Made available in DSpace on 2014-06-11T19:23:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-27Bitstream added on 2014-06-13T19:48:05Z : No. of bitstreams: 1 braga_prc_me_botfmvz.pdf: 1253627 bytes, checksum: 841018a463fea183c2e1b5ae5a1e9c0a (MD5) / Universidade Estadual Paulista (UNESP) / O presente estudo investigou a ocorrência de anticorpos contra o vírus da arterite eqüina, utilizando a técnica de soroneutralização viral em 1.400 eqüinos criados nas Mesorregiões Macro Metropolitana Paulista e Campinas, pertencentes a 42 municípios do estado de São Paulo (SP), Brasil entre os meses de janeiro de 2007 a dezembro de 2008. Do total das amostras, 80 (5,71%) apresentaram anticorpos para o vírus (títulos entre 4 e 4096). Dentre os 42 municípios amostrados, 15 (35,7%) apresentaram pelo menos um animal sororeagente. Foram analisadas 238 propriedades das quais 41 apresentaram ao menos um animal sororeagente. A ocorrência de animais reagentes foi maior em cavalos destinados ao esporte, particularmente das raças de Salto e Quarto de Milha e foi semelhante entre machos e fêmeas. A ocorrência também foi maior em animais acima de 24 meses de idade. Os resultados obtidos sugerem a circulação do vírus nos criatórios amostrados e alertam para o impacto econômico- sanitário da doença para a eqüideocultura do estado de São Paulo / This study investigate the occurence of arteritis virus antibodies using virus neutralization teste in 1,400 equines from Campinas and Macro Metropolitan Paulista Mesoregions which belong to the 42 cities located on State of Sao Paulo, Brazil between january of 2007 until december of 2008. All the samples, 80 (5.71%) showed antibodies to equine arteritis virus (titers ranging from 4 to 4096). Among the 42 cities, 15 (35.7%) presented at least one positive animal to equine arteritis virus. Were studied 238 farms, and from these 41 showed at least one soropositive animal. The prevalence was higher in sport horses like Jumping Horses, Quarter Horses and was similar between females and males. The seropositive ocurrence was higher in animals above 24 months of age. These results suggest the circulation of virus among horse population in farms sampled and alert to sanitary and economical importance of this disease for the Sao Paulo State equine husbandry

Resumo: O presente estudo investigou a ocorrência de anticorpos contra o vírus da arterite eqüina, utilizando a técnica de soroneutralização viral em 1.400 eqüinos criados nas Mesorregiões Macro Metropolitana Paulista e Campinas, pertencentes a 42 municípios do estado de São Paulo (SP), Brasil entre os meses de janeiro de 2007 a dezembro de 2008. Do total das amostras, 80 (5,71%) apresentaram anticorpos para o vírus (títulos entre 4 e 4096). Dentre os 42 municípios amostrados, 15 (35,7%) apresentaram pelo menos um animal sororeagente. Foram analisadas 238 propriedades das quais 41 apresentaram ao menos um animal sororeagente. A ocorrência de animais reagentes foi maior em cavalos destinados ao esporte, particularmente das raças de Salto e Quarto de Milha e foi semelhante entre machos e fêmeas. A ocorrência também foi maior em animais acima de 24 meses de idade. Os resultados obtidos sugerem a circulação do vírus nos criatórios amostrados e alertam para o impacto econômico- sanitário da doença para a eqüideocultura do estado de São Paulo / Abstract: This study investigate the occurence of arteritis virus antibodies using virus neutralization teste in 1,400 equines from Campinas and Macro Metropolitan Paulista Mesoregions which belong to the 42 cities located on State of Sao Paulo, Brazil between january of 2007 until december of 2008. All the samples, 80 (5.71%) showed antibodies to equine arteritis virus (titers ranging from 4 to 4096). Among the 42 cities, 15 (35.7%) presented at least one positive animal to equine arteritis virus. Were studied 238 farms, and from these 41 showed at least one soropositive animal. The prevalence was higher in sport horses like Jumping Horses, Quarter Horses and was similar between females and males. The seropositive ocurrence was higher in animals above 24 months of age. These results suggest the circulation of virus among horse population in farms sampled and alert to sanitary and economical importance of this disease for the Sao Paulo State equine husbandry / Orientador: Alexandre Secorun Borges / Coorientador: Márcio Garcia Ribeiro / Banca: Wilson Roberto Fernandes / Banca: João Pessoa Araújo Junior / Mestre

Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

A major goal of this research was to evaluate a variety of cell lines for theirpermissiveness to equine arteritis virus (EAV) infection and then identify the mechanismthat restricts EAV infection in certain cell lines. The cell lines BHK-21, RK-13, andC2C12 were found to support productive infection with EAV strain VBS53, whereasHela, Hep-2, and L-M cell lines exhibited limited susceptibility to infection with thisvirus. In the course of the study, it was found that the Hela cell line became moresusceptible to infection with EAV strain VBS53 after extended serial passage. Therespective cell lines were referred to as Hela High (passage 170-221) and Hela Low(passage 95-115) lines. While the Hela High cell line was more susceptible than the HelaLow cell line, it was still considerably less susceptible than the BHK-21 cell line to EAVinfection. Subsequent studies demonstrated that infection with EAV strain VBS53 wasrestricted at the entry step in Hela, Hep-2, and L-M cell lines.The second major goal of this research was to establish an in vitro model ofpersistent EAV infection using cell culture and then use the persistently infected culturesas a tool to study virus-host cell interactions, and to investigate virus and host cellevolution. Persistent infection was successfully established in the Hela High cell line withthe VBS53 strain of EAV. Properties of the persistently infected Hela High cell line werecharacterized. Virus evolution with respect to virus growth characteristics, ability of thevirus to initiate secondary persistent infection, and genetic changes during persistentEAV infection in Hela cells was investigated. Neutralization phenotypic changes of viruses were observed during the course of persistent EAV infection in Hela cells. Reverse genetics studies identified that amino acid 98 of the GP5 protein is a new neutralization determinant of EAV. Using an in vitro assay, it was found that EAV probably became progressively less virulent during the course of persistent infection in Hela cells. The potential changes in pathogenicity of EAV during persistent infection of Hela cells need to be verified by inoculation of horses.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV can cause abortion in pregnant mares and persistent infection in stallions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions. Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular biology techniques with dual color flow cytometric analysis to characterize the interactions of viral structural proteins and the equine peripheral blood mononuclear cells in vitro. Results from this study demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins of EAV play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. Secondly, a genome wide association study using SNP genotyping identified a common haplotype associated with the in vitro CD3+ T lymphocyte/resistance to EAV infection among four breeds of horses. Subsequently, these studies were extended to establish a possible correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Interestingly, carrier stallions with susceptible CD3+ T lymphocyte phenotype to EAV may represent those at higher risk of becoming persistently infected. Finally, the precise effect of EAV on the immune system of horses, innate and humoral immunity, was studied. Horses were shown to mount a strong humoral antibody response to nonstructural proteins (nsps) 2, 4, 5 and 12 of EAV, whereas nsps 1, 2 and 11 suppressed the type I interferon production. The data presented in this dissertation suggest new directions for future EAV research using genomic and proteomic approaches to study host cell factors involved in EAV attachment and entry and establishment of persistent infection in the stallions.

