RESUMO
Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.
Assuntos
Vírus da Febre Suína Clássica/isolamento & purificação , Peste Suína Clássica/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SuínosRESUMO
In 2002 and 2003, two successive outbreaks of classical swine fever were declared in wild boar in northern France. The first was in Moselle, near the town of Thionville and the border with Luxembourg, and the second was in the northern Vosges area, near the German border. The outbreaks were investigated by serological and virological diagnosis of dead or shot animals. Hunting restrictions were applied to limit the spread of the outbreaks. The virus was detected eight times between April and July 2002 in the Thionville area, an area well delimited by natural or artificial barriers such as rivers or highways. Cooperation between the authorities concerned was good, and hunting restrictions were applied for one year. No virus was detected after July 2002 and the Thionville outbreak was officially considered over in March 2005. In the northern Vosges the situation was different, with no barriers to animal movements, continuous forest, difficulties in establishing hunting restrictions in this huge area, and the circulation of the virus in Germany close to the frontier. Virus of a different strain from that isolated in the Thionville outbreak was still being isolated in the northern Vosges in 2004, and owing to the failure of the hunting restrictions, the French health authorities decided to vaccinate wild boar.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/epidemiologia , Peste Suína Clássica/prevenção & controle , Sus scrofa , Vacinação/veterinária , Animais , Animais Selvagens , Anticorpos Antivirais/sangue , Vírus da Febre Suína Clássica/isolamento & purificação , Surtos de Doenças/veterinária , Feminino , França/epidemiologia , Alemanha/epidemiologia , MasculinoRESUMO
Two new real-time RT-PCR kits developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF) obtained a manufacturing agreement in France during the past year. For that purpose, the Classical Swine Fever (CSF) National Reference Laboratory (NRL) planned a schedule of conditions to be fulfilled by commercial real-time RT-PCR assays. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were: sensitivity, specificity, especially "pestivirus specificity", reproducibility and easy handling, using 187 different samples distributed in four different panels. These samples were either CSFV inactivated strains or organs collected from CSF experimental SPF infected pigs, or naturally infected wild boars. All these samples were previously tested for genome detection using an RT-nested PCR assay and for virus isolation on cell culture. The LSI TaqVet kit was used for the CSF surveillance of wild boars in an area known to be infected, during the winter of 2004-2005. This field evaluation was carried out on 4710 spleen samples. In summary, the new CSF real-time RT-PCR assays have a higher predictive value than the current diagnostic standard, Virus Isolation.
Assuntos
Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/isolamento & purificação , Peste Suína Clássica/diagnóstico , Peste Suína Clássica/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Suínos/virologia , Animais , Peste Suína Clássica/epidemiologia , Surtos de Doenças/veterinária , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Organismos Livres de Patógenos EspecíficosRESUMO
This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes - H1N1, H3N2 and H1N2 - at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as 'avian-like' H1N1, 36 as human-like H3N2 and 47 as human-like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human-like HA and three H1N2 viruses an avian-like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross-hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human-like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.
Assuntos
Vírus da Influenza A , Infecções por Orthomyxoviridae/veterinária , Vigilância de Evento Sentinela/veterinária , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Animais , Europa (Continente) , Variação Genética , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Suínos , Doenças dos Suínos/transmissão , ZoonosesRESUMO
A longitudinal study was carried out in five French farrow-to-finish herds differently affected by respiratory diseases to describe the carrying and infection patterns of batches of sows to various respiratory pathogens during gestation and lactation. An entire batch of sows was followed during two successive reproduction cycles. Nasal, tonsillar and oro-pharyngeal swabs and blood samples were taken from each sow 9 and 4 weeks before farrowing and 1 and 4 weeks after farrowing. Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis were detected from swab samples using PCR assays. Blood samples were tested for antibodies against M. hyopneumoniae, A. pleuropneumoniae serotypes 1-9-11 and 2, Porcine Circovirus type-2 (PCV-2) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) by ELISA tests. Antibodies against H(1)N(1), H(1)N(2) and H(3)N(2) Swine Influenza Viruses (SIV) of European lineages were tested by hemagglutination inhibition assay. The results indicated that S. suis is widespread among sows (67.1% of PCR-positive sows). A. pleuropneumoniae, P. multocida, and H. parasuis were detected by PCR in 30.9%, 24.6% and 23.4% of the sows, respectively. Antibodies against M. hyopneumoniae were recovered from more than 55% of the sows in all herds whereas the micro-organism was detected in 2.4% of the sows. Although PCV-2 and SIV infections were highly prevalent, the PRRSV infection patterns ranged from no infection in farms mildly affected by respiratory diseases to active circulation in more severely affected herds. The sow population thus constitutes a reservoir for a continuous circulation of respiratory pathogens and needs to be properly considered in control strategies.
