Humoral immunity is not altered in overweight pregnant Crioulo mares / Imunidade humoral não é alterada em éguas Crioulas gestantes com sobrepeso

Both pregnancy and obesity can influence significant changes in the immune system. On this basis, the present study proposes to evaluate the humoral immune response of overweight pregnant mares in response to a commercial vaccine. Thirty pregnant Crioulo mares were separated according to body condition score (BCS) into overweight (BCS≥7/9) or lean-control (BCS= 5-6/9). In each group, the animals were subdivided into vaccinated and controls. The mares were vaccinated against EHV-1 in two doses spaced 21 days apart and had their blood collected monthly, for five months, for antibody evaluation. Both vaccinated groups had an increase in specific neutralizing antibodies after the vaccine. However, after the second dose, there was no increase in antibodies in any of the groups. Vaccinated overweight and lean-control mares did not differ at any time point. Therefore, this study demonstrated that obesity does not influence the humoral immune response in pregnant Crioulo mares.(AU)

Tanto a gestação quanto a obesidade podem influenciar o desenvolvimento de alterações significativas no sistema imune, portanto, o presente estudo teve como objetivo avaliar a resposta imune humoral de éguas gestantes com sobrepeso em resposta a uma vacina comercial. Trinta éguas Crioulas gestantes foram separadas de acordo com o escore de condição corporal (ECC) em éguas com sobrepeso (ECC≥7/9) e éguas controles (ECC=5-6/9) e, ainda, em cada grupo, os animais também foram separados em vacinados e controles. As éguas foram vacinadas contra o EHV-1 em duas doses com intervalo de 21 dias, sendo realizadas coletas de sangue mensalmente durante cinco meses para avaliação de anticorpos neutralizantes. Ambos os grupos vacinados tiveram aumento de anticorpos neutralizantes específicos após a vacina, porém, após a segunda dose, não foi observado aumento de anticorpos em nenhum dos grupos. Nenhuma diferença foi observada entre éguas vacinadas com sobrepeso e as éguas controles em nenhum momento. Assim, este estudo demonstrou que a obesidade não é um fator que influencia a resposta imune humoral de éguas Crioulas gestantes.(AU)

Equine herpesvirus 1 elicits a strong pro-inflammatory response in the brain of mice.

Equine herpesvirus type 1 (EHV-1) is an emerging pathogen that causes encephalomyelitis in horses and non-equid species. Several aspects of the immune response in the central nervous system (CNS), mainly regarding the role of inflammatory mediators during EHV-1 encephalitis, remain unknown. Moreover, understanding the mechanisms underlying extensive neuropathology induced by viruses would be helpful to establish therapeutic strategies. Therefore, we aimed to evaluate some aspects of the innate immune response during highly neurovirulent EHV-1 infection. C57BL/6 mice infected intranasally with A4/72 and A9/92 EHV-1 strains developed a fulminant neurological disease at 3 days post-inoculation with high viral titres in the brain. These mice developed severe encephalitis with infiltration of monocytes and CD8+ T cells to the brain. The inflammatory infiltrate followed the detection of the chemokines CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9 and CXCL-10 in the brain. Notably, the levels of CCL3, CCL4, CCL5 and CXCL9 were higher in A4/72-infected mice, which presented higher numbers of inflammatory cells within the CNS. Pro-inflammatory cytokines, such as interleukins (ILs) IL-1α, IL-1ß, IL-6, IL-12ß, and tumour necrosis factor (TNF), were also detected in the CNS, and Toll-like receptor (TLR) TLR2, TLR3 and TLR9 genes were also upregulated within the brain of EHV-1-infected mice. However, no expression of interferon-γ (IFN-γ) and IL-12α, which are important for controlling the replication of other herpesviruses, was detected in EHV-1-infected mice. The results show that the activated innate immune mechanisms could not prevent EHV-1 replication within the CNS, but most likely contributed to the extensive neuropathology. The mouse model of viral encephalitis proposed here will also be useful to study the mechanisms underlying extensive neuropathology.

Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C2254/H752 Strain in French Horses Showing no Major Impact on the Strain Behaviour.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

Neuropathogenic and non-neuropathogenic EHV-1 strains induce the accumulation of hyperphosphorylated Tau in primary murine neurons.

