RESUMEN
Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.
Asunto(s)
Encéfalo , Animales , Caballos/virología , Encéfalo/virología , Encéfalo/embriología , Encéfalo/citología , Cultivo Primario de Células/métodos , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/fisiología , Línea Celular , Neuronas/virología , Cultivo de Virus/métodos , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/veterinaria , Células Cultivadas , Replicación ViralRESUMEN
Equid Gamma herpesvirus (eGHV) infections have been reported worldwide and may be correlated with clinical signs, e.g., affecting the respiratory tract in young horses. eGHV are shed by healthy horses as well as horses with respiratory tract disease. The prevalence in healthy Swiss horses is unknown to date but this data would provide valuable information for causal diagnosis in clinical cases and formulation of biosecurity recommendations. Nasal swabs from 68 healthy horses from 12 Swiss stables and 2 stables near the Swiss border region in Germany were analyzed by panherpes nested PCR. Positive samples were sequenced. A multivariable model was used to determine if sex, age, breed, canton, or stable had a significant effect on the shedding status of each detected eGHV. Overall, the eGHV prevalence was 59% (n = 68); the prevalence for equid herpesvirus-2 (EHV-2), equid herpesvirus-5 (EHV-5) and asinine herpesvirus-5 (AHV-5) was 38%, 12% and 9%, respectively. Co-infections with multiple eGHVs were observed in 25% of the positive samples. The odds of shedding EHV-2 decreased with age (p = 0.01) whereas the odds of shedding AHV-5 increased with age (p = 0.04). Breed, sex, canton, or stable had no significant association with eGHV shedding. As EHV-2 shedding was common in healthy horses a positive PCR result must be interpreted with caution regarding the formulation of biosecurity recommendations and causal diagnosis. As EHV-5 and AHV-5 shedding was less common than EHV-2, a positive test result is more likely to be of clinical relevance. Shedding of multiple eGHV complicates the interpretation of positive test results in a horse.
Asunto(s)
Herpesvirus Équido 1/aislamiento & purificación , Nariz/virología , Enfermedades Respiratorias/veterinaria , Esparcimiento de Virus , Animales , Anticuerpos Antivirales/sangre , ADN Viral/genética , Femenino , Gammaherpesvirinae/aislamiento & purificación , Gammaherpesvirinae/fisiología , Alemania/epidemiología , Herpesvirus Équido 1/clasificación , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/fisiología , Enfermedades de los Caballos/epidemiología , Enfermedades de los Caballos/virología , Caballos , Masculino , Enfermedades Respiratorias/epidemiología , Enfermedades Respiratorias/virología , Suiza/epidemiología , ViremiaRESUMEN
Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.
Asunto(s)
Quimiocinas/inmunología , Citocinas/inmunología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Enfermedades de los Caballos/inmunología , Leucocitos Mononucleares/virología , Animales , Quimiocinas/genética , Citocinas/genética , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/genética , Enfermedades de los Caballos/genética , Enfermedades de los Caballos/virología , Caballos , Interacciones Huésped-Patógeno , Leucocitos Mononucleares/inmunología , Replicación ViralRESUMEN
In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.
Asunto(s)
Dermis/citología , Herpesvirus Équido 1/fisiología , Caballos , Mitocondrias/virología , Animales , Línea Celular , Replicación ViralRESUMEN
Equid herpesvirus-1 (EHV-1) outbreaks continue despite widely used vaccination. We demonstrated previously that an ORF1/ORF71 gene deletion mutant of the EHV-1 strain Ab4 (Ab4ΔORF1/71) is less virulent than its parent Ab4 virus. Here, we describe the Ab4 challenge infection evaluating protection induced by the Ab4ΔORF1/71 vaccine candidate. Susceptible control horses developed respiratory disease, fever, nasal shedding, and viremia. Full protection after challenge infection was observed in 5/5 previously Ab4 infected horses and 3/5 Ab4ΔORF1/71 horses. Two Ab4ΔORF1/71 horses developed short-lasting viremia and/or virus shedding. Protective immunity in the respiratory tract was characterized by pre-existing EHV-1-specific IgG4/7 antibodies, the absence of IFN-α secretion and rapidly increasing IgG4/7 upon challenge infection. Pre-existing systemic EHV-1-specific IgG4/7 highly correlated with protection. T-cell immunity was overall low. In conclusion, protective immunity against EHV-1 infection including prevention of viremia was associated with robust systemic and intranasal IgG4/7 antibodies suggesting immediate virus neutralization at the local site.
