Molecular Mechanism Of Cellular Cholesterol 25-hydroxylase Inhibiting Senecavirus A Replication

Senecavirus A(Senecavirus A,SVA),a member of the Genus Seneca of Picornaviridae,is a single-stranded positive-stranded RNA virus without envelope.Seneca virus has the closest genetic relationship with myocarditis virus,which can cause blisters and festering wounds on pig nose and hoof crown,resulting in lameness and even death.The virus was first detected in Brazil in 2015.Subsequently,the virus was reported in more and more countries,and the SVA epidemic in China has spread in many provinces,which has brought great economic losses to the pig industry.At present,the mechanisms of immunity and pathogenesis are not clear,which is not conducive to the prevention and control of the disease.Cholesterol-25-hydroxylase(CH25H)gene belongs to the family of interferon-stimulating genes,which plays a key role in inflammation,innate immunity and subsequent acquired immune response through interferon signaling.Studies have shown that CH25 H can inhibit the infection of a variety of highly pathogenic viruses.In this study,using SVA VP2 recombinant protein as immunogen,4 monoclonal antibodies against SVA were successfully prepared,which laid an important foundation for the study of the mechanism,diagnosis and prevention of the disease.We further studied the functional molecular mechanism of host CH25 H against SVA,systematically elucidated the role of CH25 H in SVA replication,enriched the theoretical basis of the virus replication mechanism,and provided new ideas for the prevention of viral infection.The contents of this research as following:1.Development of monoclonal antibody against VP2 protein of Senecavirus A.In this study,the purified recombinant VP2 protein was used as immunogen to immunize 6-week-old BALB/c female mice.The serum of 6-week-old female mice was positive by indirect ELISA detection.Cell lines stably secreting Mc Ab against SVA VP2 protein were successfully obtained by cell fusion and subcloning,which were named 1A5,9D2,1C4 and 6C12,respectively.The results of Western blot and IFA showed that the four Mc Abs could specifically recognize SVA.The development of these monoclonal antibodies has laid an important foundation for the study of the structure and function of SVA virus protein.2.The Molecular Mechanism of CH25 H Inhibits SVA Replication in BHK-21 cellsIn order to study the effect of CH25 H on SVA replication and its molecular mechanism,eukaryotic expression plasmid p CAGGS-CH25H-HA was transfected into BHK-21 and IB-RS-2 cells,respectively.It was found that CH25 H significantly inhibited the replication of SVA.At the same time,the results of si RNA interference or CRISPR/Cas9 knockout of CH25 H,which showed that SVA replication was enhanced,and CH25 H played an antiviral role mainly through its enzyme activity product 25 HC,and 25 HC could significantly inhibit the adsorption and replication of SVA.Further,we found that CH25 H mutant lacking hydroxylase activity(CH25H-M)still restricts SVA infection.CH25H-M was co-transfected with viral proteins(L-e GFP,VP1,VP2,VP3,VP4-e GFP,2B,2C,3A,3C and 3D)into cells,and it was found that CH25H-M could significantly degrade the 3A protein through ubiquitin-proteasome pathway.Co-immunoprecipitation assay showed that CH25H-M could interact with 3A protein.Altogether,these results suggest that CH25 H has an antiviral function in SVA infection and provides an alternative manner to control SVA.In summary,four monoclonal antibodies against SVA were successfully obtained,and CH25 H could inhibit SVA replication in both enzyme activity-dependent and independent ways.These results will be helpful to SVA infection and pathogenic mechanism,and lay a foundation for the development of diagnostic kits and prevention strategies.