RESUMO
The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.
Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Sequências Reguladoras de Ácido Nucleico/genética , Vacinas Atenuadas , Proteínas do Envelope Viral/imunologia , Animais , Formação de Anticorpos , Bacteriófago T7/genética , Sequência de Bases , Clonagem Molecular , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/imunologia , Epitopos , Genoma Viral , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Mutação Puntual , Análise de Sobrevida , Células Vero , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , VirulênciaRESUMO
Four monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Proteínas do Envelope Viral/genética , Replicação Viral , Aedes/microbiologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Cricetinae , Vírus da Encefalite Equina Venezuelana/química , Vírus da Encefalite Equina Venezuelana/fisiologia , Insetos Vetores/microbiologia , Cinética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.