Asunto(s)
Anticuerpos Antivirales/inmunología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/inmunología , Vacunas contra Herpesvirus/administración & dosificación , Enfermedades de los Caballos/prevención & control , Inmunoglobulina G/inmunología , Viremia/veterinaria , Administración Intranasal , Animales , Femenino , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/efectos de los fármacos , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/fisiología , Vacunas contra Herpesvirus/inmunología , Enfermedades de los Caballos/inmunología , Enfermedades de los Caballos/virología , Caballos , Masculino , Mucosa Nasal/inmunología , Mucosa Nasal/virología , Vacunación , Viremia/inmunología , Viremia/prevención & control , Viremia/virología , Esparcimiento de VirusRESUMEN
Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Herpesvirus Équido 1/patogenicidad , Enfermedades de los Caballos/virología , Neuronas/virología , Animales , Células Cultivadas , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/crecimiento & desarrollo , Caballos , Especificidad del Huésped , Ratones , Ratones Endogámicos BALB C , Pase Seriado , Virulencia , Replicación ViralRESUMEN
VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 was the same in cells infected with the parent and VP22-deficient viruses. Hence, VP22 appears to promote synthesis of ICP4 post-transcriptionally.
Asunto(s)
Herpesvirus Équido 1/fisiología , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Estructurales Virales/fisiología , Animales , Perros , Eliminación de Gen , Herpesvirus Équido 1/genética , Proteínas Inmediatas-Precoces/genética , Células de Riñón Canino Madin Darby , Transcripción Genética , Proteínas Estructurales Virales/genética , Replicación Viral/fisiologíaRESUMEN
The respiratory epithelium of humans and animals is frequently exposed to alphaherpesviruses, originating from either external exposure or reactivation from latency. To date, the polarity of alphaherpesvirus infection in the respiratory epithelium and the role of respiratory epithelial integrity herein has not been studied. Equine herpesvirus type 1 (EHV1), a well-known member of the alphaherpesvirus family, was used to infect equine respiratory mucosal explants and primary equine respiratory epithelial cells (EREC), grown at the air-liquid interface. EHV1 binding to and infection of mucosal explants was greatly enhanced upon destruction of the respiratory epithelium integrity with EGTA or N-acetylcysteine. EHV1 preferentially bound to and entered EREC at basolateral cell surfaces. Restriction of infection via apical inoculation was overcome by disruption of intercellular junctions. Finally, basolateral but not apical EHV1 infection of EREC was dependent on cellular N-linked glycans. Overall, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defence against primary alphaherpesvirus infections. In addition, by targeting a basolaterally located receptor in the respiratory epithelium, alphaherpesviruses have generated a strategy to efficiently escape from host defence mechanisms during reactivation from latency.
Asunto(s)
Alphaherpesvirinae/fisiología , Uniones Intercelulares/metabolismo , Receptores Virales/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , Animales , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Enfermedades de los Caballos/metabolismo , Enfermedades de los Caballos/virología , Caballos , Uniones Intercelulares/efectos de los fármacos , Polisacáridos/metabolismo , Receptores Virales/química , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Carga Viral , Replicación ViralRESUMEN
We describe the histopathologic, immunohistochemical, and molecular features of a case of meningoencephalitis in a Thomson's gazelle ( Eudorcas thomsonii) naturally infected with zebra-borne equid herpesvirus 1 (EHV-1) and the implications for the molecular detection of zebra-borne EHV-1. A 4-y-old female Thomson's gazelle was submitted for postmortem examination; no gross abnormalities were noted except for meningeal congestion. Microscopic evaluation demonstrated multifocal nonsuppurative meningoencephalitis with intranuclear eosinophilic and amphophilic inclusion bodies and EHV-9 antigen in neurons. PCR demonstrated the presence of a herpesvirus with a nucleotide sequence 99-100% identical to the corresponding sequences of zebra-borne EHV-1 and of EHV-9 strains. To determine whether EHV-1 or EHV-9 was involved, a PCR with a specific primer set for EHV-9 ORF59/60 was used. The sequence was identical to that of 3 recognized zebra-borne EHV-1 strains and 91% similar to that of EHV-9. This isolate was designated as strain LM2014. The partial glycoprotein G ( gG) gene sequence of LM2014 was also identical to the sequence of 2 zebra-borne EHV-1 strains (T-529 isolated from an onager, 94-137 from a Thomson's gazelle). The histologic lesions of encephalitis and antigen localization in this gazelle indicate prominent viral neurotropism, and lesions were very similar to those seen in EHV-1- and EHV-9-infected non-equid species. Histologic lesions caused by EHV-9 and zebra-borne EHV-1 are therefore indistinguishable.
