RESUMO
A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.
Assuntos
Infecções por Arterivirus/veterinária , Equartevirus/isolamento & purificação , Doenças dos Cavalos/virologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Argentina/epidemiologia , Infecções por Arterivirus/epidemiologia , Infecções por Arterivirus/virologia , Surtos de Doenças/veterinária , Equartevirus/genética , Regulação Viral da Expressão Gênica , Doenças dos Cavalos/epidemiologia , Cavalos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.
Assuntos
Antígenos Virais/imunologia , Infecções por Arterivirus/virologia , Equartevirus/imunologia , Doenças dos Cavalos/virologia , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Argentina , DNA Complementar/genética , DNA Viral/genética , Equartevirus/classificação , Equartevirus/genética , Equartevirus/isolamento & purificação , Variação Genética , Cavalos , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genéticaRESUMO
Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.
La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.
Assuntos
Animais , Antígenos Virais/imunologia , Equartevirus/imunologia , Infecções por Arterivirus/virologia , Doenças dos Cavalos/virologia , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Argentina , Antígenos Virais/genética , Equartevirus/classificação , Equartevirus/genética , Equartevirus/isolamento & purificação , DNA Complementar/genética , DNA Viral/genética , Variação Genética , Cavalos , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genéticaRESUMO
This paper describes the first isolation of equine arteritis virus (EAV) in Argentina. The virus was isolated from the semen of an imported seropositive stallion held in isolation at a breeding farm in Tandil in the Buenos Aires Province. In addition, viral nucleic acid was detected in seminal plasma using the reverse-transcription polymerase chain reaction. The isolated virus was propagated in cell cultures and confirmed as EAV by indirect immunofluorescence and virus neutralisation, using a serum specific for the reference Bucyrus strain of EAV. As far as the authors are aware, this is the first time that EAV has been isolated in South America. The equine industry is very important for Argentina and international movement of horses is very intensive. This finding may have effects on the international trade of horses and semen from Argentina.
Assuntos
Infecções por Arterivirus/veterinária , Equartevirus/isolamento & purificação , Doenças dos Cavalos/virologia , Sêmen/virologia , Animais , Antígenos Virais/análise , Argentina , Infecções por Arterivirus/virologia , Linhagem Celular , Efeito Citopatogênico Viral , DNA Complementar/análise , Equartevirus/genética , Equartevirus/imunologia , Imunofluorescência/veterinária , Cavalos , Masculino , Testes de Neutralização/veterinária , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
A total of 60 samples of dry sausages were analyzed (50 of "salami" and 10 of "chorizo" "candelario" type) obtained at random in markets authorized for their commercialization, for the purpose of evidencing the presence of bacteria of the genus Listeria (Listeria monocytogenes and Listeria spp.). The results obtained in salami were the following: 10 (20%) isolates of Listeria spp., were characterized: 1 (2%) strain as L. monocytogenes type 1, 7 (14%) strains as L. innocua, 2 (4%) strains as L. welshimeri. In chorizo candelario type 6 (60%) isolates of Listeria spp., were characterized: 2 (33%) strains as L. monocytogenes type 1 and 4 (66%) strains as L. innocua. The total percentages of isolations were: 26.6% of Listeria spp., 5% of L. monocytogenes type 1, 18.3% of L. innocua and 3.3% of L. welshimeri. In conclusion, we consider that methodologies of control must be developed and implemented in order to guarantee the inocuity of these products.
Assuntos
Contaminação de Alimentos , Microbiologia de Alimentos , Listeria/isolamento & purificação , Produtos da Carne/microbiologia , Animais , Argentina , Listeria monocytogenes/isolamento & purificação , Especificidade da Espécie , Saúde da População UrbanaRESUMO
A total of 60 samples of dry sausages were analyzed (50 of [quot ]salami[quot ] and 10 of [quot ]chorizo[quot ] [quot ]candelario[quot ] type) obtained at random in markets authorized for their commercialization, for the purpose of evidencing the presence of bacteria of the genus Listeria (Listeria monocytogenes and Listeria spp.). The results obtained in salami were the following: 10 (20
) isolates of Listeria spp., were characterized: 1 (2
) isolates of Listeria spp., were characterized: 2 (33
) strains as L. monocytogenes type 1 and 4 (66
) strains as L. innocua. The total percentages of isolations were: 26.6
of L. innocua and 3.3
of L. welshimeri. In conclusion, we consider that methodologies of control must be developed and implemented in order to guarantee the inocuity of these products.
RESUMO
A total of 60 samples of dry sausages were analyzed (50 of [quot ]salami[quot ] and 10 of [quot ]chorizo[quot ] [quot ]candelario[quot ] type) obtained at random in markets authorized for their commercialization, for the purpose of evidencing the presence of bacteria of the genus Listeria (Listeria monocytogenes and Listeria spp.). The results obtained in salami were the following: 10 (20
) isolates of Listeria spp., were characterized: 1 (2
) strain as L. monocytogenes type 1, 7 (14
) strains as L. innocua, 2 (4
) strains as L. welshimeri. In chorizo candelario type 6 (60
) isolates of Listeria spp., were characterized: 2 (33
) strains as L. monocytogenes type 1 and 4 (66
) strains as L. innocua. The total percentages of isolations were: 26.6
of Listeria spp., 5
of L. monocytogenes type 1, 18.3
of L. innocua and 3.3
of L. welshimeri. In conclusion, we consider that methodologies of control must be developed and implemented in order to guarantee the inocuity of these products.
RESUMO
In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.
Assuntos
DNA Viral/isolamento & purificação , Herpesvirus Suídeo 1/isolamento & purificação , Reação em Cadeia da Polimerase , Pseudorraiva/diagnóstico , Doenças dos Suínos/diagnóstico , Suínos/virologia , Animais , Argentina/epidemiologia , Southern Blotting , Feminino , Pseudorraiva/epidemiologia , Pseudorraiva/patologia , Pseudorraiva/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Fatores de TempoRESUMO
In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.
Assuntos
Animais , Feminino , DNA Viral , Doenças dos Suínos/diagnóstico , Herpesvirus Suídeo 1 , Reação em Cadeia da Polimerase , Pseudorraiva , Suínos/virologia , Argentina , Southern Blotting , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Pseudorraiva , Fatores de TempoRESUMO
In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.(AU)