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
Equine herpesvirus 1 (EHV1) is considered as a major pathogen of Equidae, causing symptoms from mild respiratory disease to late-term abortion and neurological disorders. Different EHV1 strains circulating in the field have been characterized to be of abortigenic or neurovirulent phenotype. Both variants replicate in a plaque-wise manner in the epithelium of the upper respiratory tract (URT), where the abortigenic strains induce more prominent viral plaques, compared to the neurovirulent strains. Considering the differences in replication at the URT, we hypothesized that abortigenic strains may show an increased ability to modulate the type I IFN secretion/signaling pathway, compared to strains that display the neurovirulent phenotype. Here, we analyze IFN levels induced by abortigenic and neurovirulent EHV1 using primary respiratory epithelial cells (EREC) and respiratory mucosa ex vivo explants. Similar levels of IFNα (~70 U/ml) were detected in explants inoculated with both types of EHV1 strains from 48 to 72 hpi. Second, EREC and mucosa explants were treated with recombinant equine IFNα (rEqIFNα) or Ruxolitinib (Rux), an IFN signaling inhibitor, prior to and during inoculation with abortigenic or neurovirulent EHV1. Replication of both EHV1 variants was suppressed by rEqIFNα. Further, addition of Rux increased replication in a concentration-dependent manner, indicating an IFN-susceptibility for both variants. However, in two out of three horses, at a physiological concentration of 100 U/ml of rEqIFNα, an increase in abortigenic EHV1 replication was observed compared to 10 U/ml of rEqIFNα, which was not observed for the neurovirulent strains. Moreover, in the presence of Rux, the plaque size of the abortigenic variants remained unaltered, whereas the typically smaller viral plaques induced by the neurovirulent variants became larger. Overall, our results demonstrate the importance of IFNα in the control of EHV1 replication in the URT for both abortigenic and neurovirulent variants. In addition, our findings support the speculation that abortigenic variants of EHV1 may have developed anti-IFN mechanisms that appear to be absent or less pronounced in neurovirulent EHV1 strains.
Asunto(s)
Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/inmunología , Interacciones Huésped-Patógeno , Evasión Inmune , Factores Inmunológicos/análisis , Interferón-alfa/análisis , Animales , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/virología , Herpesvirus Équido 1/clasificación , Caballos , Modelos Biológicos , Técnicas de Cultivo de Órganos , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , Ensayo de Placa Viral , Replicación ViralRESUMEN
Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10ß containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and Red/ET expression vector were used to develop different ORF68 mutants. Multi-step growth kinetic experiments were conducted in order to evaluate the growth properties of the constructed mutant viruses. Growth of the Ab4pΔORF68 showed the lowest titer, compared to the Ab4pΔORF68R, Ab4pΔORF68R non-sense, and the parent Ab4p viruses without any significant difference (P > 0.05). The growth of the mutant viruses was almost similar across the cell types, but viruses growth was more efficient in FHK cells as judged by the number of the obtained virus particles. The plaque size of Ab4pΔORF68 was significantly (40%) smaller than those of Ab4p (P < 0.01), Ab4pΔORF68R, and Ab4pΔORF68R non-sense viruses which confirmed the importance of ORF68 protein in the cell-to-cell transmission of EHV-1. Subcellular localization of the green fluorescent protein (GFP) ORF68 gene fusion product showed late expression with intranuclear localization of the transfected cells while immunofluorescent antibody technique (IFAT) localized it at the nucleus and nuclear membranes of the infected cells. Hence, it could be concluded that ORF68 protein may not be essential for EHV-1 Ab4p growth but plays a crucial role in virus penetration and transmission at the cellular level. Therefore, the generated EHV-1 ORF68 negative mutant could be a prospective candidate for the development of a vaccine marker.
