RESUMEN
The detection of neutralizing antibodies against vaccinia virus is a valuable tool for the investigation of previous smallpox vaccination. Compulsory smallpox vaccination ended in Brazil during the early 1970s, although the vaccine was available until the late 1970s. The threat of smallpox as a biological weapon has called the attention of public health authorities to the need for an evaluation of the immune status of the population. Based on our previous experience with a micro plaque reduction neutralization test (PRNT) for the evaluation of yellow fever immunity, a similar test was developed for the detection and quantification of vaccinia neutralizing antibodies. A cross-sectional study to test the repeatability and validity of plaque reduction neutralization test (PRNT) for vaccinia antibodies was performed in 182 subjects divided into two categories: subjects above 31 years old and the other > or = 35 years old. Cases were subjects considered to have been vaccinated with vaccinia virus if they declared vaccination history or evidenced vaccination marks. The assay is carried out in 96-well plates, provides results within 30 h, is easily performed, has good sensitivity (92.7%) and specificity (90.8), excellent repeatability (ICC 0.89 (0.88; 0.92)) and is thus suitable for use in mass screening of a population's antibody levels.
Asunto(s)
Anticuerpos Antivirales/análisis , Virus Vaccinia/inmunología , Vaccinia/inmunología , Vaccinia/virología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antivirales/biosíntesis , Línea Celular , Niño , Chlorocebus aethiops , Estudios Transversales , Humanos , Persona de Mediana Edad , Pruebas de Neutralización/métodos , Pruebas de Neutralización/normas , Reproducibilidad de los Resultados , Vacuna contra Viruela/inmunología , Vaccinia/diagnóstico , Virus Vaccinia/crecimiento & desarrollo , Ensayo de Placa Viral/normasRESUMEN
We report 2 strategies to identify Brazilian vaccinia virus (VACV) isolates related to Cantagalo virus (CTGV) based on the amplification of the hemagglutinin (HA) gene by the polymerase chain reaction (PCR). One PCR protocol was combined with restriction analysis using the endonuclease SnaB I, generating a unique digestion pattern for CTGV amplicons. The restriction profile could identify 41 CTGV-related isolates in 43 clinical specimens and clearly differentiated them from other orthopoxviruses and strains of VACV. Alternatively, we used a 1-step PCR assay with primers that specifically targeted CTGV HA sequence. This protocol produced similar results more rapidly than the 1st strategy, eliminating post-PCR procedures. The results were supported by Western blot analysis of the viral protein profile in infected cells. Both PCR-based methods enabled a fast, sensitive, and cost-effective detection of new isolates of VACV related to CTGV directly from clinical samples without requiring virus isolation.
Asunto(s)
Enfermedades Transmisibles Emergentes/virología , Hemaglutininas Virales/genética , Reacción en Cadena de la Polimerasa/métodos , Virus Vaccinia/clasificación , Virus Vaccinia/aislamiento & purificación , Animales , Brasil , Bovinos , Enfermedades de los Bovinos/virología , Cartilla de ADN , ADN Viral/análisis , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Vaccinia/virología , Virus Vaccinia/genéticaRESUMEN
The vaccinia virus F11L gene product was identified during search for additional factors involved in the control of post-replicative viral gene transcription elongation. F11L is a 1065 base pairs (354 aminoacids) gene expressed early during infection with no attributed function. The F11L gene is conserved in many but not all poxviruses. The essential presence of the F11L gene was tested using two different genetic methods. F11L gene disruption by insertion of a selectable cassette containing the Escherichia coli guanine phosphoribosyl transferase gene driven by the viral early-late 7.5K transcriptional promoter resulted exclusively in recombinant viruses containing both the wild type and disrupted alleles, indicating that the F11L gene was essential. However, an alternative test, using transient dominant selection to insert nonsense mutations into the F11L gene, proved that the F11L gene was non-essential for growth in culture. These experiments suggest that misleading results can be obtained using gene insertional mutagenesis as a test of essential presence of the gene. The experiments also provide genetic data on the probability of co-insertion of linked mutations in vaccinia virus genome using transient dominant selection.
Asunto(s)
Genes Esenciales , Proteínas Inmediatas-Precoces/genética , Selección Genética , Virus Vaccinia/crecimiento & desarrollo , Western Blotting , Medios de Cultivo , Escherichia coli , Técnicas Genéticas , Hipoxantina Fosforribosiltransferasa/genética , Proteínas Inmediatas-Precoces/metabolismo , Mutagénesis Insercional , Recombinación Genética , Virus Vaccinia/genéticaRESUMEN
Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.
Asunto(s)
Ciclofilina A/metabolismo , Virus Vaccinia/fisiología , Vaccinia/metabolismo , Virión/fisiología , Western Blotting , Línea Celular , Cromatografía de Afinidad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Fracciones Subcelulares/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Virión/genética , Virión/metabolismoRESUMEN
In the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage.