RESUMO
The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírus da Febre Amarela , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Sequência Conservada , Células HeLa , Humanos , Imunoprecipitação , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Viral , Ribonucleoproteína Nuclear Pequena U1/química , Técnicas do Sistema de Duplo-Híbrido , Células Vero , Proteínas não Estruturais Virais/química , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/metabolismoRESUMO
Concanavalin A-Sandwich ELISA (Con A-S-ELISA) was developed for the detection of infectious bronchitis virus (IBV) or chicken specific anti-viral antibodies. The antigen detection limit for the Con A-S-ELISA was 10(5,1) EID(50)/mL. Three homologous and four heterologous IBV strains were similarly detected. This assay was highly effective in detecting the virus after infected tissue homogenates were passed once in embryonated chicken eggs, showing a good agreement with virus isolation technique. The Con A-S-ELISA was also used to measure anti-IBV chicken antibodies and showed a high coefficient of correlation (r = 0.85) and an agreement of k = 0.80 with the commercially available Indirect-ELISA. The relative sensitivity and specificity between these two tests were, respectively, 92.86% and 95.65% with an accuracy of 93.39%. Thus, the Con A-S-ELISA proved to be able to detect alternatively homologous and heterologous IBV strains or specific chicken anti- IBV antibodies, using the Con A as capture reagent of this assay.
Assuntos
Anticorpos Antivirais/análise , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Bronquite Infecciosa/imunologia , Vírus da Bronquite Infecciosa/isolamento & purificação , Animais , Especificidade de Anticorpos , Galinhas , Concanavalina A , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A reverse-transcriptase PCR (RT-PCR) and a multiplex nested PCR were developed for the rapid detection and identification of 14 Brazilian alphaviruses. Using Alphavirus genus-specific primers in a RT-PCR, we obtained amplified products of 434 bp. Species-specific primers were selected and simultaneously tested in a multiplex nested PCR. The nested PCR increased the test sensitivity 1000-fold and was capable of identifying Brazilian Alphavirus showing the expected bands with diagnostic sizes for Venezuelan (400 bp), Eastern (124 bp), and Western (208 bp) equine encephalitis, Aura (86 bp), and Mayaro (270 bp) viruses. This strategy for diagnosis is fast, sensitive, specific and it can be used as a reliable alternative for routine Brazilian Alphavirus diagnosis.
Assuntos
Infecções por Alphavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Brasil , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Ágar/normas , Humanos , Reação em Cadeia da Polimerase/normas , RNA Viral/isolamento & purificação , RNA Viral/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e EspecificidadeRESUMO
RNA interference (RNAi) is a process that is induced by double stranded RNA and involves the degradation of specific sequences of mRNA in the cytoplasm of the eukaryotic cells. It has been used as an antiviral tool against many viruses, including flaviviruses. The genus Flavivirus contains the most important arboviruses in the world, i.e., dengue (DENV) and yellow fever (YFV). In our study, we investigated the in vitro and in vivo effect of RNAi against YFV. Using stable cell lines that expressed RNAi against YFV, the cell lines were able to inhibit as much as 97% of the viral replication. Two constructions (one against NS1 and the other against E region of YFV genome) were able to protect the adult Balb/c mice against YFV challenge. The histopathologic analysis demonstrated an important protection of the central nervous system by RNAi after 10 days of viral challenge. Our data suggests that RNAi is a potential viable therapeutic weapon against yellow fever.