Equid herpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Similarly, to other alphaherpesviruses, EHV-1 is neurotropic and establishes latency in the neurons of its natural host. Despite the fact that many studies have been devoted to the pathogenesis of various clinical forms of EHV-1 infection, mechanisms of the neuronal damage are still not fully understood. The aim of this study was to define the phosphorylation status of tau protein in neuronal cell culture infected with EHV-1. Phosphorylation of tau was tested at tau-ser199/ser202, tau-ser404, tau-ser262, tau-thr181, tau-thr217 and tau-thr205 sites. We described, for the first time, that EHV-1 infection leads to the accumulation of hyperphosphorylated tau in primary murine neurons. We showed that non-neuropathogenic and neuropathogenic EHV-1 strains specifically induce hyperphosphorylation of tau-ser199/ser202, tau-ser404 and tau-thr205 during long-term infection and after a controlled activation of productive infection. Keywords: tau protein; hyperphosphorylation; equid herpesvirus 1 (EHV-1); neuronal cell culture.

Detection of Coxiella burnetii and equine herpesvirus 1, but not Leptospira spp. or Toxoplasma gondii, in cases of equine abortion in Australia - a 25 year retrospective study.

Equine abortion is a cause of severe economic loss to the equine industry. Equine herpesvirus 1 is considered a primary cause of infectious abortion in horses, however other infectious agents can also cause abortion. Abortions due to zoonotic pathogens have implications for both human and animal health. We determined the prevalence of Coxiella burnetii, Leptospira spp. and Toxoplasma gondii in 600 aborted equine foetal tissues that were submitted to our diagnostic laboratories at the University of Melbourne from 1994 to 2019. Using qPCR we found that the prevalence of C. burnetii was 4%. The highest annual incidence of C. burnetii was observed between 1997-2003 and 2016-2018. The prevalence of C. burnetii in Victoria and New South Wales was 3% and 6% respectively. All the samples tested negative for Leptospira spp. and Toxoplasma gondii DNA. Equine herpesvirus 1 DNA was detected at a prevalence of 3%. This study has provided evidence for the presence of C. burnetii in equine aborted foetal tissues in Australia, but the role of C. burnetii as potential cause of abortion in Australia requires further investigation. C. burnetii is a zoonotic disease agent that causes the disease 'Q fever' in humans. We recommend that appropriate protective measures should be considered when handling material associated with equine abortions to reduce the risk of becoming infected with C. burnetii.

Equine herpesvirus-1 genotype did not significantly affect clinical signs and disease outcome in 65 horses diagnosed with equine herpesvirus-1 myeloencephalopathy.

The objective of this study was to determine if the genotype of equine herpesvirus-1 (EHV-1) impacted clinical disease and outcome of horses with laboratory confirmed equine herpesvirus myeloencephalopathy (EHM). Medical records from 65 horses diagnosed with EHM from 2011 to 2019 were reviewed for signalment, presence and severity of clinical signs (lethargy, fever, ataxia, urinary incontinence) and outcome. Horses were further grouped based on the EHV-1 genotype into neuropathic (D752) or non-neuropathic (N752) EHV-1 infection. Between the two EHV-1 genotype groups, age and sex distributions were similar, although breed distribution was different (Quarter Horses and Saddlebreds were overrepresented and Warmbloods were underrepresented in the EHV-1 D752 group compared to the EHV-1 N752 group; P = 0.009). Lethargy, fever, ataxia and outcome were not significantly different between the two EHV-1 genotype groups (P > 0.05). However, urinary incontinence was significantly more frequently reported in horses infected with the D752 genotype of EHV-1 (P=0.04). Contrary to previous studies, the present study showed no difference in frequency of genotype (D752 or N752) among 65 horses with EHM and, with the exception of urinary incontinence, no difference in clinical disease or outcome related to the EHV-1 genotype.

Susceptibility of rat immortalized neuronal cell line Rn33B expressing equine major histocompatibility class 1 to equine herpesvirus-1 infection is differentiation dependent.

