Дисертації з теми "Equid herpesvirus"

Equid alphaherpesvirus 1 (EHV-1) is an economically important virus, associated with respiratory infection, late gestation abortion, neonatal death and myeloencephalopathy in horses. The aim of the present study was to test the hypothesis that EHV-1 is present in the nasopharynx and placentae of neonatal foals in the absence of clinical signs of infection. This would suggest that vertical transmission of virus occurs in inter-epizootic periods: such information could inform foaling management and the potential eradication of the virus by vaccination. Samples were collected from animals resident on a single farm in the Western Cape Province, South Africa, which had not experienced a clinical outbreak of EHV-1 recently. Sterile swab samples from 71 post-partum Thoroughbred mares, their healthy full-term foals and fetal membranes were obtained and assayed for EHV-1 and EHV-4 nucleic acid using a duplex quantitative polymerase chain reaction (qPCR). The null hypothesis for this study was that EHV-1 was not present in the nasopharynx and placentae of new-born, viable and healthy foals. As no EHV-1 or EHV-4 nucleic acid was detected on a duplex EHV-1/EHV-4 qPCR assay from the mare and foal nasal and fetal membrane swabs, the null hypothesis was accepted. It was therefore concluded that there was no detectable EHV-1 and -4 DNA in this population at the time of sampling. It was speculated that this may have been due to the cyclical nature of EHV-1 infections. The inclusion of additional breeding seasons on additional farms would be valuable for future studies.
Dissertation (MSc)--University of Pretoria, 2017.
Production Animal Studies
MSc

Os herpesvírus equinos do tipo 1 (EHV-1) e do tipo 4 (EHV-4) são considerados os principais agentes infecciosos para a espécie equina. Dentre as doenças causadas por estes agentes, destacam-se a rinopneumonite em animais jovens, o abortamento em fêmeas no terço final da gestação, a mortalidade perinatal em potros e a mieloencefalopatia. Estudos anteriores relatam ampla disseminação do EHV-1 na população eqüina no Estado de São Paulo, entretanto a ocorrência de infecção pelo EHV-4 não possui registro. Devido à similaridade antigênica entre os dois tipos virais, a diferenciação pelos métodos de sorodiagnóstico tradicionais, como a Soroneutralização e a Reação de Fixação de Complemento, não é possível. Assim, este trabalho avaliou, pela primeira vez no Estado de São Paulo, através de um teste de ELISA indireto que emprega uma região da glicoproteína G para diferenciar o EHV-1 do EHV-4 (iELISAgG),a presença de anticorpos específicos para os dois tipos de herpesvírus equino em 512 animais de 20 municípios de 8 mesoregiões do Estado de São Paulo, dentre equinos, muares e asininos, de ambos os sexos, diferentes faixas etárias, vacinados e não vacinados. As mesmas amostras foram testadas para o EHV através do teste de soroneutralização, tradicionalmente empregado para a pesquisa de anticorpos contra o vírus. Os resultados obtidos com a soroneutralização revelam 205/512 (40,03%) animais soropositivos. Através do teste de ELISA obteve-se 3/512 (0,59%) animais positivos para o EHV-1, 347/512 (67,77%) animais positivos para o EHV-4 e 108/512 (21,09%) animais positivos para ambos. O grupo de animais não vacinados apresentou 127/352 (36,07%) soropositivos pelo teste de soroneutralização; enquanto 4/352 (1,14%) foram positivos para o EHV-1, 237/352 (67,33%) foram positivos para o EHV-4 e 69/352 (19,6%) foram positivos para ambos, pelo teste de ELISA. O grupo de animais vacinados apresentou 78/160 (48,75%) soropositivos pelo teste de soroneutralização; enquanto 1/160 (0,63%) foram positivos para o EHV-1, 112/160 (70%) foram positivos para o EHV-4 e 37/160 (23,13%) foram positivos para ambos, pelo teste de ELISA. Os resultados sugerem baixa circulação de EHV-1 e alta circulação de EHV-4, de acordo com os resultados encontrados nos animais não vacinados. A análise de correlação entre os dois testes empregados mostrou baixa concordância.
The equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) are considered the major infectious agents for the equine species. Among the diseases caused by these agents, we highlight the rinopneumonite in young animals, abortion in females in the final third of pregnancy, perinatal mortality in foals and encefalopathy. Previous studies have reported wide spread of EHV-1 equine population in the State of São Paulo, however the occurrence of infection with EHV-4 is not registered. Due to the antigenic similarity between the two virus types, the differential serodiagnosis by traditional methods such as neutralization and complement fixation reaction, it is not possible. Thus, this study evaluated the first time in São Paulo, through an indirect ELISA employing a region of glycoprotein G to differentiate EHV-1 EHV-4 (iELISAgG), the presence of specific antibodies to the two types of equine herpesvirus in 512 animals from 20 municipalities in 8 regions the State of São Paulo, among horses, mules and donkeys of both sexes, different age groups, vaccinated and unvaccinated. The same samples were tested for EHV through the neutralization test, traditionally used for the detection of antibodies against the virus. The results obtained with the neutralization revealed 205/512 (40.03%) seropositive animals. By ELISA we obtained 3/512 (0.59%) animals positive for EHV-1, 347/512 (67.77%) animals positive for EHV-4 and 108/512 (21.09% ) animals positive for both. The group of unvaccinated animals showed 127/352 (36.07%) HIV-positive by serum neutralization test, while 4/352 (1.14%) were positive for EHV-1, 237/352 (67.33%) were positive for EHV-4 and 69/352 (19.6%) were positive for both ELISA. The group of vaccinated animals showed 78/160 (48.75%) seropositive by neutralization test, while 1 / 160 (0.63%) were positive for EHV-1, 112/160 (70%) were positive for EHV-4 and 37/160 (23.13%) were positive for both ELISA. The results suggest low circulation of EHV-1 and high circulation of EHV-4 according to the results found in unvaccinated animals. The correlation analysis between the two tests employed showed poor agreement

The project was undertaken to monitor a group of 30 foals on a farm both clinically and microbiologically from birth until one year of age, to determine the aetiology of upper respiratory tract infections and to establish immune profiles of some of the known respiratory viral pathogens. One to two months prior to the birth of their foals, blood for serology was collected from the mares. The same specimens were collected from the foals just after birth, prior to suckling and a day after suckling. Thereafter the foals were examined monthly for the presence of respiratory disease and specimens taken. The following specimens were collected from each foal: three nasopharyngeal swabs, (one for virus isolation, one for bacteria and fungus isolation, and one for mycoplasma isolation) and blood that was allowed to clot. Blood was collected in heparin from sick foals with elevated rectal temperatures. Virus isolation was done on roller tube cultures of equine embryonic lung (EEL), Vero cells and rabbit kidney 13 (RK13) cells. The bacteria (including mycoplasmas) and fungi were cultured from the swabs and identified using a variety of traditional methods. The serum neutralization test (SNT) was used to detect antibodies to equid herpesvirus 1 (EHV-1), equid herpesvirus 4 (EHV-4), equine rhinovirus 1 (ERV-1), equine rhinovirus 2 (ERV-2) and equine adenovirus 1 (EAdV-1). The complement fixation test (CFT) was used to detect antibodies to EHV-1 and EHV-4 and the haemagglutination inhibition test (HIT) antibodies to equine influenzavirus (EIV). Only EHV-4 was cultured from the nasopharyngeal swabs of nine foals when they were 5 to 6 months of age and from one foal two months later. A wide variety of bacteria and fungi were cultured and it was established that coagulase-negative staphylococci, viridans streptococci, Moraxella spp. and Flavobacterium spp. predominated in most of the samples. Several potential bacterial pathogens were isolated but the most common were Streptococcus equi subsp. zooepidemicus, Actinobacillus equuland Staphylococcus aureus. Colostrum-derived antibodies were detected for all the viruses in all but two of the foals. It was found that the foals had similar or slightly higher titres than their mothers. The levels declined in direct proportion to what they initially were and were depleted by the time the foals were 2 to 7 months of age. Antibodies to natural infection was detected to EHV-4, ERV-2 and EAdV-1. A rise in antibody titres occurred when the foals were 5 to 6 months of age, two months later and when they were one year of age. Antibodies resulting from immunization was detected to EHV-1, EHV-4 and EIV. It was established that the most important virus causing upper respiratory tract disease of the foals from 5 to 12 months of age was EHV-1 with EAdV-1 playing a minor role. These viruses caused repeated bouts of infection with a two to five months interval. Streptococcus equi subsp. zooepidemicus was considered to be the most important secondary pathogen. Prior to this period most of the foals were healthy with only a few suffering from upper respiratory disease. The aetiology was not determined in these cases, but based on the bacteriology results, it was suspected that some of them were suffering from bacterial infections.
