Cloning and expression of an envelope gene of West Nile virus and evaluation of the protein for use in an IgM ELISA.
West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and epidemiologic control in areas where multiple flaviviruses are endemic. The coexistence of WNV along with other members of flaviviruses like dengue and Japanese encephalitis in India has complicated the serodiagnosis due to cross-reactive antigens. In the present study, the development and evaluation of a highly sensitive and specific IgM enzyme-linked immunosorbent assay (ELISA) using the recombinant envelope protein (rWNV-Env) for rapid, early, and accurate diagnosis of WNV are reported. The gene coding for the envelope protein of WNV was cloned and expressed in pET 28a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rWNV-Env protein was optimized having no cross reactivity with healthy human serum. Furthermore, the specificity of this assay was confirmed by cross checking with serum samples obtained from patients with dengue and Japanese encephalitis viruses. The comparative evaluation of this rWNV-Env protein-specific IgM ELISA with plaque reduction neutralization test assay using 105 acute phase of clinical samples revealed 95% concordance with sensitivity and specificity of 92% and 97%, respectively. The positive and negative predictive values of recombinant-based Env ELISA were 94% and 96%, respectively. The recombinant envelope protein-based WNV-specific ELISA reported in this study will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.
Division of Virology, Defence Research and Development Establishment, Gwalior-474002, India.
Divyasha
D
Saxena
Manmohan
M
Parida
Putcha Venkata L
PV
Rao
Jyoti S
JS
Kumar
D000914:Antibodies, Viral / Q000097:blood; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood; D007194:India; D009500:Neutralization Tests; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D014901:West Nile Fever / Q000175:diagnosis*; D014902:West Nile virus / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins