RESUMO
Zika virus (ZIKV) is an arbovirus that causes birth defects, persistent male infection, and sexual transmission in humans. The purpose of this study was to continue the development of an ovine ZIKV infection model; thus, two experiments were undertaken. In the first experiment, we built on previous pregnant sheep experiments by developing a mid-gestation model of ZIKV infection. Four pregnant sheep were challenged with ZIKV at 57-64 days gestation; two animals served as controls. After 13-15 days (corresponding with 70-79 days of gestation), one control and two infected animals were euthanized; the remaining animals were euthanized at 20-22 days post-infection (corresponding with 77-86 days of gestation). In the second experiment, six sexually mature, intact, male sheep were challenged with ZIKV and two animals served as controls. Infected animals were serially euthanized on days 2-6 and day 9 post-infection with the goal of isolating ZIKV from the male reproductive tract. In the mid-gestation study, virus was detected in maternal placenta and spleen, and in fetal organs, including the brains, spleens/liver, and umbilicus of infected fetuses. Fetuses from infected animals had visibly misshapen heads and morphometrics revealed significantly smaller head sizes in infected fetuses when compared to controls. Placental pathology was evident in infected dams. In the male experiment, ZIKV was detected in the spleen, liver, testes/epididymides, and accessory sex glands of infected animals. Results from both experiments indicate that mid-gestation ewes can be infected with ZIKV with subsequent disruption of fetal development and that intact male sheep are susceptible to ZIKV infection and viral dissemination and replication occurs in highly vascular tissues (including those of the male reproductive tract).
Assuntos
Idade Gestacional , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Autopsia , Biomarcadores , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Histocitoquímica , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Ovinos , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/transmissãoRESUMO
Congenital infections of domestic animals with viruses in several families, including Bunyaviridae, Flaviridae, Parvoviridae, and Reoviridae, are the cause of naturally occurring teratogenic central nervous system and/or musculoskeletal defects (arthrogryposis) in domestic animals. Congenital infections of ruminant livestock with bluetongue virus (BTV) and some related members of the genus Orbivirus (family Reoviridae) have clearly shown the critical role of gestational age at infection in determining outcome. Specifically, fetuses infected prior to mid-gestation that survive congenital BTV infection are born with cavitating central nervous system defects that range from severe hydranencephaly to cerebral cysts (porencephaly). Generally, the younger the fetus (in terms of gestational age) at infection, the more severe the teratogenic lesion at birth. Age-dependent virus infection and destruction of neuronal and/or glial cell precursors that populate the developing central nervous system are responsible for these naturally occurring virus-induced congenital defects of animals, thus lesions are most severe when progenitor cells are infected prior to their normal migration during embryogenesis. Whereas congenital infection is characteristic of certain BTV strains, notably live-attenuated (modified-live) vaccine viruses that have been passaged in embryonating eggs, transplacental transmission is not characteristic of many field strains of the virus and much remains to be determined regarding the genetic determinants of transplacental transmission of individual virus strains.
Assuntos
Vírus Bluetongue/genética , Bluetongue/virologia , Orbivirus/patogenicidade , Ruminantes/virologia , Viroses/complicações , Fatores Etários , Animais , Bluetongue/complicações , Bluetongue/transmissão , Vírus Bluetongue/isolamento & purificação , Vírus Bluetongue/patogenicidade , Anormalidades Congênitas/virologia , Feminino , Idade Gestacional , Transmissão Vertical de Doenças Infecciosas , Gado/virologia , Orbivirus/genética , Gravidez , Infecções por Reoviridae/complicações , Infecções por Reoviridae/virologia , Ovinos , Teratogênicos , Viroses/virologiaRESUMO
Equine herpesvirus-2 (EHV-2) infection has been implicated as a cause of a variety of clinical disorders in young horses, including upper respiratory tract disease, generalized malaise, fever, pharyngeal lymphoid hyperplasia, and lymphadenopathy. Considerable sequence heterogeneity has been demonstrated previously among EHV-2 strains, and individual horses can be concurrently infected with more than one virus strain. In this study, the temporal variation of the viral load and genomic diversity of the glycoprotein B (gB) gene of EHV-2 in the nasal secretions of a cohort of foals was characterized during the first 5 months of life. The viral load in nasal secretions of foals peaked when the foals were approximately 3 months old, and there was notable genetic heterogeneity of the gB gene, both among foals and within individuals. Furthermore, there was evidence of positive selection of EHV-2 variants with unique amino acid sequences at specific sites of gB.
