RESUMO
The Equid alphaherpesvirus type 1 (EHV-1) infection can have devastating economic consequences in the horse industry due to large-scale outbreaks of abortions, perinatal foal mortality, and myeloencephalopathy. The present study analyzed the genome of two isolates obtained from aborted fetuses in Argentina, E/745/99 and E/1297/07. The E745/99 genome shares 98.2% sequence identity with Ab4, a reference EHV-1 strain. The E/1297/07 genome shares 99.8% identity with NY03, a recombinant strain containing part of ORF64 and part of the intergenic region from Equid alphaherpesvirus-4 (EHV-4). The E/1297/07 genome has the same breakpoints as other United States and Japanese recombinants, including NY03. The recombinant regions have varying numbers of tandem repeat sequences and different minor parental sequences (EHV-4), suggesting distinct origins of the recombinant events. These are the first complete genomes of EHV-1 from Argentina and South America available in the Databases.
Assuntos
Genoma Viral , Infecções por Herpesviridae , Herpesvirus Equídeo 1 , Filogenia , Argentina , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 1/classificação , Animais , Genoma Viral/genética , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Cavalos/virologia , Recombinação Genética , Doenças dos Cavalos/virologia , Fases de Leitura Aberta/genética , Sequenciamento Completo do Genoma , DNA Viral/genéticaRESUMO
Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.
Assuntos
Variação Genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Animais , Brasil , Análise por Conglomerados , Sequência Conservada , DNA Viral/química , DNA Viral/genética , Genótipo , Infecções por Herpesviridae/virologia , Cavalos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.(AU)
Assuntos
Animais , Variação Genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Brasil , Análise por Conglomerados , Sequência Conservada , DNA Viral/química , DNA Viral/genética , Genótipo , Infecções por Herpesviridae/virologia , Cavalos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.
Assuntos
Animais , Variação Genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Brasil , Análise por Conglomerados , Sequência Conservada , DNA Viral/química , DNA Viral/genética , Genótipo , Cavalos , Infecções por Herpesviridae/virologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Equid herpesvirus 1 (EHV-1) infection has a signifcant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identifcation of specifc EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-fve EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplifed and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.
La infección por Herpesvirus equino 1 (EHV-1) tiene un signifcativo impacto económico en la producción equina mundial al causar abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos. La identifcación de genes específcos relacionados con la virulencia y patogenicidad de este virus ha sido el propósito de varios grupos de investigación. En este trabajo se analizaron diferentes regiones genómicas de cepas argentinas de EHV-1 para determinar la posible relación entre la estructura genómica y la virulencia o los signos clínicos producidos. Veinticinco cepas aisladas de diferentes casos clínicos observados entre los años 1979 y 2007 y dos cepas de referencia fueron amplifcadas y secuenciadas. El alineamiento de las secuencias se realizó con el programa Clustal X versión 1.92; el programa Bio-Edit versión 7.05 permitió deducir la secuencia de aminoácidos. Solo se observaron cambios menores, no se encontraron variaciones que pudieran estar relacionadas con la diferencia de virulencia observada previamente en el modelo ratón. No se hallaron variantes genómicas. Las regiones genómicas analizadas no permitieron diferenciar cepas abortigénicas de aquellas aisladas de muertes neonatales.
Assuntos
Animais , Camundongos , Genoma Viral , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Sequência de Aminoácidos , Aborto Animal/epidemiologia , Aborto Animal/virologia , Argentina/epidemiologia , Sequência de Bases , DNA Viral/genética , Genes Virais , Cavalos , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/epidemiologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Virulência/genéticaRESUMO
Equid herpesvirus 1 (EHV-1) infection has a significant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identification of specific EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-five EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplified and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.
Assuntos
Genoma Viral , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Aborto Animal/epidemiologia , Aborto Animal/virologia , Sequência de Aminoácidos , Animais , Argentina/epidemiologia , Sequência de Bases , DNA Viral/genética , Genes Virais , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/epidemiologia , Cavalos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Virulência/genéticaRESUMO
Equid Herpesvirus 1 (EHV-1) has long been causally implicated in the occurrence of abortion, neonatal death, respiratory disease, and neurological disorders in horses. This study analyzed for the first time the characteristics of the genomic section of Argentinian EHV-1 strains and reconstructed the phylogeny in order to establish their origin. The phylogenetic dataset included 22 Argentinian strains and four additional reference strains isolated in other countries. The intergenic region between ORF 62 and ORF 63 was amplified by PCR and sequenced. The phylogenetic analysis carried out by parsimony algorithms showed that six of the Argentinian strains had the same origin as British and Japanese strains. The mapping of symptoms caused by EHV-1 suggested that neonatal disease developed through convergent evolution, which would constitute an adaptation mechanism of the virus. This study constitutes the first analysis carried out in South-American strains that establishes the phylogenetic relationship between Argentinian strains and rebuilds the evolutionary history of symptoms. This study focuses on a very important aspect of evolution of Herpesviridae infecting perissodactyls and attempts to shed light on the evolution of symptoms, an issue of high clinical interest.