Asunto(s)
Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/genética , Proteínas Virales/genética , Animales , Línea Celular , Núcleo Celular/virología , Cromosomas Artificiales Bacterianos , Escherichia coli/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Caballos , Microscopía Fluorescente , Membrana Nuclear/virología , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Carga Viral , Ensayo de Placa Viral , Proteínas Virales/análisisRESUMEN
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
Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other researchers. We found that no progeny viruses were produced by transfection of fetal equine kidney cells and rabbit kidney (RK-13) cells with the UL11 null mutant and truncation mutant BAC DNAs. However, mutant viruses were generated after transfection of RK13-UL11 cells constitutively expressing EHV-1 UL11 with the mutant BAC DNAs. In conclusion, UL11 of EHV-1 Ab4p is essential for replication in cultured cells.
Asunto(s)
Células Epiteliales/virología , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/patogenicidad , Sistemas de Lectura Abierta , Proteínas Estructurales Virales/genética , Replicación Viral , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/ultraestructura , Núcleo Celular/virología , Cromosomas Artificiales Bacterianos/química , Cromosomas Artificiales Bacterianos/metabolismo , Células Epiteliales/ultraestructura , Expresión Génica , Aparato de Golgi/ultraestructura , Aparato de Golgi/virología , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/metabolismo , Caballos , Riñón/citología , Riñón/virología , Mutación , Conejos , Proteínas Estructurales Virales/metabolismo , VirulenciaRESUMEN
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
Factors identified that affect the sensitivity of microorganisms to polyhexamethyleneguanidine (PHMG). Salts of PHMG chloride, valerate, maleate, succinate was to use. Test strains of Esherichia coli, Staphylococcus aureus, Bacillus cereus, Leptospira interrogans, Paenibacillus larvae, Mycobacterium bovis, M. avium, M. fortuitum, Aspergillus niger and some strains of viruses are taken as objects of research. We have determined that the cytoplasm membrane phospholipids is main "target" for the polycation molecules of PHMG. A differential sensitivity of the microorganisms to this drug is primarily determined by relative amount of lipids in membrane and their accessibility. Such trends exist: increase the relative contents of anionic lipids and more negative surface electric potential of membrane, and reduction of the sizes fat acid remainder of lipids bring to increase of microorganism sensitivity. Types of anion salt PHMG just have a certain value. Biocide activity of PHMG chloride is more, than its salts with organic acid. Feasibility of combining PHMG with other biocides in the multicomponent disinfectants studied and analyzed. This combination does not lead to a significant increase in the sensitivity of microorganisms tested in most cases. Most species of pathogenic bacteria can be quickly neutralized by aqueous solutions of PHMG in less than 1% concentrations.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antivirales/farmacología , Desinfectantes/farmacología , Guanidinas/farmacología , Antibacterianos/administración & dosificación , Antibacterianos/química , Antifúngicos/administración & dosificación , Antifúngicos/química , Antivirales/administración & dosificación , Antivirales/química , Aspergillus niger/efectos de los fármacos , Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/metabolismo , Desinfectantes/administración & dosificación , Desinfectantes/química , Relación Dosis-Respuesta a Droga , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Bacterias Grampositivas/metabolismo , Guanidinas/administración & dosificación , Guanidinas/química , Herpesvirus Équido 1/efectos de los fármacos , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/metabolismo , Pruebas de Sensibilidad Microbiana , Fosfolípidos/metabolismo , Especificidad de la EspecieRESUMEN
The equine herpesvirus type 1 (EHV-1) open reading frame 34 (ORF34) is predicted to encode a polypeptide of 161 amino acids. We show that an ORF34 deletion mutant exhibited a significant growth defect in equine peripheral blood mononuclear cells taken directly ex vivo during early but not late times of infection. ORF34 protein (pORF34)-specific antibodies specifically reacted with a 28-kDa early polypeptide present in the cytosol of infected cells. From 10h post infection, multiple smaller pORF34-specific protein moieties were detected indicating that expression of a late viral gene product(s) caused pORF34 degradation. Proteasome inhibitors blocked pORF34 degradation as did treatment of infected cells with a ubiquitin-activating enzyme (E1) inhibitor. Finally, kinetic studies showed that pORF34 is modified by addition of multiple copies of ubiquitin. Taken together, our findings suggest that the ubiquitin proteasome pathway is required for pORF34 degradation that may modulate protein activity in the course of infection.