Assuntos
Heterogeneidade Genética , Variação Genética , Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/virologia , Rhadinovirus/genética , Sequência de Aminoácidos , Animais , Infecções por Herpesviridae/virologia , Cavalos , Dados de Sequência Molecular , Filogenia , Rhadinovirus/classificação , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Carga Viral/veterináriaAssuntos
Equidae/virologia , Gammaherpesvirinae/patogenicidade , Infecções por Herpesviridae/veterinária , Animais , Anticorpos Antivirais/sangue , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologiaRESUMO
Bluetongue virus (BTV) is the cause of bluetongue (BT), an emerging, arthropod-transmitted disease of ungulates. The cellular tropism of BTV in ruminants includes macrophages, dendritic cells and endothelial cells (ECs), and fulminant infection is characterized by lesions consistent with those of so-called viral hemorrhagic fevers. Specifically, BT is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema. To further investigate the pathogenesis of vascular injury in BT, we evaluated the responses of cultured bovine pulmonary artery EC (bPAEC) and monocyte-derived macrophages (bMDM) to BTV infection by measuring transcript levels of genes encoding molecules important in mediating EC activation and/or endothelial barrier dysregulation. The data confirm that BTV infection of bPAEC resulted in increased transcription of genes encoding chemokine ligand 2 (CCL2) and E-selectin, and BTV infection of bMDM resulted in increased transcription of genes encoding TNF-alpha, IL-1beta, IL-8, and inducible nitric oxide synthase (iNOS). The data from these in vitro studies provide further evidence that cytokines and other vasoactive substances produced in macrophages potentially contribute to vascular injury in BTV-infected ruminants, along with direct effects of the virus itself on ECs.
Assuntos
Vírus Bluetongue/imunologia , Bluetongue/virologia , Doenças dos Bovinos/virologia , Artéria Pulmonar/virologia , Transcrição Gênica/imunologia , Animais , Bluetongue/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Células Endoteliais/virologia , Cinética , Macrófagos/imunologia , Macrófagos/virologia , Microscopia de Fluorescência , Artéria Pulmonar/citologia , Artéria Pulmonar/imunologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.
Assuntos
Vírus Bluetongue/patogenicidade , Bluetongue/patologia , Bluetongue/fisiopatologia , Junções Aderentes/patologia , Animais , Bluetongue/etiologia , Bovinos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Octoxinol/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Replicação ViralRESUMO
Comparative sequence analysis of a series of strains of equine arteritis virus (EAV) of defined virulence for horses, ranging from the horse-adapted virulent Bucyrus (VB) strain to a fully attenuated vaccine strain derived from it, identified 13 amino acid substitutions associated with attenuation. These include 4 substitutions in the replicase proteins and 9 in the structural proteins. Using reverse genetic techniques, these amino acid substitutions were introduced into a virulent infectious cDNA clone pEAVrVBS derived from the VB strain of EAV. Inoculation of horses with the recombinant viruses clearly demonstrated that changes in either the replicase (nsp1, nsp2 and nsp7) or structural proteins (GP2, GP4, GP5 and M) resulted in attenuation of the virulent VB strain. The recombinant virus with substitutions in the structural proteins was more attenuated than the recombinant virus with substitutions only in the replicase proteins.
Assuntos
Substituição de Aminoácidos/genética , Infecções por Arterivirus/veterinária , Equartevirus/genética , Equartevirus/patogenicidade , Doenças dos Cavalos/virologia , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genética , Animais , Anticorpos Antivirais/sangue , Temperatura Corporal , Cavalos , Leucócitos/virologia , Contagem de Linfócitos , Dados de Sequência Molecular , Cavidade Nasal/virologia , Testes de Neutralização , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Vacinas Atenuadas/genética , Ensaio de Placa Viral , Vacinas Virais/genéticaRESUMO
Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 (AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.