Dissertation (MSc (Veterinary Science))--University of Pretoria, 2005.
Veterinary Tropical Diseases
unrestricted

Orientador: Clarice Weis Arns
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O herpesvírus eqüino do tipo 1 (EHV-1) é um membro da subfamília Alfaherpesvirinae, implicado no surgimento de distúrbios respiratórios, reprodutivos e nervosos em cavalos. A principal forma de contaminação dos animais é através do contato direto com secreções contaminadas pelo vírus. No eqüino, a disseminação do vírus ocorre pela transposição da infecção respiratória a outros órgãos e sistemas através da corrente sanguínea. Pouco se sabe sobre a ocorrência do EHV-1 no Brasil. Dessa forma, este estudo teve por objetivo o isolamento do EHV-1 a partir de material biológico e produção e análise de dados moleculares de isolados brasileiros de EHV-1. Durante este estudo, foi realizado o isolamento de uma amostra de EHV-1 a partir da inoculação de material clínico em células de derme eqüina (ED). Este isolado foi diagnosticado como EHV-1 através da reação em cadeia da polimerase (PCR) para o gene da timidina quinase (tk). Neste trabalho, foram também realizados os seqüenciamentos de fragmentos de PCR derivados do isolado aqui descrito, de uma outra amostra brasileira de EHV-1 e de duas amostras estrangeiras do vírus para análise filogenética. A análise comparativa entre seqüências permitiu inferências sobre o nível de divergência entre os vírus estudados, além da listagem de seqüências regulatórias para atividade gênica em um sítio do genoma localizado próximo ao gene tk. Na região genômica reportada foram contextualizadas ao menos três genes (ORF 38, ORF 37 e ORF 36). Os dados levantados com o seqüenciamento de amostras de EHV-1 de origens geográficas distintas (Brasil, Europa e América do Norte) não mostraram divergências, o que pode estar associado a um processo seletivo constritivo, que impediria a fixação de novas mutações naquela região. A ausência de divergências também pode estar associada à importância dessa região na regulação gênica do EHV-1. Também é um indicativo para a fidelidade dos mecanismos de replicação envolvidos na síntese do DNA viral, o que sugere a importância da região estudada na regulação da expressão gênica do EHV-1
Abstract: Equine Herpesvirus Type 1 (EHV-1) is a member of Alphaherpesvirinae subfamily implicated with abortions, respiratory and neurological disturbs in horses. The principal mode of viral transmission is through close contact virus-containing secretions of infected horses. Systemic pathogenesis in which this virus is implicated combines primary respiratory infection and spread of viral particles through the circulatory/lymphatic system. Until today, there are only few studies involving the isolation of this virus in Brazil. Thus, the main goal of this study was the isolation of EHV-1 from biological material and the production and analysis of molecular data derived from Brazilian EHV-1 isolates. Clinical samples were screened by inoculation into Equine Dermis (ED) cells monolayers, searching for the characteristic citopathic effect produced by EHV-1. Inoculation of one tissue sample has presented a suggestive citopathic effect. Re-inoculation of the original tissue homogenate in a second, independent experiment reproduced the same positive result. Following these observations, infection agent diagnostic was done by PCR for thymidine kinase (tk) gene. The results demonstrated that sample was EHV-1 positive. In this work, it was done either the sequencing of PCR fragments derived from two Brazilian and two foreign samples of EHV-1 for filogenetic and genomic analyses purposes. It was assigned at least three Open Reading Frames contexts (ORF 38, ORF 37, ORF 36). The data do not show genetic variation between sequences. The high level of genetic conservation for this region, despite the distinct geographic origins (Brazil, Europe and North America) of EHV-1 samples studied, indicates a strong selection process against the fixation of new mutations. It also highlights a high level of fidelity for DNA replication and strongly suggests the importance of the studied region for EHV-1 gene regulation
Doutorado
Microbiologia
