Establishment Of A Reverse Genetics System For BmCPV And Vaccine Design Against Avian Influenza

Bmbyx mori cypovirus(BmCPV),a member of the reovirus family,is a dsRNA virus consisting of ten dsRNA virus segments,each with its own complete open reading frame(ORF).It encodes 6 structural proteins,(VP1,VP2,VP3,VP4,VP6,VP7),and 3 nonstructural proteins(p101(NSP5),p44(NSP8),NS5(NSP9)),and one polyhedrin.Due to the particularity of BmCPV structure and the lack of susceptible cell lines,the study of functional fragments of BmCPV was hindered.In order to solve this problem,the study aims to establish a reverse genetics system,which can lay the foundation for the further study of BmCPV.This study is based on the constructed ten segments BmCPV plasmid containing T7 RNA polymerase,The recombinant plasmids were linearized and co-transfected into BmN cells which expressing T7 RNA polymerase stably,the results showed that the cells showed obvious pathological changes.Further,the co-transfected cultured cells supernatant transferred in BmN cells,the expression of vp1 gene in BmCPV was detected by Real-time PCR,Western blot assay and immunofluorescence assay showed that BmCPV proteins can be detected in BmN cells which infected by rBmCPV.These results sugeested that the recombinant virus was rescued successful.In order to learn the infectivity of recombinant virus rBmCPV to silkworm,we can observe free polyhedral in the midgut through microscope by adding the recombinant virus to it.Real-time PCR showed that the recombinant virus could be reproduced in the midgut,which can prove that the rescued recombinant virus rBmCPV could infect and replicate the midgut of silkworm cells.Avian influenza virus(AIV)is a influenza A virus which has eight different negative RNA fragments and belongs to Orthomyxoviridae.After the influenza virus infects the host cell,the antigen epitope of the virus is recognized by the host's immune cell receptors,creating an immune response.We have predicted the amino acid sequences of T cells and Th and B cell epitopes of different subtypes of AIV based on computer software,optimized with reference to the dominant baculovirus codons,and synthetically obtained the corresponding nucleotide sequences CTLT and THB.The P10 promoter controls the CTLT sequence and the CMV promoter controls the THB sequence.Recombinant baculovirus rBac-CMV-THB-CTLT was constructed by using Bac-to-Bac system.Based on this,PCR,Western blotting,and cell immunofluorescence demonstrated that the antigenic epitope CTLT can be expressed in silkworm cells which infected with recombinant baculovirus;rBac-CMV-THB-CTLT can enter mammalian HEK293 t cells,and express the antigen epitope THB.Further,the recombinant virus rBac-CMV-THB-CTLT was used to immunize chickens and mice.The results of indirect ELISA,immunohistochemistry,T-lymphocyte proliferation,and organ coefficient showed that rBac-CMV-THB-CTLT could cause Specific humoral and cellular responses to chicken and mice by H7 antigen.The research results laid the foundation for the application of the multi-antigen oral vaccine for AIV.