Preparation Of Polyclonal Antibodies Against IBV Nsp4 Protein And Effects Of Nsp4 And Viperin On IBV Replication

Avian infectious bronchitis virus(IBV)has caused serious economic losses to the poultry industry.Research on the pathogenesis and immune mechanism of IBV is very important for the prevention and control of this disease.In the early stage of virus infection,the innate immune response is the first line of defense of the host against virus invasion,and Viperin is an antiviral protein induced by innate immune response.Studies have shown that nonstructural protein 4(nsp4)encoded by IBV after infection of host cells is involved in the formation of replicative organelles to evade host recognition,antagonize host innate immune responses,and promote effective replication of the virus.Our previous studies showed that Viperin had an inhibitory effect on IBV,but the molecular mechanism of Viperin inhibiting IBV is unknown,in particular,the correlation mechanism of Viperin,nsp4 and replicator organelles needs to be studied.In view of this,this research focused on Viperin inhibiting viral replication by interfering with the formation of replicating organelles through acting on IBV nsp4,with the purpose of clarifying the pathogenesis and immune mechanism of IBV,and providing a clue for the development of new vaccines and drugs.The specific research contents are as follows:1.Prokaryotic expression of IBV nsp4 and preparation of polyclonal antibodiesIn order to further study the biological function of IBV nsp4,an IBV nsp4 full sequence eukaryotic expression vector was constructed,and the fragments with good antigenicity of IBV nsp4 were screened for prokaryotic expression,the recombinant truncated nsp4 protein was immunized with healthy rabbits and chickens to prepare polyclonal antibodies against nsp4,and the biological functions of the polyclonal antibodies were studied in the present study.The results showed that the recombinant nsp4 truncated protein with a molecular mass of 13.6 k D was successfully expressed,and two prepared polyclonal antibodies had high titers and could react with nsp4 expressed after infected with different serotypes of IBV strains or nsp4 expressed in transfection state.2.Molecular mechanism study on the effect of Viperin and IBV nsp4 on IBV replicationA fluorescence quantitative PCR detection method for IBV nsp4 was established,and a Viperin eukaryotic expression vector was constructed.Viperin and nsp4 eukaryotic expression vectors were transfected separately or co-transfected into cells,and then infected with IBV to study the role of Viperin and IBV nsp4 in virus replication.The results showed that overexpression of nsp4 protein could promote IBV replication;overexpression of Viperin could effectively inhibit IBV replication;simultaneous overexpression of Viperin and nsp4 protein could not restore IBV replication.Research showed that Viperin could inhibit the promoting effect of nsp4 on IBV replication.3.The key domains of Viperin inhibiting IBV replication and affecting on the replication organelles induced by IBVTo further investigate the molecular mechanism and key functional domains of Viperin inhibiting IBV replication,three Viperin domain deletion mutants eukaryotic expression vectors were constructed,and their effects on IBV replication were observed in transfected cells;The co localization of them with nsp4 protein and their effect on replication organelle induced by IBV were also studied.The results showed that the key domain of Viperin inhibiting IBV replication was the SAM domain;The N domain of Viperin was the key domain for co localization with the IBV nsp4;Viperin could affect the formation of zippered endoplasmic reticulum in replicate organelle,but the Viperin domain deletion mutants had no effect on the formation of replicated organelle.4.Transcriptome study on HD11 cells infected with IBVTranscriptome sequencing was performed after infecting HD11 cells with IBV Beaudette strain,and the enriched differential genes and related signal pathways were analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Ten differential genes were randomly selected for fluorescence quantitative PCR validation.The results showed that after 48 hours of infection with IBV in HD11 cells,619 genes were upregulated and265 genes were downregulated.GO analysis showed that differential genes were mainly significantly enriched in biological processes,including lymphocyte chemotaxis,monocyte chemotaxis,chemokine-mediated signaling pathway,positive regulation of inflammatory response,cellular response to interleukin-1,etc;KEGG analysis showed that differential genes were mainly significantly enriched in endocytosis,Herpes simplex virus 1 infection,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,etc;The fluorescent quantitative PCR verification showed that the transcriptome sequencing results were accurate and reliable.In conclusion,IBV nsp4 rabbit and chicken polyclonal antibodies with good reactivity and specificity were prepared;Viperin can inhibit the promotion of nsp4 protein on IBV replication,and Viperin inhibits IBV replication by affecting the formation of zippered endoplasmic reticulum in replicative organelle through the action on IBV nsp4 protein;IBV infection could cause changes in endocytosis,Toll like receptor signaling pathways,and NOD like receptor signaling pathways.This study provides a theoretical basis for clarifying the pathogenesis and immune mechanism of IBV,and provides new ideas for the development of new drugs and vaccines.