Asunto(s)
Antílopes , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Meningoencefalitis/veterinaria , Animales , Animales de Zoológico , Equidae/virología , Femenino , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/clasificación , Herpesvirus Équido 1/genética , Louisiana , Meningoencefalitis/patología , Meningoencefalitis/virología , Filogenia , Análisis de Secuencia de ADN/veterinariaRESUMEN
Carbon-based architectures, especially graphene and its derivatives, have recently attracted much attention in the field of biomedicine and biotechnology for their use as pathogen inhibitors or biosensors. One of the major problems in the development of novel virus inhibitor systems is the adaption of the inhibitor to the size of virus particles. We here report the synthesis and biological testing of carbon-based inhibitors differing in size for evaluating the potential size effect on the inhibition of virus entry and replication. In this context, different sized nanomaterials were functionalized with polygylcerol through a "grafting from" polymerization to form new polyvalent nanoarchitectures which can operate as viral inhibitor systems after post-modification. For this purpose a polysulfation was carried out to mimic the heparan sulfates present on cell surfaces that we reasoned would compete with the binding sites of herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1), which both cause major global health issues. Our results clearly demonstrate that the inhibitory efficiency is regulated by the size of the polymeric nanomaterials and the degree of sulfation. The best inhibiting graphene sheets were â¼300 nm in size and had a degree of sulfation of â¼10%. Furthermore, it turned out that the derivatives inhibited virus infection at an early stage during entry but did not affect cell-to-cell spread. Overall, tunable polyvalent nanomaterials are promising and efficient virus entry inhibitors, which can likely be used for a broad spectrum of enveloped viruses.
Asunto(s)
Grafito , Herpesvirus Équido 1/fisiología , Herpesvirus Humano 1/fisiología , Nanoestructuras , Internalización del Virus , Animales , Células Cultivadas , Chlorocebus aethiops , Caballos , Polímeros , Piel/citología , Células VeroRESUMEN
Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a+ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse's body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a+ cells. Here we characterize the in vitro replication kinetics of two EHV-1N strains in CD172a+ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1N replication was restricted to 7-8% in CD172a+ cells compared to 100% in control RK-13 cells. EHV-1N replication was not delayed in CD172a+ cells but virus production was significant lower (103.0 TCID50/105 inoculated cells) than in RK-13 cells (108.5 TCID50/105 inoculated cells). Approximately 0.04% of CD172a+ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a+ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a+ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.
Asunto(s)
Herpesvirus Équido 1/fisiología , Monocitos/virología , Replicación Viral , Animales , Células Cultivadas , Herpesvirus Équido 1/patogenicidad , CaballosRESUMEN
The NucleoCounter NC-3000, a portable high-speed cell counting device based on the principle of fluorescence microscopy, provides the alternative method for standard flow cytometry. The main objective of the study was to apply an efficient technique for the assessment of the primary murine neurons culture infected with either neuropathogenic or non-neuropathogenic strains of Equine Herpesvirus type 1 (EHV-1). Using the NucleoCounter NC-3000 we have observed a decrease in mitochondrial potential and reduction in cells viability but we have not observed changes in the cell cycle of cultured neurons infected with all EHV-1 strains.
Asunto(s)
Herpesvirus Équido 1/fisiología , Neuronas/virología , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Células Cultivadas , Potencial de la Membrana Mitocondrial , RatonesRESUMEN
Equid herpesvirus type 1 (EHV-1) is a major pathogen of horses with a worldwide distribution, which can cause various clinical signs ranging from mild respiratory disease to neurological disorders. To initiate an effective infection, EHV-1 evolved a broad spectrum of mechanisms exploiting the host cell, including its actin filaments. An actin-myosin-driven transport has been described to precede cellular entry of different viruses. Therefore, in the present study we investigated the role of actin motor protein - myosin, during replication of two EHV-1 strains: Jan-E (wild-type EHV-1 strain isolated from aborted equine fetus) and Rac-H (attenuated strain highly adapted in cell cultures in vitro) in primary murine neurons. In order to investigate this, we used two inhibitors: blebbistatin (BLB; non-muscle myosin II inhibitor) and 2,3-butanedione monoxime (BDM; inhibitor of myosin ATPase). Our results demonstrated that limitation of Jan-E EHV-1 replication occurred in cells treated with myosin inhibitor, which confirmed the important role of actin motor proteins during the entry and egress of EHV-1 virions. Application of blebbistatin did not affect Rac-H EHV-1 replication, while BDM caused reduction of replication in murine neurons. Based on these results it can be assumed that EHV-1 virion movement was myosin-dependent.
