RESUMO
According to the data of the Federal Service on Customers' Rights Protection and Human Well-being Surveillance, the enzyme-linked immunosorbent assay (ELISA) detects the tick-borne encephalitis virus (TBEV) in ticks more often than the polymerase chain reaction (PCR). The goal of this work was to compare TBEV detection efficiency in the ixodid ticks of different species with the commercial kits based on ELISA and real-time PCR. Ticks of five species were parenterally infected with 2-6 IgPFU of the European or Siberian TBEV subtypes. We formed randomized and encoded series of infected and intact ticks of different species, and in "blind" experiment analyzed the ticks on the TBEV presence with the kits based on ELISA and real-time PCR. ELISA and real-time PCR effectiveness of the TBEV detection in ticks was not affected by gender, species of ticks or presence of blood meal. The kits based on ELISA were less sensitive than those based on real-time PCR. ELISA effectiveness depended on the TBEV subtype. The presence of the false positive reactions and sensitivity of ELISA were affected by the protocols of reaction. The problem of the different TBEV prevalence in the field-collected ticks obtained with various methods remains to be studied.
Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/normas , Ixodidae/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Animais , Antígenos Virais/genética , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Reações Falso-Positivas , Feminino , Masculino , Camundongos , Coelhos , Sensibilidade e Especificidade , Carga ViralRESUMO
Several distinct methods currently used for the Crimean-Congo Hemorrhage Fever diagnosis (CCHF) were suggested in this work. We demonstrated that the ELISA-based diagnostic kits, which are based on CCHFV recombinant antigens produced in E. coli cells, still possessed a few substantial shortcomings, which are yet to be addressed. In this work we presented the development of the unique CCHFV detection system fully based on reverse transcription--nested two-step polymerase chain reaction (RT-PCR). Our RT-PCR-based diagnostic kit for the CCHFV detection is now commercially available. We also developed a simple screening method for the samples, potentially containing CCHFV, which is based on restriction fragment length polymorphism (RFLP) amplicons analysis and allows for preliminary genotyping of the CCHFV isolates.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Febre Hemorrágica da Crimeia/diagnóstico , Proteínas do Nucleocapsídeo/isolamento & purificação , Escherichia coli , Genótipo , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/patologia , Humanos , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Polimorfismo de Fragmento de RestriçãoRESUMO
The significant antigenic crossovers between West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) make the immunological diagnosis of these diseases difficult. The avidicity index of virus-specific class G immunoglobulins (IgG) was used as a criterion for the differentiation of an immune response to WNV or TBEV in patients and convalescents. The panels of the sera sampled from patients with tick-borne encephalitis and convalescents in the Novosibirsk and Tomsk Regions and in the Primorye Territory and from those with West Nile fever and convalescents in the Volgograd Region. The determination of the avidicity index could establish that in the convalescents' sera, the avidicity index of virus-specific IgG was much higher than that in the patients' sera in the acute phase of infection. In relation to heteroantigen, the avidicity index and the positivity coefficient were substantially less than those in the reaction with homoantigen. The findings have indicated that the determination of the value of the avidicity index of virus-specific IgG and the positivity coefficient makes it possible to differentiate West Nile fever and tick-borne encephalitis with confidence on the basis of solid-phase enzyme immunoassay in determining virus-specific IgG in the sera of patients and convalescents in different regions of Russia.
Assuntos
Anticorpos Antivirais/sangue , Afinidade de Anticorpos , Encefalite Transmitida por Carrapatos/sangue , Encefalite Transmitida por Carrapatos/diagnóstico , Imunoglobulina G/sangue , Febre do Nilo Ocidental/sangue , Febre do Nilo Ocidental/diagnóstico , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Reações Cruzadas , Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Estudos Retrospectivos , Febre do Nilo Ocidental/imunologia , Vírus do Nilo OcidentalAssuntos
Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/sangue , DNA Bacteriano/genética , Eletroforese/métodos , Reação em Cadeia da Polimerase/métodos , Doadores de Sangue , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodosRESUMO
The primary and secondary structures of the pp65 phosphoprotein of human cytomegalovirus coded by the UL83 gene were studied by the methods of computer-aided analysis. An immunodominant protein fragment with 3 antigenic determinant was detected. The UL83 fragment coding the selected region was amplified and cloned in bacterial expressing vector. The recombinant protein was obtained and purified. On the basis of ELISA findings it was acknowledged as possible to use the pp65 recombinant protein jointly with pp150 and p52 in the diagnosis of antibodies specific to human cytomegalovirus.
Assuntos
Citomegalovirus/metabolismo , Fosfoproteínas/genética , Proteínas da Matriz Viral/genética , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismoRESUMO
The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.
Assuntos
Epitopos/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Epitopos/genética , Epitopos/isolamento & purificação , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.
Assuntos
Antígenos Virais/isolamento & purificação , Citomegalovirus/imunologia , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Virais/isolamento & purificação , Sequência de Aminoácidos , Antígenos Virais/química , Antígenos Virais/genética , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , DNA Viral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Reactivities toward the alkylating reagent 4-(N-2-chloroethyl-N-methylamino)benzylamine of guanosine residues in the tRNAPhe free and bound to the phenylalanyl-tRNA synthetase were determined. This highly efficient reagent modifies mainly guanosine in single-stranded as well as in helical regions of tRNAs. The reaction is sensitive to the elements of the macrostructure of tRNAs. It is found the phenylalanyl-tRNA synthetase protects the D-stem of the tRNAPhe (guanosines G10, G22, G24) from alkylation.