Equine herpesvirus-1 (EHV-1), which causes encephalomyelitis in horses, shows endotheliotropism in the central nervous system of horses, and generally does not infect neurons. However, little is known about the mechanism underlying the resistance of neuron to EHV-1, due to the lack of convenient cell culture systems. In this study, we examined EHV-1 infection in immortalized Rn33B rat neuronal cells, which differentiate into neurons when cultured under nonpermissive conditions. Because murine cell lines are resistant to EHV-1 infections due to the lack of functional entry receptors for EHV-1, we used an Rn33B-derived cell line that stably expresses the equine MHC class 1 molecule, which acts as EHV-1 entry receptor (Rn33B-A68B2M cells). EHV-1 infected undifferentiated Rn33B-A68B2M cells more efficiently than differentiated cells, resulting in the production of progeny virus in the former but not in the latter. By contrast, both differentiated and undifferentiated cells infected with herpes simplex virus-1 produced infectious viral progeny. While EHV-1 infection induced stronger expression of IFN alpha gene in differentiated cells than in undifferentiated cells, downstream IFN responses, including phosphorylation of STAT1 (signal transducer and activator of transcription 1) and expression of IFN-stimulated genes, were not activated regardless of whether cells were differentiated or not. These results suggest that neuronal differentiation of RN33B-A68B2M cells reduced their susceptibility to EHV-1, which is not due to different IFN responses. This culture system may be useful as an in vitro model for studying neuron-specific resistance to EHV-1, by investigating viral and host factors responsible for the difference in susceptibility between differentiated and undifferentiated cells.

Respuesta inmunoprotectora inducida por la glicoproteina D recombinante en el modelo de infección respiratoria de ratones BALB/c por alfaherpesvirus equino 1 / Immunoprotective response induced by recombinant glycoprotein D in the BALB/c respiratory mouse model " of Equid alphaherpesvirus 1 infection

Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host.

La infección con alfaherpesvirus equino 1 (EHV-1) causa abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos en equinos. La infección natural y las vacunas disponibles solo proporcionan protección parcial y de corta duración contra las reinfecciones. En el presente estudio se analizó la inducción de inmunidad protectiva de la glicoproteina D (gD) expresada en baculovirus y purificada al ser administrada por diferentes rutas en ratones BALB/c desafiados con la cepa AR8 de EHV-1. Los signos clínicos fueron variables entre los grupos de ratones inmunizados por rutas parenterales y, aunque la gD indujo respuesta especifica de IgG en suero, no logró prevenir la llegada del virus al pulmón. En los ratones inmunizados intranasalmente no se observaron signos clinicos ni lesiones histopatológi-cas, y el aislamiento viral y la detección de antigenos por inmunohistoquímica en pulmón fueron negativos. Además, por esta ruta la gD no estimuló la producción de IgG y de IgA en suero. Sin embargo se confirmó la respuesta de IgA especifica en el tracto respiratorio de ratones inmunizados intranasalmente. Esta respuesta inmune mucosal podría haber reducido la unión inicial del virus a la célula huésped y, de este modo, prevenir la llegada del virus al pulmón. Nuestros hallazgos proporcionan un aporte para continuar estudiando nuevas estrategias de inmunización en el huésped natural.

Pollens destroy respiratory epithelial cell anchors and drive alphaherpesvirus infection.

Pollens are well-known triggers of respiratory allergies and asthma. The pollen burden in today's ambient air is constantly increasing due to rising climate change and air pollution. How pollens interact with the respiratory mucosa remains largely unknown due to a lack of representative model systems. We here demonstrate how pollen proteases of Kentucky bluegrass, white birch and hazel selectively destroy integrity and anchorage of columnar respiratory epithelial cells, but not of basal cells, in both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC). In turn, this pollen protease-induced damage to respiratory epithelial cell anchorage resulted in increased infection by the host-specific and ancestral alphaherpesvirus equine herpesvirus type 1 (EHV1). Pollen proteases of all three plant species were characterized by zymography and those of white birch were fully identified for the first time as serine proteases of the subtilase family and meiotic prophase aminopeptidase 1 using mass spectrometry-based proteomics. Together, our findings demonstrate that pollen proteases selectively and irreversibly damage integrity and anchorage of columnar respiratory epithelial cells. In turn, alphaherpesviruses benefit from this partial loss-of-barrier function, resulting in increased infection of the respiratory epithelium.

