La fièvre aphteuse (FMD pour Foot-and-mouth disease en anglais) est une maladie très contagieuse touchant les animaux biongulés. Elle provoque des dégâts économiques considérables sur toute la surface du globe. La fièvre aphteuse est provoquée par un virus, le FMDV. Il s'agit d'un virus à ARN simple brin, de polarité positive appartenant au genre Aphtovirus dans la famille Picornaviridae. Ce virus se réplique et se propage dans l'hôte très rapidement. Dans les zones infectées, les deux principales stratégies de contrôle utilisées sont l'abattage systématique des animaux infectés et la vaccination. A l'inverse, les vaccins ne sont pas utilisés dans les zones sans FMDV, mais l'apparition d'une épidémie nécessite des stratégies pour arrêter ou au moins limiter la diffusion du virus. Actuellement, les vaccins inactivés sont les vaccins les plus utilisés pour prévenir la maladie. Cependant, ils requièrent une production à grande échelle du virus, et malgré les mesures mises en place (laboratoire sécurisé, etc), des épidémies ont été provoquées par le passé du fait de la fuite de virus FMDV. De plus, il est difficile de distinguer les animaux infectés des vaccinés (DIVA). Il est donc nécessaire de développer de nouveaux vaccins. Au cours de l'infection, la polyprotéine du virus est clivée par des protéases virales en précurseurs structural (P1) et non structuraux (P2 et P3). La protéase 3C est responsable de la majorité des clivages ; ainsi, le précurseur P1 est clivé par la 3C en trois protéines structurales, VP1, VP3 et VP0, formant le protomère de FMDV, l'unité de base de la capside virale. La protéine VP1 joue des rôles importants dans l'attachement, la protection et le sérotypage. Du fait de la présence d'un site antigénique linéaire suffisant à la protection par production d'anticorps neutralisants, VP1 est considérée comme la protéine la plus immunogénique du virus. Dans cette étude, nous avons développé un nouveau vaccin contre la FMD, basé sur l'adénovirus canin de type 2 (Cav2). L'évaluation du transfert de gène médié par Cav2 chez le porc et le bétail in vitro montre des résultats prometteurs pour le développement de vaccins pour ces espèces, notamment l'expression des antigènes de FMDV par les candidats vaccins Cav2-FMDV. L'immunogénicité de ces candidats vaccins a été montrée chez les modèles murins et cobayes. De plus, des résultats encourageants ont été observés chez le cobaye, suggérant que la réponse immunitaire élicitée par les vecteurs recombinants pouvait conduire à une protection partielle des animaux après épreuve. Cependant, une optimisation de l'immunisation doit être faite dans le but de confirmer ces résultats. Ce type de vaccin peut de plus être utilisé comme vaccin marqueur, car il ne contient aucune protéine non structurale. / Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. Foot-and-mouth disease virus (FMDV) is the causative agent of FMD and one of the most infectious known animal viruses. FMDV is a positive-sense, single-stranded RNA virus belonging to the Aphthovirus genus in the Picornaviridae family. FMDV replicates and spreads in the host extremely rapidly. Slaughter and vaccination are the two major strategies used to control FMD in infected countries. In FMDV-free countries, vaccines are not used, and once the disease breaks out in these areas, strategies are required to stop or at least slow the spread of the virus. Currently, inactivated vaccines are by far the most commonly used vaccines to prevent FMD. Such vaccines, however, require large-scale production of virus, and despite the use of bio-safety facilities, vaccine production has led to inadvertent virus release and FMDV outbreak. Another limitation of inactivated vaccines is the difficulty in distinguishing between infected and vaccinated animals (DIVA). Therefore, improved vaccines need to be developed.During infection, the FMDV polyprotein is cleaved into structural (P1) and non-structural (P2 and P3) precursors by a viral protease. The non-structural 3C protein is the protease that is responsible for most of the maturation events. The P1 precursor is processed by 3C protease into three structural proteins, VP1, VP3 and VP0, forming the FMDV protomer. The VP1 protein plays important roles in attachment, protective immunity and serotype specificity. VP1 is considered to be the major immunogenic protein, as it contains a linear antigenic site that is able to induce neutralizing antibodies that suffice to protect animals against the disease.In this project, we developed a novel vaccine against FMD, based on canine adenovirus type 2 (Cav2). In vitro evaluation of Cav2 mediated gene transfer in pigs and cattle showed that the Cav2 vector holds promise for the development of vaccines for pigs and cattle. Study of these recombinant viruses indicated that Cav2-FMDV supported expression of FMDV capsid proteins in vitro. The immunogenicity of these recombinant viruses was evidenced in mouse and guinea pig models, and encouraging results in guinea pigs suggested that the immune response elicited against FMD by recombinant virus could afford partial protection against FMDV challenge. In the future, immunization with Cav2-derived vector should be optimized to confirm these preliminary results. This type of vaccine, when designed to express capsid but not non-structural proteins of FMDV, can serve as a marker vaccine against FMD.