Asunto(s)
Herpesvirus Équido 1/fisiología , Enfermedades de los Caballos/enzimología , Miosinas/metabolismo , Neuronas/enzimología , Internalización del Virus , Liberación del Virus , Animales , Células Cultivadas , Herpesvirus Équido 1/genética , Enfermedades de los Caballos/genética , Enfermedades de los Caballos/virología , Caballos , Interacciones Huésped-Patógeno , Ratones , Miosinas/genética , Neuronas/virologíaRESUMEN
This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.
Asunto(s)
Aborto Veterinario/patología , Muerte Celular , Proliferación Celular , Citocinas/genética , Infecciones por Herpesviridae/veterinaria , Aborto Veterinario/virología , Animales , Citocinas/metabolismo , Femenino , Citometría de Flujo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/fisiología , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos BALB C , Placenta/patología , Placenta/virología , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Organismos Libres de Patógenos Específicos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Útero/patología , Útero/virologíaRESUMEN
The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells.
Asunto(s)
Actinas/metabolismo , Herpesvirus Équido 1/enzimología , Herpesvirus Équido 1/fisiología , Interacciones Huésped-Patógeno , Proteínas Serina-Treonina Quinasas/metabolismo , Liberación del Virus , Línea Celular , Técnicas de Inactivación de Genes , Herpesvirus Équido 1/crecimiento & desarrollo , Humanos , Proteínas Serina-Treonina Quinasas/genética , Ensayo de Placa Viral , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.
Asunto(s)
Citoesqueleto/ultraestructura , Herpesvirus Équido 1/fisiología , Citometría de Barrido por Láser/métodos , Microscopía Confocal/métodos , Neuronas/virología , Animales , Células Cultivadas , Dineínas/ultraestructura , Caballos , Procesamiento de Imagen Asistido por Computador/métodos , Filamentos Intermedios/ultraestructura , Filamentos Intermedios/virología , Ratones , Microtúbulos/ultraestructura , Microtúbulos/virología , Replicación ViralRESUMEN
Equine herpesvirus myeloencephalopathy (EHM), a disease caused by equine herpesvirus type 1 (EHV-1), is characterized by severe inflammation, thrombosis, and hypoxia in central nervous system (CNS) endothelial cells, which can result in a spectrum of clinical signs including urinary incontinence, ataxia, and paralysis. Strains of EHV-1 that contain a single point mutation within the viral DNA polymerase (nucleotide A2254>G2254: amino acid N752âD752) are isolated from EHM afflicted horses at higher frequencies than EHV-1 strains that do not harbor this mutation. Due to the correlation between the DNA Pol mutation and EHM disease, EHV-1 strains that contain the mutation have been designated as neurologic. In this study, we measured virus replication, cell to cell spread efficacy, and host inflammatory responses in equine endothelial cells infected with 12 different strains of EHV-1. Two strains, T953 (Ohio 2003) (neurologic) and Kentucky A (KyA) (non-neurologic), have well described disease phenotypes while the remaining strains used in this study are classified as neurologic or non-neurologic based solely on the presence or absence of the DNA pol mutation, respectively. Results show that the neurologic strains do not replicate better or spread more efficiently in endothelial cells. Also, the majority of the host inflammatory genes were modulated similarly regardless of EHV-1 genotype. Analyses of host gene expression showed that a subset of pro-inflammatory cytokines, including the CXCR3 ligands CXCL9, CXCL10, and CXCL11, as well as CCL5, IL-6 and TNF-α were consistently up-regulated in endothelial cells infected with each EHV-1 strain. The identification of specific pro-inflammatory cytokines in endothelial cells that are modulated by EHV-1 provides further insight into the factors that contribute to the immunopathology observed after infection and may also reveal new targets for disease intervention.