The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. RESULTS: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. CONCLUSIONS: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract - Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

Absence of relationship between type-I interferon suppression and neuropathogenicity of EHV-1.

Equine herpesvirus-1 (EHV-1) infection is an important and highly prevalent disease in equine populations worldwide. Previously we have demonstrated that a neuropathogenic strain of EHV-1, T953, suppresses the host cell's antiviral type-I interferon (IFN) response in vitro. Whether or not this is unique to EHV-1 strains possessing the neuropathogenic genotype has been undetermined. Here, we examined whether there is any direct relationship between neuropathogenic genotype and the induced IFN-ß response in equine endothelial cells (EECs) infected with 10 different strains of EHV-1. The extent of virus cell-to-cell spread following infection in EECs was also compared between the neuropathogenic and the non-neuropathogenic genotype of EHV-1. We then compared IFN-ß and the total type-I IFN protein suppression between T953, an EHV-1 strain that is neuropathogenic and T445, an EHV-4 strain mainly associated only with respiratory disease. Data from our study revealed no relationship between the neuropathogenic genotype of EHV-1 and the induced IFN-ß mRNA by the host cell. Results also indicate no statistically significant difference in plaque sizes of both genotypes of EHV-1 produced in EECs. However, while the T953 strain of EHV-1 was able to suppress IFN-ß mRNA and type-I IFN biological activity at 12 h post-infection (hpi), EHV-4 weakly induces both IFN-ß mRNA and type-I IFN biological activity. This finding correlated with a statistically significant difference in the mean plaque sizes produced by the two EHV subtypes in EECs. Our data help illuminate how EHV-1, irrespective of its genotype, evades the host cell's innate immune response thereby enabling viral spread to susceptible cells.

Equine herpesvirus type 1 ORF51 encoding UL11 as an essential gene for replication in cultured cells.

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.

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons-the possible effects of the multiple passages of EHV-1 on its neurovirulence.

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.

Low occurrence of equine herpesvirus 1 (EHV-1) as cause of abortion and perinatal mortality in Brazil / Baixa ocorrência de herpes-vírus equino 1 (HVE-1) como causa de abortamento e mortalidade perinatal no Brasil

Equine herpesvirus type 1 (EHV-1) is an important pathogen that causes abortion, neonatal disease, respiratory disorders, and neurological syndrome in equine populations worldwide. To evaluate EHV-1 as a cause of abortion and perinatal mortality in Brazil, tissue samples from 105 aborted equine fetuses, stillbirths, and foals up to one month of age were examined using virus isolation, immunohistochemistry (IHC), histopathology, and nested polymerase chain reaction (PCR). Two fetuses were positive for EHV-1 by PCR, one of which showed syncytia and eosinophilic intranuclear inclusion bodies in bronchial epithelia, but it was negative by virus isolation. The other showed no characteristic histological lesions, but it was positive by viral isolation. No sample was positive by IHC. The results presented low occurrence of EHV-1 in the studied population and suggested that the use of a combination of techniques increases the likelihood of an accurate diagnosis of EHV-1.(AU)

O herpes-vírus equino tipo 1 (HVE-1) é um importante agente patogênico causador de aborto, doença neonatal, distúrbios respiratórios e síndrome neurológica em populações de equinos em todo o mundo. Para avaliar a ocorrência do HVE-1 como agente causal de abortamento e mortalidade perinatal no Brasil, foram examinadas amostras de 105 fetos equinos abortados, natimortos e potros de até 1 mês de idade, utilizando as técnicas de isolamento viral, imuno-histoquímica (IHQ), histopatologia e reação em cadeia da polimerase aninhada (nested-PCR). Dois fetos foram positivos na análise de PCR, e um deles apresentou corpúsculos de inclusão viral eosinofílicos e sincícios no epitélio brônquico, porém foi negativo na análise de isolamento viral. O outro feto não apresentou lesões histológicas características de infecção herpética, mas foi positivo na análise de isolamento viral. Nenhuma amostra apresentou resultado positivo pela análise de IHQ. Os resultados demonstraram baixa ocorrência de HVE-1 na população estudada e que o uso de diferentes técnicas diagnósticas aumenta a probabilidade de um diagnóstico preciso para o HVE-1.(AU)

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Viral genes and cellular markers associated with neurological complications during herpesvirus infections.