Assuntos
Equidae/virologia , Gammaherpesvirinae/classificação , Gammaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/veterinária , Reação em Cadeia da Polimerase/veterinária , Animais , Infecções por Herpesviridae/virologia , FilogeniaRESUMO
Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.
Assuntos
Gammaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/virologia , Animais , DNA Viral/química , DNA Viral/genética , Gammaherpesvirinae/genética , Gammaherpesvirinae/ultraestrutura , Glicoproteínas/química , Glicoproteínas/genética , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/virologia , Doenças dos Cavalos/sangue , Cavalos , Masculino , Microscopia Eletrônica de Transmissão/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions.
Assuntos
Equartevirus/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Epitopos/imunologia , Equartevirus/isolamento & purificação , Soros Imunes , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Neutralização , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Recombinação Genética , Alinhamento de Sequência , Especificidade da Espécie , Proteínas do Envelope Viral/genéticaRESUMO
Jamestown Canyon (JC) and Saint Louis encephalitis (SLE) viruses are mosquito-transmitted viruses that have long been present in California. The objective of this study was to determine the seroprevalence of these two viruses in horses prior to the introduction of West Nile (WN) virus. Approximately 15% of serum samples collected in 1998 from 425 horses on 44 equine operations horses throughout California had serum antibodies to JC virus, whereas antibodies were not detected to SLE virus. The results indicate that horses in California were commonly infected prior to 1998 with mosquito-transmitted Bunyaviruses that are identical or closely related to JC virus, but not with SLE virus. The different seroprevalence of SLE and JC viruses in horses likely reflects the unique ecology of each virus, and it is predicted that WN virus will have a wider distribution in California than closely related SLE virus.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Encefalite da Califórnia/imunologia , Vírus da Encefalite de St. Louis/imunologia , Encefalite de St. Louis/veterinária , Doenças dos Cavalos/virologia , Infecções por Polyomavirus/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , California/epidemiologia , Vírus da Encefalite da Califórnia/isolamento & purificação , Vírus da Encefalite de St. Louis/isolamento & purificação , Encefalite de St. Louis/epidemiologia , Encefalite de St. Louis/imunologia , Encefalite de St. Louis/virologia , Feminino , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/imunologia , Cavalos , Modelos Logísticos , Masculino , Testes de Neutralização/veterinária , Infecções por Polyomavirus/epidemiologia , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/virologia , Estudos Soroepidemiológicos , Inquéritos e Questionários , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologiaRESUMO
Equine arteritis virus (EAV) infects endothelial cells (ECs) and macrophages in horses, and many of the clinical manifestations of equine viral arteritis (EVA) reflect vascular injury. To further evaluate the potential role of EAV-induced, macrophage-derived cytokines in the pathogenesis of EVA, we infected cultured equine alveolar macrophages (AMphi), blood monocyte-derived macrophages (BMphi), and pulmonary artery ECs with either a virulent (KY84) or an avirulent (CA95) strain of EAV. EAV infection of equine AMphi, BMphi, and ECs resulted in their activation with increased transcription of genes encoding proinflammatory mediators, including interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha. Furthermore, the virulent KY84 strain of EAV induced significantly higher levels of mRNA encoding proinflammatory cytokines in infected AMphi and BMphi than did the avirulent CA95 strain. Treatment of equine ECs with the culture supernatants of EAV-infected AMphi and BMphi also resulted in EC activation with cell surface expression of E-selectin, whereas infection of ECs with purified EAV alone caused only minimal expression of E-selectin. The presence of TNF-alpha in the culture supernatants of EAV-infected equine AMphi, BMphi, and ECs was confirmed by bioassay, and the virulent KY84 strain of EAV induced significantly more TNF-alpha in all cell types than did the avirulent CA95 strain. Thus, the data indicate that EAV-induced, macrophage-derived cytokines may contribute to the pathogenesis of EVA in horses, and that the magnitude of the cytokine response of equine AMphi, BMphi, and ECs to EAV infection reflects the virulence of the infecting virus strain.