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/metabolismo , Enfermedades de los Caballos/virología , Sistemas de Lectura Abierta , Proteínas Virales/metabolismo , Animales , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/química , Herpesvirus Équido 1/genética , Caballos , Cinética , Proteolisis , Ubiquitinación , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Equine herpesvirus type 1 (EHV-1) ORF59 is predicted to encode a protein consisting of 180 amino acids. To determine whether ORF59 in fact encodes a protein, sequences encoding an HA epitope (YPYDVPDYA) was inserted at the carboxyterminus of the ORF59 protein in EHV-1 strain Ab4. Using anti-HA monoclonal antibodies, a 21-kDa band was specifically detected by western blot analysis in lysates of cells infected with a recombinant EHV-1 from strain Ab4 that carries the pORF59-HA but not in cells infected with parental Ab4. Further characterization of the protein using immunofluorescence and fractionation studies showed that pORF59 is an early protein that localizes to the cytosol in virus-infected cells. Recombinant EHV-1 lacking ORF59 (rAb4∆59) exhibited a small-plaque phenotype and could not be propagated. Our findings suggest that the ORF59 protein plays a major role in EHV-1 replication in vitro and likely in vivo.
Asunto(s)
Citosol/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/metabolismo , Enfermedades de los Caballos/virología , Sistemas de Lectura Abierta , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/genética , Caballos , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Virales/genéticaRESUMEN
Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell culture in vitro. Immunofluorescent labeling and confocal microscopy revealed the formation of long, thin projections in neurons infected with the Jan-E strain, which was probably associated with enhanced intracellular spread of the virus. The EHV-1 Rac-H strain caused disruption of the microfilaments system and general depolymerization of actin, but treatment of neurons with cytochalasin D or latrunculin A resulted in limitation of viral replication. It can therefore be assumed that actin filaments are required only at the early stages of infection. Our results allow us to suggest that the actin cytoskeleton participates in EHV-1 infection of primary murine neurons but is not essential, and that other components of the cytoskeleton and/or cellular mechanisms may be also involved during EHV-1 infection.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Herpesvirus Équido 1/fisiología , Interacciones Huésped-Patógeno , Neuronas/virología , Animales , Células Cultivadas , Herpesvirus Équido 1/crecimiento & desarrollo , RatonesRESUMEN
Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to α4ß1 or α4ß7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that α4ß1 and α4ß7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/fisiología , Herpesvirus Équido 4/fisiología , Enfermedades de los Caballos/virología , Integrinas/metabolismo , Proteínas del Envoltorio Viral/genética , Animales , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 4/genética , Herpesvirus Équido 4/crecimiento & desarrollo , Caballos , Especificidad del Huésped , Proteínas del Envoltorio Viral/metabolismo , Ensayo de Placa Viral/veterinaria , Replicación ViralRESUMEN
EUL47 is a major component of the tegument of equine herpesvirus 1 (EHV-1). To determine its function, we used Red/ET cloning to delete its gene (gene 13) from EHV-1 strain Ab4p inserted into a bacterial artificial chromosome (BAC), yielding Ab4pattBΔ13. We also examined the reverted virus (Ab4pattB13R). Ab4pattBΔ13 replicated in rabbit kidney (RK)-13 cells, indicating that ORF13 is dispensable for virus replication in cell culture. Its intracellular and extracellular titers were about 10- and 100-fold lower than those of the revertant and parent strains, respectively. In addition, the plaque size was half the plaque sizes of the other two strains. The particle-to-plaque forming unit ratio of Ab4pattBΔ13 was 21-fold greater than the ratios of the revertant and parent strains. No enveloped virions were detected in the cytoplasm of Ab4pattBΔ13-infected cells by transmission electron microscopy. In hamster, Ab4pattBΔ13 caused clinical signs and weight loss after only 1 day, but induced less severe neurological signs than did the revertant and parent strains. These results indicate that EUL47 is structurally required for normal virus replication, viral morphogenesis and viral infectivity, and that loss of EUL47 moderately attenuates the neuropathogenicity of EHV-1 in the hamster model.
Asunto(s)
Herpesvirus Équido 1/fisiología , Herpesvirus Équido 1/ultraestructura , Proteínas Estructurales Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Cricetinae , Modelos Animales de Enfermedad , Eliminación de Gen , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/patogenicidad , Mesocricetus , Microscopía Electrónica de Transmisión , Conejos , Carga Viral , Ensayo de Placa Viral , Proteínas Estructurales Virales/genética , Virión/ultraestructura , VirulenciaRESUMEN
The 150 kbp genome of equine herpesvirus-1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7 kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus.
Asunto(s)
ADN Viral/genética , Herpesvirus Équido 1/fisiología , Secuencias Invertidas Repetidas , Eliminación de Secuencia , Replicación Viral , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/patogenicidad , Pulmón/virología , Ratones , Ratones Endogámicos CBA , Análisis de Supervivencia , Ensayo de Placa Viral , Proteínas Virales/biosíntesis , VirulenciaRESUMEN
Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N-->D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D(752) or N(752), to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N(752) isolates and at 24 h p.i. for all D(752) isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D(752) isolates compared with N(752) isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/patogenicidad , Caballos/virología , Mucosa Nasal/virología , Factores de Virulencia/genética , Replicación Viral , Animales , Bélgica , ADN Polimerasa Dirigida por ADN/fisiología , Herpesvirus Équido 1/aislamiento & purificación , Monocitos/virología , Mutación Missense , Técnicas de Cultivo de Órganos , Linfocitos T/virología , Factores de Tiempo , Proteínas Virales/genética , Proteínas Virales/fisiología , Virulencia , Factores de Virulencia/fisiologíaRESUMEN
Equine herpesvirus 1 (EHV-1) bacterial artificial chromosome clone (Ab4p BAC) was established based on neuropathogenic strain Ab4p. ORF37 encoding UL24 was replaced with a selection cassette, rpsL-neo gene, to produce an ORF37 deletion mutant, Ab4pORF37. Transfection of RK-13 cells with Ab4pORF37 genome DNA produced infectious virus, indicating that ORF37 is not essential for EHV-1 replication in cell culture. Deletion of ORF37 had no effect on the transcript expression of neighboring genes, ORF36 and ORF38, and the growth activity in MDBK cells. Ab4pDeltaORF37 lost neuropathogenicity in CBA/N1 mice as indicated by the absence of any neurological disorders and death. The growth of Ab4pDeltaORF37 in cultivated neural cells was one order of magnitude lower than that of parental and revertant viruses. These results indicated that the ORF37 is a neuropathogenicity determinant of EHV-1 in the mouse encephalitis model.
Asunto(s)
Encefalitis Viral/virología , Herpesvirus Équido 1/patogenicidad , Proteínas Virales/fisiología , Factores de Virulencia/fisiología , Animales , Peso Corporal , Encéfalo/patología , Encéfalo/virología , Bovinos , Línea Celular , Encefalitis Viral/patología , Técnicas de Inactivación de Genes , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/crecimiento & desarrollo , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos CBA , Conejos , Carga Viral , Ensayo de Placa Viral , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection.
