Full-length cDNA clones of the nucleoprotein (NP) genes of influenza A/Ann Arbor/6/60 and B/Ann Arbor/1/86 viruses were constructed from virion RNA and subsequently expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector, Autographa californica nuclear polyhedrosis virus. Western blot analysis of lysates prepared from Sf9 cells infected with the recombinant viruses confirmed that the baculo- virus-expressed NP antigens were reactive with monoclonal antibodies specific for either type A or B NP and with anti-NP antibodies in human serum samples. Electrophoretic analysis indicated that the expressed NP antigens comigrated with NP purified from influenza A or B virions and that the recombinant NP antigens represented greater than 10% of total protein in infected cells. Dilutions of clarified Sf9 cell lysates were used as antigens in a standard enzyme immunoassay to detect serum antibody specific for influenza A or B viruses. The results from assays using the baculovirus-expressed NP antigens showed good correlation with the results obtained using bacterially expressed NP antigen as well as complement fixation. Therefore, baculovirus-expressed NP antigens have the potential to be used to develop reproducible and routine assays for the serodiagnosis of influenza virus infections as an alternative to the complement fixation or haemagglutination inhibition tests.
CoxN. J.,
BaiZ. S.,
KendalA. P.1983; Laboratory-based surveillance of influenza A (H1N1) and (H3N2) viruses in 19801981: antigenic and genomic analyses. Bulletin of the World Health Organization 61:143–152
HarmonM. W.,
JonesI. A.,
ShawM. W.,
KeitelW.,
ReimerC. B.,
HalonenP.,
KendalA. P.1989; Immunoassay for serologic diagnoses of influenza type A using recombinant DNA- produced nucleoprotein antigen and monoclonal antibody to human IgG. Journal of Medical Virology 27:25–30
LanfordR. E.,
LuckowV.,
KennedyR. C.,
DreesmanG. R.,
NotvallL.,
SummersM. D.1989; Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system. Journal of Virology 63:1549–1557
LapeyreB.,
AmalricF.1985; A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100 kdalnucleolar protein. Gene 37:215–220
MatsuuraY.,
PosseeR. D.,
OvertonH. A.,
BishopD. H. L.1987; Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. Journal of General Virology 68:1233–1250
MurphyC. I.,
WeinerB.,
BikelI.,
Piwnica-WormsH.,
BradleyM. K.,
LivingstonD. M.1988; Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells. Journal of Virology 62:2951–2959
OvertonH. A.,
FijiiY.,
PriceI. R.,
JonesI. M.1989; The protease and gag products of the human immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 170:107–116
RyanK. W.,
MackowE. R.,
ChanockR. M.,
LaiC.-J.1986; Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA. Virology 154:144–154
SchmaljohnC. S.,
SugiyamaK.,
SchmaljohnA. L.,
BishopD. H. L.1988; Baculovirus expression of the small genome segment of Hantaan virus and potential use of the expressed nucleocapsid protein as a diagnostic antigen. Journal of General Virology 69:777–786
SchmaljohnC. S.,
ParkerM. D.,
EnnisW. H.,
DalrympleJ. M.,
CollettM. S.,
SuzichJ. S.,
SchmaljohnA. J.1989; Baculovirus expression of the M genome segment of Rift Valley fever and examination of antigenic and immunologic properties of the expressed proteins. Virology 170:184–192
SummersM. D.,
SmithG. E.1987; A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin1555
TownsendA. R. M.,
SkehelJ. J.1984; The influenza virus nucleoprotein gene controls the induction of both subtype specific cross-reactive T cells. Journal of Experimental Medicine 160:552–563
TsangV. C. W.,
PeraltaJ. M.,
SimonsA. R.1983; Enzyme-linked immunotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods in Enzymology 92:377–391
Van WykeK. L.,
HinshawV. S.,
BeanW. J.,
WebsterR. G.1980; Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies. Journal of Virology 35:24–30
WallsH.,
HarmonM.,
SlagleJ.,
StocksdaleC.,
KendalA.1986; Characterization and evaluation of monoclonal antibodies developed for typing influenza A and B viruses. Journal of Clinical Microbiology 23:240–245
WraithC. D.,
VesseyA. E.,
AskonasB. A.1987; Purified influenza virus nucleoprotein protects mice from lethal infection. Journal of General Virology 68:433–440