Assuntos
Citocinas/metabolismo , Equartevirus/imunologia , Equartevirus/patogenicidade , Macrófagos Alveolares/virologia , Macrófagos/virologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/virologia , Inflamação , Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Monócitos/imunologia , Monócitos/virologia , Artéria Pulmonar , Fator de Necrose Tumoral alfa/genéticaRESUMO
Bluetongue is an insect-transmitted disease of sheep and wild ruminants that is caused by bluetongue virus (BTV). Cattle are asymptomatic reservoir hosts of BTV. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of BTV infection of both sheep and cattle, but it is uncertain as to why sheep are highly susceptible to BTV-induced microvascular injury, whereas cattle are not. Thus, to better characterize the pathogenesis of bluetongue, the transcription of genes encoding a variety of vasoactive and inflammatory mediators was quantitated in primary ovine lung microvascular ECs (OLmVECs) exposed to BTV and/or inflammatory mediators. BTV infection of OLmVECs increased the transcription of genes encoding interleukin- (IL) 1 and IL-8, but less so IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. In contrast, we previously have shown that transcription of genes encoding all of these same mediators is markedly increased in BTV-infected bovine lung microvascular ECs and that BTV-infected bovine ECs produce substantially greater quantities of prostacyclin than do sheep ECs. Thus, sheep and cattle were experimentally infected with BTV to further investigate the role of EC-derived vasoactive mediators in the pathogenesis of bluetongue. The ratio of thromboxane to prostacyclin increased during BTV infection of both sheep and cattle, but was significantly greater in sheep (P = 0.001). Increases in the ratio of thromboxane to prostacyclin, indicative of enhanced coagulation, coincided with the occurrence of clinical manifestations of bluetongue in BTV-infected sheep. The data suggest that inherent species-specific differences in the production and activities of EC-derived mediators contribute to the sensitivity of sheep to BTV-induced microvascular injury.
Assuntos
Bluetongue/imunologia , Endotélio Vascular/imunologia , Epoprostenol/sangue , Tromboxanos/sangue , Vasoconstritores/sangue , Vasodilatadores/sangue , Animais , Biomarcadores , Bluetongue/sangue , Bluetongue/fisiopatologia , Bluetongue/virologia , Vírus Bluetongue/imunologia , Bovinos , Células Cultivadas , Ciclo-Oxigenase 2 , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Interleucina-1/genética , Interleucina-6/genética , Interleucina-8/genética , Isoenzimas/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Prostaglandina-Endoperóxido Sintases/genética , Ovinos , Transcrição GênicaRESUMO
An imported carrier stallion (A) from Europe was implicated in causing an extensive outbreak of equine viral arteritis (EVA) on a Warmblood breeding farm in Pennsylvania, USA. Strains of equine arteritis virus (EAV) present in the semen of two carrier stallions (A and G) on the farm were compared to those in tissues of foals born during the outbreak, as well as viruses present in the semen of two other stallions that became persistently infected carriers of EAV following infection during the outbreak. The 2822 bp segment encompassing ORFs 2-7 (nt 9807-12628; which encode the G(S), GP3, GP4, G(L), M and N proteins, respectively) was directly amplified by RT-PCR from semen samples and foal tissues. Nucleotide and phylogenetic analyses confirmed that virus present in the semen of stallion A initiated the outbreak. The genomes of viruses present in most foal tissues (10/11) and serum from an acutely infected mare collected during the outbreak were identical to that of virus present in the lung of the first foal that died of EVA. Virus in the placenta of one foal differed by one nucleotide (99.9% identity) from the predominant outbreak virus. The relative genetic stability of viruses that circulated during the outbreak contrasts markedly with the heterogeneous virus populations variously present in the semen of persistently infected stallions on the farm. These findings are consistent with the hypothesis that the carrier stallion can be a source of genetic diversity of EAV, and that outbreaks of EVA can be initiated by the horizontal aerosol transmission of specific viral variants that occur in the semen of particular carrier stallions.