Despite the importance of neurological disorders associated with herpesviruses, the mechanism by which these viruses influence the central nervous system (CNS) has not been definitively established. Owing to the limitations of studying neuropathogenicity of human herpesviruses in their natural host, many aspects of their pathogenicity and immune response are studied in animal models. Here, we present an important model system that enables studying neuropathogenicity of herpesviruses in the natural host. Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes a devastating neurological disease (EHV-1 myeloencephalopathy; EHM) in horses. Like other alphaherpesviruses, our understanding of virus neuropathogenicity in the natural host beyond the essential role of viraemia is limited. In particular, information on the role of different viral proteins for virus transfer to the spinal cord endothelium in vivo is lacking. In this study, the contribution of two viral proteins, DNA polymerase (ORF30) and glycoprotein D (gD), to the pathogenicity of EHM was addressed. Furthermore, different cellular immune markers, including alpha-interferon (IFN-α), gamma-interferon (IFN-γ), interleukin-10 (IL-10) and interleukin-1 beta (IL-1ß), were identified to play a role during the course of the disease.

Equine herpesvirus type 1 induces both neurological and respiratory disease in Syrian hamsters.

The equine herpesvirus type 1 (EHV-1) is an important cause of myeloencephalopathy and respiratory disease in horses. Animal models for EHV-1 infection have been specially developed using mice and Syrian hamsters (Mesocricetus auratus). However, few studies have attempted to evaluate the pathogenesis of EHV-1 infection in the central nervous system (CNS) and respiratory system of hamsters. Therefore, the aim of this study was to evaluate the pathogenesis of four Brazilian EHV-1 strains within the CNS and lungs of Syrian hamsters. Hamsters intranasally infected with A4/72, A9/92, A3/97, and Iso/72 EHV-1 strains developed severe neurological and respiratory signs and died during acute EHV-1 infection within 3 to 5days post-inoculation. However, neurological signs were more severe in A4/72 and A9/92-infected hamsters, whereas respiratory signs were more prominent in A3/97 and Iso/72-infected hamsters. In the latter, lesions in the CNS were predominantly inflammatory, whereas in A4/72 and A9/92-infected hamsters, neuronal and liquefactive necrosis were the predominant lesions. EHV-1 infected hamsters also developed an interstitial pneumonia with infiltration of alveolar septa by macrophages, neutrophils, and lymphocytes, with the exception of A9/92-infected hamsters, which developed severe hemorrhages within the airways. EHV-1 antigens were detected along with CNS and pulmonary lesions. EHV-1 was also recovered from CNS of all infected hamsters, whereas the virus was recovered from the lungs of A4/72, A9/92, and Iso/72-infected hamsters. Brazilian EHV-1 strains caused both severe CNS and respiratory disease in hamsters, thus making this species an interesting model for EHV-1 infection in the CNS and respiratory system.

Long term stability and infectivity of herpesviruses in water.

For viruses to utilize environmental vectors (hard surfaces, soil, water) for transmission, physical and chemical stability is a prerequisite. There are many factors including pH, salinity, temperature, and turbidity that are known to contribute to the ability of viruses to persist in water. Equine herpesvirus type-1 (EHV-1) is a pathogenic alphaherpesvirus associated with domestic horses and wild equids. EHV-1 and recombinants of EHV-1 and EHV-9 are able to cause infections in non-equid animal species, particularly in captive settings. Many of the captive non-equid mammals are not naturally sympatric with equids and do not share enclosures, however, in many cases water sources may overlap. Similarly, in the wild, equids encounter many species at waterholes in times of seasonal drought. Therefore, we hypothesized that EHV-1 is stable in water and that water may act as a vector for EHV-1. In order to establish the conditions promoting or hindering EHV-1 longevity, infectivity and genomic stability in water; we exposed EHV-1 to varied water environments (pH, salinity, temperature, and turbidity) in controlled experiments over 21 days. The presence and infectivity of the virus was confirmed by both qPCR and cell culture experiments. Our results show that EHV-1 remains stable and infectious under many conditions in water for up to three weeks.

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells.

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.