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1.
Vet J ; 257: 105448, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32546352

RESUMO

The major control methods for Aujeszky's Disease (AD) involve SHV1 gE gene-deleted vaccines and ELISA for detection of specific gE antibodies in infected animals, distinguishing infected animals from vaccinated animals (DIVA). This work aimed to develop a DIVA ELISA recombinant gE (gErec) for AD diagnosis using recombinant gE fused to thioredoxin protein. The analytical sensitivity and specificity were assessed with World Organisation for Animal Health (OIE) AD serum and sera from specific pathogen free (SPF), vaccinated SPF and AD-vaccinated SPF animals. The OIE serum reacted up to the recommended limit of detection and the other sera presented negative results. The cut-off point, diagnostic sensitivity and diagnostic specificity were determined by receiver operating curve analysis. This cut-off value corresponded to a diagnostic sensitivity of 97.60% and diagnostic specificity of 96.42%. Furthermore, two other cut-off points were chosen to discuss the ELISAgErec as a screening test in AD-endemic and free areas.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/química , Ensaio de Imunoadsorção Enzimática/veterinária , Herpesvirus Suídeo 1/imunologia , Pseudorraiva/diagnóstico , Doenças dos Suínos/diagnóstico , Vacinação/veterinária , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/química , Sensibilidade e Especificidade , Suínos , Tiorredoxinas/química
2.
Arch Virol ; 163(10): 2871-2875, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29982961

RESUMO

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anemia Infecciosa Equina/virologia , Imunodifusão/métodos , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Proteínas Ligantes de Maltose/análise , Proteínas do Core Viral/análise , Animais , Ensaio de Imunoadsorção Enzimática/instrumentação , Anemia Infecciosa Equina/diagnóstico , Anemia Infecciosa Equina/imunologia , Cavalos , Imunodifusão/instrumentação , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia
3.
Arch Virol ; 162(3): 817-822, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27815697

RESUMO

The classical swine fever (CSF) is a highly contagious viral disease of pigs and wild boar. The CSF causes great economic losses for pork production and the occurrence of the disease is notifiable to the OIE. The objective of this work was to identify and characterize CSF virus isolates from Brazil. Seven viral isolates were obtained and the full-length E2 sequences were analyzed. Phylogenetic analysis revealed a different segregation pattern between Brazilian isolates and members of subgenotype 1.1, forming a separate group within genotype 1. Genetic distance analysis suggested the existence of two new subgenotypes, designated subgenotypes 1.5 and 1.6.


Assuntos
Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/isolamento & purificação , Peste Suína Clássica/virologia , Animais , Animais Domésticos/virologia , Brasil , Vírus da Febre Suína Clássica/classificação , Genótipo , Filogenia , Sus scrofa/virologia , Suínos , Proteínas Virais/genética
4.
J Virol Methods ; 207: 226-31, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25066279

RESUMO

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS). The PCV2 capsid (Cap) protein is a leading antigen candidate for vaccine and serological diagnostic testing, due to its immunogenic properties. In this study, the codon-optimized PCV2 Cap gene was cloned into a pPICZαA vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The screening of recombinant yeasts was followed by detection of the recombinant Cap (rCap) protein by Western blot, using sera from pigs naturally infected with PCV2. The rCap secreted protein was used without prior purification as a coating antigen in the ELISA test, with high discrimination between PCV2-positive and negative sera. These results reveal a high confidence in the specific immunoreactivity of the secreted antigen and show the antigenicity of the recombinant protein. The feasibility of the P. pastoris expression system for the production of PCV2 Cap as secreted protein and its apparent bioactivity, suggests there are good prospects for the use of this antigen in the investigation of PCV2 infections and testing for vaccine purposes.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Circovirus/genética , Pichia/genética , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Western Blotting , Clonagem Molecular , DNA Viral/química , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , Análise de Sequência de DNA , Suínos
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