RESUMO
Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.
Assuntos
Infecções por Herpesviridae/diagnóstico , Herpesvirus Bovino 2/genética , Herpesvirus Bovino 2/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , DNA Viral/análise , DNA Viral/genética , Infecções por Herpesviridae/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e EspecificidadeRESUMO
Transmission of ovine herpesvirus-2 (OvHV-2) in sheep via natural contact and nasal secretions was examined. OvHV-2-free lambs were produced by separating newborn lambs from their mothers within 5 days of birth and raising them in an isolation facility. Transmission experiments via natural contact were conducted by keeping OvHV-2-free lambs with OvHV-2-infected sheep of different ages. Six of the infected ewes in this experiment were pregnant and gave birth during the experimental period. OvHV-2 was not transmitted from the adult sheep, though viral DNA was consistently detected in their peripheral blood leukocytes (PBL). On the other hand, OvHV-2 was transmitted from recently infected lambs to sheep at 10 or 12 weeks after the onset of contact. In addition, we attempted the experimental transmission of OvHV-2 via nasal secretions, by transferring nasal washings from infected sheep to the nostrils of uninfected sheep. Sheep receiving the nasal washings from infected adult sheep maintained their negative status for 15 months, whereas sheep receiving nasal washings from recently infected lambs acquired OvHV-2 by 8 months. The results of these experiments support that OvHV-2 is more easily transmitted to negative sheep by recently infected lambs than by adult sheep. Further, it is supposed that the nasal cavity is a portal for entry and shedding of infectious OvHV-2 in sheep.
Assuntos
Transmissão de Doença Infecciosa/veterinária , Gammaherpesvirinae/genética , Febre Catarral Maligna/transmissão , Doenças dos Ovinos/transmissão , Doenças dos Ovinos/virologia , Animais , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Reação em Cadeia da Polimerase , Ovinos , Eliminação de Partículas ViraisRESUMO
We have characterized a novel, captured and fully functional viral interleukin (IL)-10 homologue ((OvHV)IL-10) from the gammaherpesvirus ovine herpesvirus 2. Unlike IL-10 homologues from other gammaherpesviruses, the (OvHV)IL-10 peptide sequence was highly divergent from that of the host species. The (OvHV)IL-10 gene is unique amongst virus captured genes in that it has precisely retained the original cellular exon structure, having five exons of similar sizes to the cellular counterparts. However, the sizes of the introns are dramatically reduced. The (OvHV)IL-10 protein was shown to be a non-glycosylated, secreted protein of M(r) 21 000 with a signal peptidase cleavage site between amino acids 26 and 27 of the nascent peptide. Functional assays showed that (OvHV)IL-10, in a similar way to ovine IL-10, stimulated mast cell proliferation and inhibited macrophage inflammatory chemokine production. This is the first example of a captured herpesvirus gene retaining the full cellular gene structure.
Assuntos
Éxons/genética , Gammaherpesvirinae/genética , Interleucina-10 , Proteínas/química , Proteínas/genética , Proteínas Virais , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Interleucina-10/química , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-10/metabolismo , Mastócitos/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Ovinos/virologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Ovine herpesvirus 2 (OvHV-2) is endemic in sheep populations worldwide and causes malignant catarrhal fever (MCF), a lymphoproliferative disease, in cattle, bison and deer. OvHV-2 has been placed in the gammaherpesvirus subfamily and is related closely to Alcelaphine herpesvirus 1 (AlHV-1). Here, the cloning, sequencing and analysis of the complete OvHV-2 genome derived from a lymphoblastoid cell line from an affected cow (BJ1035) are reported. The unique portion of the genome consists of 130,930 bp, with a mean G+C content of 52 mol%. The unique DNA is flanked by multiple copies of terminal repeat elements 4205 bp in length, with a mean G+C content of 72 mol%. Analysis revealed 73 open reading frames (ORFs), the majority (62) of which showed homology to other gammaherpesvirus genes. A further subset of nine ORFs is shared with only the related AlHV-1. Three ORFs are entirely unique to OvHV-2, including a spliced homologue of cellular interleukin-10 that retains the exon structure of the cellular gene. The sequence of OvHV-2 is a critical first step in the study of the pathogenesis and treatment of MCF.