Cloning and expression of domain III of the envelope gene of Japanese encephalitis virus: Evaluation for early clinical diagnosis by IgM ELISA
Autor(es): Shukla Jyoti, Bhargava Rakesh, Dash Paban Kumar, Parida Manmohan, Tripathi Nagesh, Rao P V Lakshmana
Resumo: Japanese encephalitis virus (JEV) is the single largest cause of childhood viral encephalitis in the world with an estimated 50,000 cases - 10,000 deaths annually. The laboratory diagnosis is based essentially on IgM ELISA owing to low transient viremia making virus isolation difficult. In addition the requirement of cerebrospinal fluid (CSF) sample for confirmatory molecular diagnosis by reverse transcription-polymerase chain reaction (RT-PCR) makes IgM ELISA the test of choice for early clinical diagnosis. The development - evaluation of a highly sensitive - specific IgM ELISA using the recombinant domain III envelope protein (rJEV-DIII) for rapid, early - accurate diagnosis of JEV is reported in the present study. The gene coding for the envelope protein of JEV was cloned - expressed in pET 30a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rJEV-DIII was optimized that had no reactivity with healthy persons. The comparative evaluation accomplished with the JE-Dengue IgM Combo ELISA (PanBio, Brisbane, Australia) - JEV Chex (XCyton Diagnostic Ltd., Bangalore, India) ELISA kits, by subjecting 120 acute phase of clinical samples revealed more than 95% accordance. The rJEV-DIII ELISA - the PanBio ELISA were found to have a sensitivity - specificity of 98% - 96%, respectively. The compared positivity of the rJEV-DIII ELISA - SYBR green-I based real-time RT-PCR assay in CSF samples revealed higher positivity. The specificity of this assay was confirmed with serum samples obtained from patients with dengue - chikungunya. The recombinant domain III envelope protein based JEV specific ELISA will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.
Palavras-Chave: Japanese encephalitis; Recombinant IgM ELISA; Clinical diagnosis; Indirect ELISA
Imprenta: Journal of Virological Methods, v. 158, n. 1-2, p. 165-170, 2009
Identificador do objeto digital: 10.1016/j.jviromet.2009.02.018
Descritores: Chikungunya virus - Flaviviridae ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Proteins ; Chikungunya virus - Antibodies ; Chikungunya virus - Viral infections ; Chikungunya virus - Molecular methods ; Chikungunya virus - Real Time PCR ; Chikungunya virus - RT-PCR ; Chikungunya Virus - Virus ; Chikungunya virus - Molecular screening ; Chikungunya virus - Dengue ; Chikungunya virus - Immunology ; Chikungunya virus - Public health
Data de publicação: 2009