Asunto(s)
Células Endoteliales/virología , Regulación de la Expresión Génica/fisiología , Herpesvirus Équido 1/fisiología , Caballos , Inflamación/metabolismo , Animales , Línea Celular , Células Endoteliales/metabolismo , Conejos , Ensayo de Placa Viral , Replicación ViralRESUMEN
Equine herpesvirus 1 (EHV-1) and equine arteritis virus (EAV) induce respiratory problems and abortion in horses and are considered as two serious threats to equine industry. Both EHV-1 and EAV misuse patrolling leukocytes in the upper respiratory tract to breach the basement membrane (BM) and to migrate to blood vessels. So far, the behavior and impact of a double infection in the respiratory mucosa of a horse are unknown. In the present study, the outcome of double infections with EHV-1 and the low virulent EAV strain 08P187 (superinfection with an interval of 12h or co-infection) were compared with single infections in fully susceptible RK-13 cells and equine upper respiratory mucosa explants. When RK-13 cells were inoculated with either EHV-1 or EAV 12h prior to the subsequent EAV or EHV-1 inoculation, the latter EAV or EHV-1 infection was clearly suppressed at 24hpi or 36hpi, respectively, without EHV-1 and EAV co-infecting the same RK-13 cells. After simultaneous infection with EHV-1 and EAV, higher numbers of EAV infected cells but similar numbers of EHV-1 infected cells were found compared to the single infections, with a low number of EHV-1 and EAV co-infected RK-13 cells at 48hpi and 72hpi. In the upper respiratory mucosa exposed to EAV 12h prior to EHV-1, the number and size of the EHV-1-induced plaques were similar to those of the EHV-1 single infected mucosa explants. In nasal and nasopharyngeal mucosae, EAV and EHV-1 pre-infections slightly reduced the number of EHV-1 and EAV infected leukocytes compared to the single infections and co-infection. In double EAV and EHV-1 infected explants, no co-infected leukocytes were detected. From these results, it can be concluded that EAV and EHV-1 are only slightly influencing each other's infection and that they do not infect the same mucosal leukocytes.
Asunto(s)
Infecciones por Arterivirus/veterinaria , Equartevirus/fisiología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Enfermedades de los Caballos/virología , Mucosa Respiratoria/virología , Animales , Infecciones por Arterivirus/virología , Línea Celular , Coinfección , Células Epiteliales/virología , Equartevirus/patogenicidad , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/patogenicidad , Caballos , Leucocitos/virología , Técnicas de Cultivo de Tejidos , Carga Viral , Replicación ViralRESUMEN
Replication kinetics and invasion characteristics of equine herpesvirus-1 and -3 (EHV-1/-3) in nasal and vaginal mucosae were compared using explants. The explants were cultured during 96 h with little change in viability. The tissues were inoculated with EHV-1 03P37 (neuropathogenic), 97P70 (abortigenic) and EHV-3 04P57, collected at 0, 24, 48 and 72 h post inoculation (pi) and stained for viral antigens. Both EHV-1 and EHV-3 replicated in a plaquewise manner. The plaques were already observed at 24 h pi, their size increased over time and did not directly cross the basement membrane (BM). However, EHV-1 infected the monocytic cells (MC) and hijacked these cells to invade the lamina propria. In contrast, EHV-3 replication was fully restricted to epithelial cells; the virus did not breach the BM via a direct cell-to-cell spread nor used infected MC. EHV-1-induced plaques were larger in nasal mucosa compared to vaginal mucosa. The opposite was found for EHV-3-induced plaques. Both EHV-1 strains replicated with comparable kinetics in nasal mucosa. However, the extent of replication of the abortigenic strain in vaginal mucosa was significantly higher than that of the neuropathogenic strain. Two-to-five-fold lower numbers of EHV-1-infected MC underneath the BM were found in vaginal mucosa than in nasal mucosa. Our study has shown that (i) EHV-1 has developed in evolution a predisposition for respiratory mucosa and EHV-3 for vaginal mucosa, (ii) abortigenic EHV-1 replicates better in vaginal mucosa than neuropathogenic EHV-1 and (iii) EHV-3 demonstrated a strict epithelial tropism whereas EHV-1 in addition hijacked MC to invade the lamina propria.
Asunto(s)
Herpesvirus Équido 1/fisiología , Herpesvirus Équido 3/fisiología , Membrana Mucosa/virología , Replicación Viral/fisiología , Animales , Femenino , Caballos , Membrana Mucosa/citología , Nariz , Técnicas de Cultivo de Tejidos , VaginaRESUMEN
Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) was manifested by changed morphology of cells, destruction of actin cytoskeleton and nuclei degeneration, which led to focal degeneration. Moreover, EHV-1 26 strain caused fusion of the infected cells to form syncytia in culture. Real-time PCR analysis demonstrated that both neuropathogenic and non-neuropathogenic EHV-1 strains replicated in neurons and ED cells (equine dermal cell line) at a similar level. We can assume that a point mutation in the EHV-1 polymerase does not affect viral replication in this cell type.