Assuntos
Infecções Bacterianas/veterinária , Infecções Respiratórias/veterinária , Doenças dos Suínos/epidemiologia , Viroses/veterinária , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/transmissão , Cruzamento , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Testes de Inibição da Hemaglutinação/veterinária , Estudos Longitudinais , Gravidez , Prevalência , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/transmissão , Suínos , Doenças dos Suínos/transmissão , Fatores de Tempo , Viroses/epidemiologia , Viroses/transmissãoRESUMO
Swine influenza is a highly contagious respiratory viral infection of pigs that has become enzootic in areas densely populated with pigs. Like other influenza A viruses, swine influenza virus (SIV) is genetically unstable and able to accumulate antigenic drifts and/or antigenic shifts. The pig is susceptible to both avian and human influenza viruses and can serve as an intermediate host in influenza virus ecology. Zoonotic agents may emerge in pigs following the modification of an established swine strain, adaptation of a strain of avian origin to the mammalian host, or reassortment between human and avian influenza viruses. Three different subtypes, H1N1, H3N2 and H1N2, are at present circulating in Europe. They differ from those found in North America and Asia and various lineages can be distinguished within each subtype. To date, European SIVs have not produced a global outbreak of influenza in humans but sporadic cases of SIV infection have been reported. This review presents a historical record of the genetic and antigenic evolution of SIVs in Europe. Our present understanding of the transmission of European SIVs from pigs to other animal species and to humans, together with the factors that limit inter-species transmission, is described.
Assuntos
Variação Genética , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Europa (Continente) , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/epidemiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , ZoonosesRESUMO
Valproic acid (VPA), a simple branched-chain fatty acid having anticonvulsant activity and used in the treatment of many forms of epilepsy, markedly stimulated human cytomegalovirus (HCMV) replication in human fibroblasts (MRC-5 cells). The maximum level of stimulation was reached when cells were treated for 24 h before infection. The enhancement of virus replication correlated with an increase in the number of immediate early (IE) and early (E) antigen-positive cells. VPA also induced expression of IE antigens after transfection of fibroblasts with a plasmid containing the entire IE1-2 region. Moreover, VPA stimulated the HCMV IE1-2 promoter/enhancer-mediated expression of beta-galactosidase in a stably transfected Jurkat T cell line. Recently, VPA was shown to inhibit glutathione reductase in human red blood cells, but an action through the glutathione metabolic pathway can be eliminated in this case, since VPA decreased the intracellular level of glutathione in Jurkat T cells but not in MRC-5 cells. The ability of VPA to stimulate HCMV replication provides an attractive model for studying the molecular mechanism of the regulation of HCMV IE1-2 gene expression.
