The pCAG M3 and pEFHAσNS plasmids were transfected into the BHK cells separately and co-transfected with Lipo3000 transfection reagent. Immunofluorescence method was used to detect the expressions and location of μNS and σNS proteins in the cells，NBV was used to infect the BHK cells， and Western blotting method was used to detect the expressions of μNS and σNS proteins in the transfected cells.A plasmid with 10－60 amino acid（aa） residues deleted from the N-terminal of σNS protein was constructed.Immunofluorescence method was used to detect the co-localized binding region of μNS and σNS proteins in the cells. Yeast two-hybrid experiment was used to verify the binding region of μNS and σNS protein interaction.

The subcellular location results showed that the μNS protein of NBV formed an inclusion body structure in the absence of other viral proteins， while the σNS protein was diffusely distributed throughout the cytoplasm.The Western blotting results confirmed that μNS and σNS proteins were expressed in the infected cells. Immunostaining was carried out with the conjugated antibody. It was observed under confocal microscope that the μNS and σNS proteins co-localized in the cytoplasm. The results of yeast two-hybrid experiment showed that the basic region of the σNS protein interacting with the μNS protein was located at 60 aa in the N-terminal region of the σNS protein within the residue region.

NBV autophagy-related protein σNS can be co-expressed in the viral inclusion bodies by interacting with μNS protein. The binding region of σNS and μNS protein is located within the 60 aa residue region of the N-terminal of σNS protein.