Doutor em Genetica e Biologia Molecular

EHV-1 is a double-stranded DNA virus whereas EAV is a positive sense, single-stranded RNA virus. Therefore, genetically, they are very different from one another. However, both these viruses are endotheliotropic and thus, infect and replicates in equine endothelial cells resulting in vasculitis. Vasculitis is central to the pathogenesis of these two viruses. Thus, the main objective of this thesis was to investigate the inflammatory and innate immune responses of EECs that contribute towards the development of vasculitis following infection with EHV-1 and EAV in-vitro. Since proinflammatory cytokines and chemokines produced by endothelial cells play a significant role in the development of vasculitis, we investigated their gene expression as well as secretion. Results from this study showed that the proinflammatory response of EECs induced by EAV is relatively less when compared with the corresponding results from EHV-1 infected EECs. Furthermore, EAV elicits a lower type I interferon response in EECs when compared with EHV-1. Further investigations revealed an active role played by TLR 3 in inducing the proinflammatory response in EHV-1 infected EECs during the first 6 hours of infection but not in EAV infected EECs. Analyzing the whole transcriptome of EHV-1 and EAV infected EECs revealed a complex pattern of gene regulation and cellular pathways related to cellular immune, inflammatory and apoptotic responses. Finally, we investigated host genetic factors associated with EHV-1 induced myeloencephalopathy but found no evidence for a recessive allele influencing the development of EHM following EHV-1 infection for any genetic locus was identified. However, more complex host-pathogen interactions are possible.

Neuf herpèsvirus sont connus pour infecter les équidés. Parmi eux, l’herpèsvirus équin 1 (HVE-1) induit les formes de la maladie les plus graves. En effet, ce virus provoque des troubles respiratoires, des avortements, des morts néonatales et des troubles nerveux qui mènent souvent à l’euthanasie de l’animal. La prophylaxie, reposant sur les bonnes pratiques sanitaires et la vaccination, demeure le meilleur moyen de lutte contre l’ensemble des herpèsvirus équins. Des vaccins qui réduisent efficacement les troubles respiratoires et la dissémination de l’HVE-1 ont été développés. Cependant, ces derniers ne préviennent pas les avortements et n’ont aucun effet démontré contre la forme nerveuse. De plus, la couverture vaccinale demeure insuffisante en France. Les traitements aux antiviraux constituent donc une approche complémentaire dans la lutte contre l’HVE-1. Cependant, trop peu d’études ont évalué l’effet de molécules contre ce virus, limitant les perspectives d’utilisation. Pour répondre à cette problématique, nous avons développé un protocole de criblage à moyen/haut débit à l’aide de la technologie RTCA xCELLigence®, basée sur la mesure d’impédance cellulaire. Suite au criblage de 2891 molécules, 21 candidats ont été identifiés pour leur efficacité contre l’HVE-1. Parmi ceux-ci, l’aphidicoline, la décitabine, le ganciclovir, l’idoxuridine, le pritelivir et le valganciclovir ont présenté les meilleurs effets. L’activité de ces molécules a été confirmée sur différents modèles cellulaires en présence de différentes souches d’HVE-1. Cette étude a conduit à l’identification et à l’étude du mode d’action d’une nouvelle molécule efficace contre l’HVE-1, la décitabine. Cet analogue de la déoxycitidine a également montré un effet synergique in vitro lorsqu’elle est associée au valganciclovir. Lors de la seconde phase de ce travail, nous avons testé l’efficacité d’un traitement au valganciclovir lors d’un challenge expérimental avec infection par nébulisation d’une souche d’HVE-1 (C2254) récemment isolée au cours de l’épizootie de 2018. Cette étude a permis de démontrer qu’une dose de 6,5 mg/kg de valganciclovir, administrée 2 fois par jour, permettait de maintenir un bon niveau de protection avant la mise en place de la réponse immunitaire humorale. En effet, ce traitement permet de réduire les signes cliniques, l’excrétion virale et la virémie cellulaire induits par l’HVE-1. Ces travaux réalisés in vivo démontrent l’efficacité du valganciclovir contre l’HVE-1 et le criblage réalisé in vitro ouvre de nouvelles perspectives de traitement, en particulier avec des associations de molécules