Assuntos
Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Filogenia , Animais , Argentina , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Herpesvirus Equídeo 1/isolamento & purificação , Cavalos , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
To determine the genomic variation of equine herpesviruses (EHVs) isolated in Argentina between 1979 and the first half of 2004, DNA sequences from all 69 strains isolated were analysed. Sixty strains were recovered from aborted fetuses, one from leucocyte-rich plasma from a horse with respiratory signs and eight from cases of neonatal disease. The DNA was extracted from rabbit kidney epithelial (RK13) cells infected with each strain and digested with three restriction endonucleases (BamHI, Bg/II and KpnI). Two strains could be differentiated using BamHI restriction and were assigned to the EHV-1 1B prototype group. Only one of these two strains was typed EHV-1 1B with Bg/II. DNA digestion with KpnI was ineffective. The results obtained in this study demonstrate that the EHV-1 1B genome has been present in Argentina since at least 1996. The finding of two strains with this electropherotype suggests that there is genomic heterogeneity among Argentinian isolates.
Assuntos
Aborto Animal/virologia , DNA Viral/análise , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Complicações Infecciosas na Gravidez/veterinária , Animais , Argentina/epidemiologia , Sequência de Bases , Enzimas de Restrição do DNA , Feminino , Variação Genética , Genoma , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/epidemiologia , Cavalos , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/virologia , Mapeamento por Restrição/métodos , Mapeamento por Restrição/veterináriaRESUMO
In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.
Assuntos
DNA Viral/isolamento & purificação , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Varicellovirus/isolamento & purificação , Animais , Primers do DNA , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Cavalos , Sensibilidade e Especificidade , Varicellovirus/classificação , Varicellovirus/genéticaRESUMO
Lytic plaques produced by three different strains of EHV 1 were morphologically compared. The local isolated SP1 strain was compared with the tissue culture adapted Kentucky B (Ky Bcc) strain, and the through hamster passages modified Kentucky B (Ky Ba) strain, both reference strains. Ky Bcc produced small, irregular in size and shape, lytic plaques of around 1 mm. In the case of Ky Ba, the plaques were uniform in size and shape, with a diameter of 2 to 3 mm. SP1 strain originated lytic plaques similar to those produced by Ky Bcc. It is concluded that SP1 is a wild abortigenic strain, similar to Ky Bcc regarding its lytic properties.
Assuntos
Herpesviridae/crescimento & desenvolvimento , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Ensaio de Placa Viral , Animais , Herpesvirus Equídeo 1/classificaçãoRESUMO
Se comparó la morfología de las placas de lisis producidas pro tres cepas de virus Herpes equino tipo 1: SP1, aislada en nuestro laboratorio, y dos cepas de referencia, Kentucky B adaptada a cultivos celulares (Ky Bcc) y Kentucky B atenuada por pasajes en hamster (Ky Ba). Se observó que la cepa Ky Bcc formó placas pequeñas de tamaño variable, de aproximadamente 1 mm de diámetro y de bordes irregulares. La cepa Ky Ba formó placas de tamaño uniforme de aproximadamente 2-3 mm y bordes lisos. La morfología de las placas formadas por la cepa SP1 fue coincidente con Ky Bcc. Se concluye que la cepa SP1 es una cepa salvaje de origen abortigénico y semejante a la cepa Ky Bcc en su propiedad de formar placas líticas
Assuntos
Animais , Herpesviridae/crescimento & desenvolvimento , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Ensaio de Placa Viral , Herpesvirus Equídeo 1/classificaçãoRESUMO
Se comparó la morfología de las placas de lisis producidas pro tres cepas de virus Herpes equino tipo 1: SP1, aislada en nuestro laboratorio, y dos cepas de referencia, Kentucky B adaptada a cultivos celulares (Ky Bcc) y Kentucky B atenuada por pasajes en hamster (Ky Ba). Se observó que la cepa Ky Bcc formó placas pequeñas de tamaño variable, de aproximadamente 1 mm de diámetro y de bordes irregulares. La cepa Ky Ba formó placas de tamaño uniforme de aproximadamente 2-3 mm y bordes lisos. La morfología de las placas formadas por la cepa SP1 fue coincidente con Ky Bcc. Se concluye que la cepa SP1 es una cepa salvaje de origen abortigénico y semejante a la cepa Ky Bcc en su propiedad de formar placas líticas (AU)