Asunto(s)
Caveolina 1/metabolismo , Endocitosis , Herpesvirus Équido 1/fisiología , Internalización del Virus , Animales , Caveolina 1/genética , Línea Celular , Células Endoteliales/virología , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/crecimiento & desarrollo , Enfermedades de los Caballos/virología , Caballos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Proteínas Tirosina Quinasas/metabolismo , TransgenesRESUMEN
IR4, an early regulatory protein of equine herpesvirus 1 (EHV-1), is not a DNA-binding protein, but interacts with the sole immediate-early protein (IEP) to increase both IEP site-specific DNA-binding and IEP-mediated trans-activation of EHV-1 promoters. To investigate the biological properties of IR4 and ascertain whether this regulatory protein is essential for virus growth, bacterial artificial chromosome methods were employed to generate an IR4-null EHV-1. The IR4 gene was dispensable for EHV-1 growth in non-immortalized equine NBL-6 cells, but virus replication was delayed and was reduced by greater than 10-fold. In addition, replication of the IR4 mutant was abrogated in all other cell types tested, including equine ETCC tumor cells and cells of mouse, rabbit, monkey, and human origin. Further, in contrast to the highly pathogenic parent virus, the IR4 deletion mutant failed to cause disease in the CBA mouse as judged by assessing body weight and clinical signs and was unable to replicate in the murine lung. To define the nature of the block in the replication of the IR4-null virus, molecular analyses were carried out in RK-13 rabbits' cells infected with the IR4-deleted virus and revealed that: 1) the synthesis of the sole IEP was not inhibited; 2) the synthesis of early viral proteins examined was either not affected or was delayed to late times; 3) viral DNA replication was inhibited by more than 99.9%; and 4) synthesis of essential late proteins such as glycoprotein D and glycoprotein K was prevented. These findings indicate that the IR4 protein is required for EHV-1 DNA replication in non-permissive cells, and, like its homologues in other alphaherpesviruses, contributes a function required for virus replication in a variety of cell types.
Asunto(s)
Herpesvirus Équido 1/fisiología , Herpesvirus Équido 1/patogenicidad , Proteínas Virales/fisiología , Factores de Virulencia/fisiología , Animales , Peso Corporal , Línea Celular , Cromosomas Artificiales Bacterianos , Eliminación de Gen , Haplorrinos , Infecciones por Herpesviridae , Herpesvirus Équido 1/crecimiento & desarrollo , Caballos , Humanos , Pulmón/virología , Ratones , Ratones Endogámicos CBA , Conejos , Índice de Severidad de la Enfermedad , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Factores de Virulencia/genética , Replicación ViralRESUMEN
We compared the growth kinetics of neuropathogenic and nonneuropathogenic equine herpesvirus type 1 (EHV-1) strains in mouse cerebral cortex cells and investigated the relevance of the D/N amino acid change at position 752 of ORF30 in Japanese isolates. Neuropathogenic electropherotype P strains 01c1 and 89c25 exhibited similar growth kinetics to nonneuropathogenic P strain 90c16 in cultured neurons; however, the growth ability of type B strain 97c7 was lower than those of the other strains tested. The amino acid encoded at 752 of ORF30 in 01c1 was asparagic acid; asparagine was encoded in the other EHV-1 strains isolated from Japanese horses. The D/N(752) difference in ORF30 may not be related to replication ability in neurons.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/genética , Neuronas/citología , Neuronas/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Corteza Cerebral/citología , ADN Polimerasa Dirigida por ADN/genética , Regulación Viral de la Expresión Génica , Herpesvirus Équido 1/patogenicidad , Cinética , Ratones , Datos de Secuencia Molecular , MutaciónRESUMEN
In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.
Asunto(s)
Línea Celular/virología , Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 4/crecimiento & desarrollo , Enfermedades de los Caballos/virología , Animales , Línea Celular/citología , Células Clonales , Efecto Citopatogénico Viral , Embrión de Mamíferos , Herpesvirus Équido 1/aislamiento & purificación , Herpesvirus Équido 4/aislamiento & purificación , Caballos , Riñón/citologíaRESUMEN
Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11DeltagG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11DeltagG in cell culture when compared to parental virus or a rescuant virus vL11DeltagGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11DeltagGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.