Nine herpesviruses are known to infect the equine population. Among them, the equid herpesvirus 1 (EHV-1) induces the most severe forms of diseases. Indeed, this virus causes respiratory symptoms, abortions, neonatal foal deaths and nervous diseases, often leading to their euthanasia. Prophylaxis, relying on good sanitary practices and vaccination remains the best way to avoid epizooties of herpesviruses. Vaccines reducing efficiently respiratory disorders and EHV-1 dissemination are currently available. However, they do not prevent abortions and have no proven effect against nervous symptoms. In addition, the vaccine coverage is insufficient in France. Antiviral therapy is therefore an interesting complementary approach in the fight against EHV-1. However, there is a lack of studies evaluating the antiviral effect of compounds against EHV-1, limiting the prospects of use. To resolve this issue, we have developed a medium/high throughput screening protocol using the RTCA xCELLigence® technology based on cell impedance measurements. Following the screening of 2891 compounds, 21 candidates were identified for their efficacy against EHV-1. Among them, aphidicolin, decitabine, ganciclovir, idoxuridine, pritelivir and valganciclovir showed the best efficacy. The activity of these compounds was confirmed on different cell lines in the presence of different EHV-1 strains. This study led to the identification and the understanding of the mode of action of decitabine. This deoxycitidine analogue, also showed a synergistic effect when combined with valganciclovir. In the second part of this work, we evaluated the effect of valganciclovir treatment during an experimental infection by nebulisation with a new EHV-1 strain (C2254) recently isolated during the epizootic of 2018. This study demonstrated that a dose of 6.5 mg/kg body weight of valganciclovir, administrated orally twice a day, allowed to maintain a good protection prior the establishment of the humoral immune response. Indeed, this treatment allows to reduce significantly clinical signs, viral excretion and cell-associated viremia induced by EHV-1 on ponies. This work carried out in vivo demonstrated the efficiency of valganciclovir treatment against EHV-1, while the in vitro screening opens up new perspectives of treatment, in particular with compounds association

Les virus influenza équin (EIV) et herpèsvirus équin-1 (EHV-1) sont fréquemment décrits dans de nombreux pays et sont deux pathogènes endémiques au sein de la population équine française. Ces maladies infectieuses ont d’importantes conséquences aussi bien au niveau santé et bien-être animal qu’en terme d’impact économique. La lutte contre ces virus repose essentiellement sur la mise en place de mesures préventives telles que la vaccination. Malgré cela, les épizooties d’EIV et d’EHV-1 sont régulièrement observées en France et dans le monde. Les anticorps neutralisants, synthétisés en réponse à l’infection ou après immunisation, représentent une ligne de défense majeure. L’amélioration des vaccins et l’enrichissement du panel d’outils de mesure des anticorps neutralisants peuvent constituer un apport stratégique dans la lutte contre ces virus. Afin d’améliorer l’efficacité de la réponse vaccinale en amplitude et dans le temps, l’utilisation d’adjuvants constitue une des voies de recherche prometteuse. Ce travail de thèse a consisté, dans un premier temps, à établir la preuve de concept de l’utilisation de l’iPPVO en tant qu’adjuvant innovant in vivo chez le cheval dans le cadre de la vaccination contre l’EIV. Pour cela, les anticorps ont été mesurés par SRH, méthode pour laquelle des corrélats de protection sont associés au taux d’anticorps mesuré. L’ajout d’iPPVO lors de la vaccination a permis d’augmenter significativement le niveau d’anticorps contre l’EIV ainsi que le niveau de protection des chevaux jusqu’à 6 mois après l’immunisation. Dans un second temps, une méthode de mesure des anticorps anti-EIV faisant appel à l’impédancemétrie a été développée afin d’enrichir les méthodes actuelles et faciliter l’analyse à haut débit. Cette méthode de séroneutralisation a présenté une bonne corrélation avec les valeurs de titres SRH. Une seconde étude a testé, le potentiel adjuvant de l’iPPVO in vivo chez le cheval dans un modèle de vaccination contre l’EHV-1,4. La réponse en anticorps mesurée par séroneutralisation a été augmentée jusqu’à 5 mois suivant l’immunisation. Enfin, des résultats préliminaires sur le mécanisme d’action de l’iPPVO sur les cellules mononucléées du sang périphérique a permis de mettre en évidence l’importance de la réponse interféron
Equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1) are frequently described in many countries and are two endemic pathogens in the French equine population. These infectious diseases have important consequences both in terms of animal health and welfare and in terms of economic impact. The fight against these viruses is essentially based on the implementation of preventive measures such as vaccination. Despite this epizootics of EIV and EHV-1 are regularly declared in France and throughout the world. Neutralising antibodies, synthesised in response to infection or after immunisation, represent the main line of defence against these viruses. Improved vaccines and a wider range of tools to measure neutralising antibodies can be a valuable strategy in the fight against these viruses. In order to improve the efficacy of the vaccine response, both in magnitude and duration, the use of adjuvants is one way to improve immunogenicity. This thesis consisted, in the first instance, in establishing the proof of concept of the use of iPPVO as an innovative adjuvant in vivo in horses in the context of vaccination against EIV. For this purpose, antibodies were measured by SRH, a method for which the correlates of protection are well defined. The addition of iPPVO at vaccination significantly increased the antibody level to EIV and protection in horses up to 6 months after immunisation. In a second step, a new method for measuring EIV antibodies in serum based on impedancemetry was developed to improve on current methods and facilitate high throughput analysis. This neutralisation test correlated well with SRH test. Another study was performed, which demonstrated the adjuvant potential of iPPVO in horses during vaccination against EHV-1,4. The antibody response measured by serum neutralisation increased up to 5 months after immunisation. Finally, preliminary results on the mechanism of action of iPPVO on peripheral blood mononuclear cells demonstrated the importance of the interferon

This thesis describes the development, optimisation and use of assays to measure equine herpes virus-specific proliferative and cytotoxic Tlymphocyte (CTL) responses in the blood of horses. Equine T cell blast cells stimulated from peripheral blood mononuclear cells (PBMC) with pokeweed mitogen were found to perform best as targets for CTL in "Cr release assays. CTL induced in vitro with an abortigenic strain of EHV-1 (EHV-1/Ab4) were shown to be antigen specific, genetically restricted and predominantly of the CD4' CD8+ phenotype. Cross-reactive CTL were induced in vitro with live EHV-4 virus, which killed EHV-1 infected blast cells. A proportion of EHV-1 induced CTL were shown to be directed against the immediate early gene products. A proliferative LDA was used to determine whether the frequency of precursor T cells detected before challenge with EHV-1 correlated with immune status. The precursor frequency of antigen-specific T cells increased in 3 out of 4 horses after infection. However, there was no correlation between precursor frequency and outcome of infection. A LDA was developed and used to evaluate the precursor frequencies of EHV-1 and EHV-4 induced CTL after infection with these viruses. Pre-infection CTLp frequencies in susceptible animals were < 1/150,000. CTLp frequencies in animals which were immune to EHV-1 were between 1/10,000 and 1/20,000. To my knowledge this is the first report of the use of LDA techniques in the horse. The development and use of CTL LDA assays have provided new information on CTL responses in horses after EHV-1 and EHV-4 infection.