There is an urgent need for detection of viral respiratory pathogens to identify the causal agent(s) involved and to prevent the spread of related diseases. The first part of this dissertation focuses on development, optimization and validation of Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the detection of several common equine viral pathogens: equine arteritis virus (EAV), equine influenza virus and equine rhinitis viruses A and B. Emphasis of the second part of this dissertation is on studying the role of EAV envelope proteins in virus attachment and entry. Using an infectious cDNA clone of EAV and reverse genetics, a panel of chimeric viruses was generated by swapping the N-terminal ectodomains and full-lengths of the two major envelope proteins (GP5 and M) from porcine reproductive and respiratory syndrome virus (PRRSV). The recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 or M or both (GP5ecto, Mecto, and GP5&Mecto, respectively) in an EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a crippled phenotype. Interestingly, the three chimeric viruses could only infect EAV susceptible cell lines but not the PRRSV susceptible cell line. Therefore, the exchange of GP5 and/or M protein N-terminal ectodomains from PRRSV did not alter the cellular tropism of the chimeric viruses. We also investigated the role of one of the minor envelope proteins (E) of EAV in virus attachment and entry. The results showed that EAV infection of equine endothelial cells is heparin-dependent and the Cterminus of the E protein contains a putative heparin-binding domain. We generated a panel of arginine to glycine mutations in the conserved region of both the full-length EAV infectious cDNA clone and individual E protein expression vectors. The triple mutation R52,60,65G construct grew significantly slower and produced much smaller plaques. The double mutant R52,60G completely blocked the interaction between E protein and heparin. Taken together, these data indicated that E protein interacts with heparin to facilitate virus attachment and plays a major role in EAV infection.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a disease of equids. Following EAV infection, up to 70% of stallions may become carriers and continuously shed the virus in their semen for varying time periods. The long-term carrier stallion has an important role in the transmission and maintenance of EAV in horse populations. Recently, it has been demonstrated a correlation between in vitro susceptibility of CD3+ T lymphocytes to EAV infection and establishment of long-term persistent infection among stallions following natural infections. In this study, we investigated whether stallions with in vitro EAV susceptible CD3+ T lymphocytes are at higher risk of becoming long-term carriers compared to those with the resistant phenotype following experimental infection with the KY84 strain of EAV. Furthermore, we investigated whether there is a significant effect of EAV infection on semen quality during acute phase of the infection. The data suggested that the establishment of the long-term carrier state seems to be associated with the in vitro CD3+ T lymphocyte susceptible phenotypes and that reduced semen quality resulted from the combined effect of fever and scrotal edema observed following EAV infection rather than the direct effect of the virus.

The objectives of this thesis were: (1) to evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal (HPG) axis by a GnRH antagonist (acyline) based upon endocrine, seminal, testicular and behavioral effects, and (2) to assess recovery after treatment. Stallions were treated for 50 days (n=4; 330µg/kg acyline q 5d) and controls (n=4) received vehicle alone. Stallions were assessed pre-treatment and for 72 days after last treatment. Treatment induced declines (p<0.05) in FSH, LH, testosterone (to castrate levels) and estrone sulfate. Gonadotropins and testosterone returned to control values within nine days and estrone sulfate by 14 days after treatment discontinuation. Acyline-treated stallions failed to respond with FSH, LH and testosterone increase after exogenous GnRH stimulation (25µg gonadorelin, IV) compared to pre-treatment and control stimulation. Total sperm numbers and motility were reduced in acyline-treated stallions, as well as total seminal plasma protein and testicular volume (p<0.05). Time to ejaculation was increased in acyline group (p<0.5). Testicular, sexual behavior and most seminal parameters regained normal levels within 72 days after treatment ceased. Sperm output of acyline-treated stallions was regained within seven months after ending treatment. Acyline reversibly suppressed the stallion HPG axis, thus has potential for treating the androgen-dependent Equine-Arteritis-Virus carrier state and as behavior modulator.

