RESUMO

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Assuntos

Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Vírus da Encefalite de St. Louis/imunologia , Encefalite de St. Louis/diagnóstico , Proteínas não Estruturais Virais/imunologia , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/imunologia , Animais , Especificidade de Anticorpos , Antígenos Virais/biossíntese , Antígenos Virais/genética , Argentina , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Reações Cruzadas , Diagnóstico Diferencial , Vírus da Encefalite de St. Louis/química , Vírus da Encefalite de St. Louis/genética , Encefalite de St. Louis/imunologia , Encefalite de St. Louis/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Corpos de Inclusão/química , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Solubilidade , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/química , Vírus do Nilo Ocidental/genética