Assuntos
Anticonvulsivantes/farmacologia , Citomegalovirus/fisiologia , Ácido Valproico/farmacologia , Replicação Viral/efeitos dos fármacos , Antígenos Virais/biossíntese , Antimetabólitos/farmacologia , Butionina Sulfoximina , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos , Expressão Gênica , Glutationa/metabolismo , Humanos , Proteínas Imediatamente Precoces/biossíntese , Cinética , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , Linfócitos T , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The linear single-stranded DNA genome of the minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate. Amplification of this RF is initiated by the folding-back of palindromic sequences serving as primers for strand-displacement synthesis and formation of dimeric RF DNA. Using an in vitro replication assay and a cloned MVM DNA template, we observed hairpin-primed DNA replication at both MVM DNA termini, with a bias toward right-end initiation. Initiation of DNA replication is favored by nuclear components of A9 cell extract and highly stimulated by the MVM nonstructural protein NS1. Hairpin-primed DNA replication is also observed in the presence of NS1 and the Klenow fragment of the Escherichia coli DNA polymerase I. Addition of ATPgammaS (adenosine 5'-O-(thiotriphosphate)) blocks the initiation of DNA replication but not the extension of pre-existing hairpin primers formed in the presence of NS1 only. The NS1-mediated unwinding of the right-end palindrome may account for the recently reported capacity of NS1 for driving dimer RF synthesis in vitro.
Assuntos
Replicação do DNA , DNA Viral/química , Conformação de Ácido Nucleico , Telômero , Proteínas não Estruturais Virais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Camundongos , Vírus Miúdo do Camundongo/metabolismo , Dados de Sequência Molecular , Origem de ReplicaçãoRESUMO
Minute virus of mice (MVM) shows an oncotropic behavior reflected by its ability to amplify its genome more efficiently in a number of transformed versus normal cells. In vivo and in vitro studies revealed that the major effect of cell transformation on MVM DNA replication occurs at the level of double-stranded replicative-form amplification. In particular, resolution of MVM DNA concatemers into monomers was found to be highly sensitive to neoplastic transformation.
Assuntos
Replicação do DNA , DNA Viral/química , Vírus Miúdo do Camundongo/genética , Replicação Viral , Transformação Celular Neoplásica , Amplificação de Genes , HumanosRESUMO
As a result of differential splicing, one subunit of the nuclear factor Y (NF-Y) consists of two major isoforms designated short (NF-YaS) and long (NF-YaL). In proliferating normal human fibroblasts, NF-YaL is by far the more expressed isoform. Surprisingly, NF-YaS was found by immunoblotting to be as prominent as NF-YaL in simian virus 40 (SV40)-transformed cell derivatives. As a consequence, two NF-Y/DNA complexes, one containing the long and the other the short isoform, were formed with extracts from transformed cells and a target promoter element in electrophoretic mobility-shift assays. Only the complex containing NF-YaL was detected with extracts from normal fibroblasts. Furthermore, the NF-Y recognition motif contributed to promoter activation in SV40-transformed cells but not in normal, cells. Our finding links transcription stimulation in transformed cells to quantitative changes in the expression of an NF-Ya subunit.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Viral/genética , Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Isoformas de Proteínas/biossíntese , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular Transformada , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Humanos , Proteína Oncogênica p21(ras)/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Splicing de RNA , Ratos , Ratos Endogâmicos F344 , Vírus 40 dos Símios , TemperaturaRESUMO
Since modulation of the glutathione (GSH) level has been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression, we have undertaken an analysis of the effect of sodium valproate (VPA) on HIV-1 replication. VPA, which is an anti-epileptic drug in widespread use in clinical medicine, has been shown to depress the activity of GSH reductase, an enzyme required for maintaining high cellular levels of reduced GSH. The effect of this drug on HIV-1 replication has been studied in primary infected cells, i.e. peripheral blood mononuclear cells (PBMC) and monocyte/macrophages, in the CEM-SS cell line, and in chronically infected stimulated and non-stimulated U1 cells. We have shown that VPA markedly enhanced viral replication in all infected cells tested. Virus production was induced in U1 cells by VPA treatment and the stimulatory effects of tumour necrosis factor-alpha, interleukin-6 and granulocyte/macrophage colony-stimulating factor were augmented. The LTR-driven gene expression in Jurkat T cells was increased. However, the elevated viral production did not correlate with the effect of VPA on the intracellular GSH level. Thus, VPA stimulated in vitro HIV-1 replication in acutely and chronically infected cells and enhanced LTR-driven gene expression. These effects were observed for concentrations that are reached in the plasma of VPA-treated patients. Therefore, although the clinical significance of these data remains to be demonstrated, these results should be considered in the choice of an anticonvulsant drug in HIV-infected individuals.