Le virus de l’Artérite Virale Équine (EAV) est un virus à ARN simple brin positif, appartenant à la famille des Arteriviridae, dans l’ordre des Nidovirales. C’est un virus spécifique des équidés qui peut être transmis par voies respiratoire et vénérienne. Lors de la primo-infection, l’EAV peut entraîner des signes cliniques grippaux, mais de façon plus grave, il peut aussi provoquer l’avortement des juments gestantes ainsi que la mort des nouveau-nés. L’EAV représente donc un enjeu économique majeur pour la filière équine. Suite à la primo-infection, ce virus peut persister dans de l’appareil reproducteur de certains étalons. Les mécanismes de cette persistance ne sont pas connus.Au cours de cette thèse, le premier modèle in vitro d’infection non lytique d’une lignée issue de l’appareil reproducteur mâle par l’EAV a été développé. L’infection de ces cellules de Leydig a montré une induction de l’expression de nombreux gènes de l’immunité innée dont ceux codant pour des cytokines pro-inflammatoires et des chimiokines qui permettraient le recrutement de cellules de l’immunité innée au niveau des testicules, et qui pourraient expliquer l’orchite observée chez certains étalons lors de la phase aiguë de l’infection. Pour les étalons infectés de façon persistante, la castration et les traitements anti-GnRH peuvent permettre la suppression de la persistance du virus, suggérant ainsi une implication de la testostérone dans la persistance du virus. Les cellules TM3 exprimant le récepteur aux androgènes, des essais de traitements ont été réalisés. Les premiers résultats préliminaires semblent indiquer que les cellules TM3 ne répondent pas ou peu au stimulus hormonal. Cependant, des tests de prétraitement par la testostérone seraient à envisager afin d’en étudier les conséquences sur le cycle viral. Ce modèle d’infection non lytique reste cependant un modèle intéressant pouvant être utilisé afin d’étudier les relations hôte-pathogène et pouvant aider à comprendre les mécanismes impliqués dans la persistance de l’EAV. / Equine Arteritis Virus (EAV) is a positive-strand RNA virus, which belongs to the Arteriviridae familly, in the Nidovirales order. It is an equid specific virus that can be transmitted by respiratory and venereal routes. During primary infection, EAV can induce flu-like clinical signs, but worse, it may also cause the abortion of pregnant mares and newborn foal death. EAV is therefore a main economic challenge for the horse industry. Following primary infection, this virus is able to persist in the reproductive tract of some stallions. The mechanisms of this persistence remain unknown.During this thesis, the first in vitro model of an EAV non-lytic infection of a male reproductive tract cell line has been developed. EAV infection of these Leydig cells induced the expression of numerous innate immune genes including those coding for pro-inflammatory cytokines and chemokines, which could recruit innate immune cells to testicles and which could explain the orchitis observed in some stallions during primary infection.For persistently infected stallions, castration and anti-GnRH treatments can suppress EAV persistence, suggesting an involvement of testosterone in the virus persistence. Since TM3 cells express the androgen receptor, treatment trials have been performed. The first preliminary results suggest TM3 cells do not respond to the hormonal stimulus, or only a little. However, pretreatment trials should be realized to study the consequences on the viral cycle.Nevertheless, this non-lytic infection model is still an interesting model that can be used to study the host-pathogen relationship and that could help understanding the mechanisms involved in EAV persistence.

Equine arteritis virus (EAV) has a global impact on the equine industry being the causative agent of equine viral arteritis (EVA), a reproductive, respiratory, and systemic disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in the reproductive tract of stallions and is continuously shed in the semen (carrier state). Recent studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S). However, the cellular and molecular mechanisms underlying the establishment and maintenance of persistent infection are yet to be determined. The studies were undertaken herein unequivocally demonstrated that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes) and that EAV has specific tropism for stromal cells and CD8+ T and CD21+ B lymphocytes but not glandular epithelium in the reproductive tract. Furthermore, persistent EAV infection is associated with a significant humoral, mucosal antibody and inflammatory response at the site of persistence, characterized by induction of high levels of neutralizing antibodies (IgG1), mucosal anti-EAV-specific IgA, IgG1, IgG3/5, and IgG4/7 with variable neutralizing efficacy; and moderate, multifocal lymphoplasmacytic ampullitis, with significant infiltration of T lymphocytes (mainly CD8+ and low numbers of FOXP3+ lymphocytes), CD21+ B lymphocytes, diverse Ig-secreting plasma cells, and Iba-1+ and CD83+ tissue macrophages/dendritic cells. Moreover, EAV long-term persistent infection is associated with a CD8+ T lymphocyte transcriptional profile with upregulation of T-cell exhaustion-related transcripts and homing chemokines/chemokine receptors (CXCL9-11/CXCR3 and CXCL16/CXCR6), orchestrated by a specific subset of transcription factors (EOMES, PRDM1, BATF, NFATC2, STAT1, IRF1, TBX21), which are associated with the presence of the susceptibility allele (CXCL16S). Finally, these studies have determined that long-term EAV persistence is associated with the downregulation of a specific seminal exosome-associated miRNA (eca-mir-128) along with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. These findings provide evidence that this miRNA plays a crucial role in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. The findings presented herein suggest that complex host-pathogen interactions shape the outcome of EAV infection in the stallion and that EAV employs complex immune evasion mechanisms favoring persistence in the reproductive tract. Further studies to identify specific mechanisms mediating the modulation of the CXCL16/CXCR6 axis and viral immune evasion in the reproductive tract of the EAV long-term carrier stallion are warranted.