indicated that the isolated GAstV is the causative pathogen for gout in goslings and provides an alternative strain for the developing vaccine in the future.2.Establishment and application of fluorescent quantitative SYBR Green I PCR based on GAstV epidemic strainIn order to establish a method for rapid etiological diagnosis of GAstV,specific primers were designed based on the conserved region of GAstV ORF2 gene.The target fragment of 288 bp was amplified by RT-PCR and ligated into pMD18-T vector,and the pMD-18T-GAstV-ORF2 plasmid was successfully constructed.Subsequently,taking the plasmid as template,the reaction conditions such as primer concentration and annealing temperature were optimized gradually,and the specificity,sensitivity,and repeatability were tested.The results showed that the reaction had a good amplification efficiency when the final concentration of primers was 200 nmol/L and the annealing temperature was 61℃.There was a good linear relationship between the labeled curve Cq value and the plasmid concentration.The correlation coefficient R2 was 0.994,and the amplification efficiency was 101.6%.The specificity was evaluated by using several common clinical pathogens of goose disease,including FAdV,GPV,GciV,AIV,and NDV.It was found that a high and smooth amplification curve was observed only by using GAstV cDNA as a template,indicating that the specificity was good.The established method in this study is 10 times more sensitive than conventional RT-PCR,and the coefficients of variation of inter-group and intra-group repetitive experiments are less than 2.5%,indicating that this method had high repeatability and good stability.This established method and conventional RT-PCR were used to detect 30 randomly collected cloacal swabs,and results showed that the positive rate was 83.3%and 66.7%,respectively,and the overall coincidence rate was 83.33%.The above results indicate that the fluorescent quantitative PCR method of SYBR Green I for GAstV ORF2 gene has been successfully established,which can be used in the epidemiological investigation of GAstV and lay a solid foundation for the evaluating immune efficacy of vaccine.3.Establishment and application of indirect ELISA antibody detection method based on GAstV Cap proteinIn order to establish a specific serological diagnosis method for GAstV,the antigenic epitopeson Cap protein of GAstV HNXX-6/China/2020 strain were predicted by IEDB and BepiPred2.0 bioinformatics software.It was found that there were 6 potential B cell epitopes at position 371 to 604.Three-dimensional structural analysis showed that the 6 epitopes formed a dense antigenic epitope region on the surface of Cap protein and were highly conserved in GAstV.Therefore,specific primers were designed for this region,and the amplicons were inserted into the pET-32a vector to construct the recombinant plasmid pET-32a-shCap.After transformed into BL21(DE3)competent cells,the recombinant shCap protein about 43 kDa was obtained by ultrasonic fragmentation,purification,and Western-blot.Using it as a coating antigen,an indirect ELISA was established.The parameters via chessboard method showed that the optimal coating concentration of recombinant shCap protein was 2 mg/L,the blocking condition was 5 mg/mL BSA of 5 mg/mL at 4℃ for 12 h,the serum dilution was 1:50 at 37℃ for 30 min,the concentration of enzyme labeled secondary antibody was 1:10000 at 37℃ for 30 min,and the TMB coloration time was 15 min.20 GAstV negative sera were detected under the optimized conditions,and the cut-off value of this method was 0.379.Then,according to the established indirect ELISA method,the specificity,sensitivity,and coincidence rate test were carried out.The results showed that the established method did not react with specific sera for other common goose pathogen,and its sensitivity was 1:800,which was 4 times higher than that of Western-blot.73 goose serum samples were detected by this method and Western-blot.Results showed that the positive rate of was 27.3%and 23.2%,respectively,and the overall coincidence rate was 93.5%.The above results show that the indirect ELISA method established in this study has strong specificity,high sensitivity,and good repeatability,and it can be used for large-scale serological investigation and provide technical support for the prevention and control.4.Preliminary preparation and efficacy Evaluation of GAstV Cap protein subunit VaccineBased on experiment 3,we optimized the parameters of prokaryotic expression and protein purification to obtain a great amounts of recombinant proteins.After mixing with adjuvants to prepare subunit vaccine,and its immunogenicity was evaluated.The results showed that the optimal IPTG concentration was 0.4mM/mL,the induction temperature was 27℃,and the induction time was 8 h.The protein was eluted with 100,250 and 350 mM/mL Imidazole,respectively.SDS-PAGEshowed that the greatest amount of recombinant protein waseluted with 350 mM/mL Imidazole.The purified and renatured recombinant protein was fully mixed with adjuvant at 1:1 to prepare subunit vaccine.GAstV negative goslings were immunized with different doses for different times.The serum was collected every week after immunization,and antibody titer was determined by neutralization test.The results showed that two weeks after immunization,animals immunized with 50 μg and 100 μg recombinant protein produced specific antibodies,and the titer was up to 2 log2.After the second immunization,the antibody level of 50 μg immunized ones increased slowly compared with that of 100 μg group,but the final serum antibody level was almost the same,reaching 5 log2.The results of IFA showed that the serum had a good inhibitory effect on the virus even if the dilution ration was 1:25,indicating that serum neutralization test was consistent with those of IFA.At the same time,it indicated that the inactivated vaccine prepared in this study could effectively induce specific neutralizing antibodies against GAstV,which laid the foundation for the development of related vaccines.