Production of recombinant hantavirus nucleocapsid protein expressed in silkworm larvae and its use as a diagnostic antigen in detecting antibodies in serum from infected rats.

The recombinant nucleocapsid protein (rNP) of Hantaan virus was expressed by a baculovirus vector in silkworm hemolymph and was used as an antigen in western blotting (WB). The rNP is expressed in insoluble form in hemolymph; therefore simple washing of the insoluble fraction with phosphate-buffered saline by low-speed centrifugation allowed preparation of purified antigen for WB. The rNP had strain-specific and hantavirus-common epitopes similar to the authentic NP antigen of hantavirus and was stable after transfer to membrane. For detection of antibody in serially obtained sera from experimentally infected rats, WB enabled detection of IgM antibodies 3 days after infection, which was at least 2 days earlier than detection by the indirect immunofluorescent antibody test (IFA). Thus WB had a higher sensitivity than the IFA for detection of hantavirus antibody in the serum of experimentally infected rats. The WB-determined IgG antibody titer was about 10 times higher than that determined by the IFA. No background staining was observed by WB even at a 1:10 dilution of serum. The selected rat sera with strong background staining or confusing staining patterns by IFA, but not focus reduction neutralization test titers, could be interpreted as test-negative because they did not have a specific reaction to virus antigen by WB. Thus the specificity of WB was higher than that of the IFA. Moreover, WB can distinguish specific from nonspecific reactions by the detection of the specific antigen on the WB membrane. Therefore the IFA or enzyme-linked immunosorbent assay followed by WB is recommended for serologic confirmation of hantavirus infection.