Research Of The Pathogenic Mechanism Of ORF2-mediated Infection Of Goose Astrovirus Type 2 And Its Application In Immune Control

Since 2015,a gosling gout disease caused by GAstV-2 has been endemic in China.By 2017,the gout disease has spread to most provinces of China with high morbidity(80-90%)and mortality(20-70%),and caused significant economic losses to the goose industry.However,little is known about the infection mechanism of GAstV-2,and there is no commercial serological technique for the detection of the specific antibody against GAstV-2.In this study,the specific monoclonal antibody against the ORF2 protein of GAstV-2 was generated and used,GAstV-2 and ORF2 protein effectively induced the expression of OASL was found,the protein DDX1 interacted with ORF2 was identified,and the recombinant FAdV-4 virus FA4-ORF2_C expressing the C-terminus of GAstV-2 ORF2 protein was developed by CRISPR-Cas9 gene editing technology.1.Preparation and application of monoclonal antibodies against the ORF2 of GAstV-2 In order to develop monoclonal antibodies against GAstV-2 ORF2 protein,in this study,three mAbs against ORF2 of GAstV-2,designated as 3G6,5H7,and 6C6,were generated using the purified His fusion protein with ORF2_C protein(P2+ACTD domain)of GAstV-2 as immunogen.Three mAbs all exhibit good reactive characteristics of IFA,Western blot and IP.Epitope identification revealed that mAbs 3G6,5H7,and 6C6 recognized 695-704aa,685-694 aa and 635-644aa of ORF2,respectively.Further sequence alignments revealed that all three epitopes were highly conserved across GAstV-2 strains and less conserved in other AAstV members.At the same time,the sandwich ELISA established here could efciently react with GAstV-2 with high specifcity and low LOD,which would supply a convenient,cheap and high throughput serological method for GAstV-2 detection.Eighteen clinical samples were tested using this method,and,upon comparison with the results of IFA,the total coincidence rates were found to be 94.4%.This shows that this assay has a high degree of accuracy and can be used for GAstV-2 detection.Moreover,the sandwich ELISA could recognize GAstV-2 but not with GAstV-1,which will lay the foundation for diferentiation of two serotypes of GAstV in clinical samples.Furthermore,in this study,one novel peptide in the ORF2 recognized by monoclonal antibody 6C6 was used as an antigen to develop an efficient ELISA for detection of antibodies against GAstV-2.Specificity analysis showed that the ELISA only reacted with sera against GAstV-2,but not with sera against other pathogens tested.The sensitivity of the ELISA in detecting positive sera was higher than that of the IFA.The coefficients of variation(CV)of the intra-assay and inter-assay were both<10%,indicating that the reproducibility of ELISA was good.For detection of clinical samples,the ELISA had 87.5%concordance with the IFA,highlighting the feasibility of the pELISA for clinical application.The development of mAbs against the ORF2 of GAstV-2 and the related characteristics laid the foundation for further exploring the role of ORF2 in infection of GAstV-2,identifying interaction proteins with ORF2 and developing serological detection technology for GAstV-2.And the established indirect ELISA assay and sandwich ELISA assay provide technical support for diagnosis of clinical GAstV-2 infection and monitoring the efficacy of GAstV-2 vaccination.2.GAstV-2 and its ORF2 protein efficiently induce the expression of OASLIn this study,GAstV-2 was found to exhibit self-limiting replication in the LMH cell line.By testing the levels of several cytokines associated with innate immunity in LMH cells infected with GAstV-2,it was found that GAstV-2 could activate high levels of OASL in LMH cells via ORF2.The OASL could also be efficiently activated in the liver and kidney from the geese infected with GAstV-2.By truncating ORF2,the truncated ORF2 without the acidic domain could attenuate the activation of OASL compared with the intact ORF2.The results of the dual luciferase reporter assay showed that ORF2 could significantly activate the luciferase activity under the control of the OASL promoter in comparison with that in the control.Moreover,the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-2,respectively.OASL could be a vital host factor to restrict the viral replication of GAstV-2 in LMH cells.Also,ORF2 was found to be efficiently restrict the viral replication of both GAstV-2 and H9N2 avian influenza virus in vitro.All data not only provide novel insights for elucidating self-limiting infection of GAstV-2,but also lay the foundation for further studies on the molecular mechanisms between GAstV-2 and the host.3.Screening and identification of interaction proteins of GAstV-2 ORF2 proteinIn order to identify host proteins that interact with ORF2 and whether they play an important role in GAstV-2 infection and pathogenesis,in this study,mAb 6C6 specific to ORF2 was used to immunoprecipitate LMH cell lysates infected GAstV-2,and then the silver stain,mass spectrum and western blot analysis were carried out to screen and identify the host proteins that interact with ORF2.Finally,the interaction between GAstV-2 ORF2 and the host protein DDX1 was identified by immunoprecipitation and co-localisation assays.Then,the effect of knockdown and overexpression of DDX1 on GAstV-2 replication and the effect of GAstV-2 replication on the expression of endogenous DDX1 were detected.The results showed that GAstV-2 infection could promote endogenous expression of DDX1.Meanwhile,interference with DDX1 in LMH cells significantly inhibited GAstV-2 replication.This study provides a new host target to further investigate the replication and infection mechanism of GAstV-2.4.Generation and in vitro characteristics of recombinant FAdV-4 virus expressing ORF2_C of GAstV-2In order to prevent and control the infection of GAstV-2 and FAdV-4 at the same time,this study successfully developed a recombinant FAdV-4 virus expressing ORF2_C protein of GAstV-2,FA4-ORF2_C,based on the CRISPR/Cas9 gene-editing technology and Cre-loxP recombinant systems,using the EGFP-expressing recombinant FAdV-4 constructed previously by our group as a template and editing the Fiber-2 gene of FAdV-4.The results of the in vitro characterization of the virus indicated that FA4-ORF2_C could stably replicate in LMH cells,and the virus titer could reach 106 TCID50/mL,but the replication ability was lower than the wild type FAdV-4.All these demonstrate that the reorganized virus FA4-ORF2_C provides candidate vaccine and new vaccine ideas for simultaneous prevention and control of GAstV-2 and FAdV-4,but also prove that FAdV-4 can be used as a vaccine vector to construct polyvalent or multiplex vaccines,laying the foundation for further development of the poultry virus vector vaccines.