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The respiratory viruses of dogs are associated with a disease called canine infectious respiratory disease (CIRD). The main etiological agents of CIRD are canine distemper virus (CDV), canine parainfluenza virus (cPIV), canine adenovirus type 2 and canid herpesvirus type 1 (CaHV-1), which may cause single or mixed infections. CIRD occurs most frequently in places with high animal density and constant movement. CDV, cPIV, CAdV-2 and CaHV-1 infections have been described worldwide, however, few reports of molecular identification of these viruses are available in Brazil. Thus, the objective of this study was to investigate the occurrence of respiratory viruses in dogs in Santa Maria, RS, and in dog shelters in RS, trying to correlate their occurrence with the environmental conditions. Nasal secretions were collected from dogs with respiratory signs submitted to veterinary clinics in Santa Maria; and from dogs of three shelters of RS (Cachoeira do Sul [shelters #1 and #2] and Passo Fundo [shelter #3]). Viral detection/identification was performed by polymerase chain reaction (PCR) for CDV, cPIV, CAdV-2 and CaHV-1. Positive samples were sequenced and, for some viruses, phylogenetic analysis was performed, comparing with sequences deposited in GenBank. Samples of shelters #1 and #3 were obtained during the cold season. Shelter #1 presented poor sanitary and nutrition conditions, high animal density and constant direct contact among dogs. In this shelter 78% (58/74) of the respiratory samples were positive for at least one virus. The single infections were caused by cPIV in 30% (22/74) of the samples and CAdV-2 in 5% (4/74). Coinfections represented 23% (cPIV and CAdV-2); 13% cPIV, CDV and CAdV-2; 4% cPIV-2 and CDV; and 3% CDV and CAdV-2. Shelters #2 and #3 presented satisfactory sanitary and nutrition conditions, with large outdoors exercise areas (#2) and animal separation by groups (#3). In shelter #2, 8% (5/35) of the samples were positive to cPIV and 6% to CaHV-1; in shelter #3, 8% (7/77) of the samples were positive to CAdV-2 and 1% to CDV. Of samples obtained in Santa Maria, 40% (10/25) were positive for virus, being 28% (7/25) for cPIV, and 4% (1/25) to each of the other viruses. Thus, the results obtained demonstrate that infections and coinfections by respiratory viruses are common in shelter dogs in RS, and their occurrence is related to population density, health and nutritional conditions and season. These viruses are also circulating in domestic dogs in Santa Maria, associated with respiratory disease. This study reinforces the importance of preventive measures such as vaccination and good environmental conditions to prevent/reduce infections caused by respiratory viruses in dogs. / Os vírus respiratórios de cães estão associados com uma enfermidade denominada doença respiratória infecciosa canina (canine infectious respiratory disease - CIRD). Os principais agentes da CIRD são o vírus da cinomose (canine distemper virus - CDV), vírus da parainfluenza canina tipo 2 (canine parainfluenza virus - cPIV), adenovírus canino tipo 2 (canine adenovirus type 2 - CAdV-2) e herpesvírus canino tipo 1 (canid herpesvirus 1 - CaHV-1), que podem causar infecções simples ou mistas. A CIRD ocorre com maior frequência em locais com alta densidade populacional e constante fluxo de animais. Infecções pelo CDV, cPIV, CAdV-2 e CaHV-1 tem sido descritas em vários países, contudo, são escassos os relatos da identificação molecular desses agentes no Brasil. Além disso, há falta de estudos relacionados aos fatores que favorecem a ocorrência e disseminação desses agentes em cães de abrigos. Desta forma, o objetivo do presente estudo foi investigar a ocorrência de vírus respiratórios em cães do município de Santa Maria, Rio Grande do Sul, e em cães de abrigos, buscando-se associar a ocorrência das infecções com as condições ambientais. Para isso, foram coletadas secreções nasais de cães com sinais respiratórios em clínicas veterinárias de Santa Maria; e de cães de três abrigos do estado do RS (dois em Cachoeira do Sul [#1 e #2] e um em Passo Fundo [#3]). A identificação viral foi realizada por reação em cadeia de polimerase (PCR) para o CDV, cPIV, CAdV-2 e CaHV-1. As amostras positivas foram sequenciadas e, para alguns vírus, foi realizada a análise filogenética, comparando-se com sequências depositadas no GenBank. As amostras dos abrigos #1 e #3 foram obtidas durante épocas de baixas temperaturas. O abrigo #1 apresentava condições sanitárias e nutricionais precárias, além de alta densidade populacional e constante contato entre os cães. Neste abrigo, 78% (58/74) das amostras foram positivas para, pelo menos, um dos vírus investigados. As infecções simples foram causadas pelo cPIV em 30% (22/74) das amostras e CAdV-2 em 5% (4/74). As coinfecções totalizaram 23% (17/74) para o cPIV e CAdV-2; 13% (10/74) para o cPIV, CDV e CAdV-2; 4% (3/74) para o cPIV e CDV; e 3% (2/74) para o CDV e CAdV-2. Os abrigos #2 e #3 eram higienizados corretamente e os cães recebiam alimentação adequada, sendo que no abrigo #2 os animais possuíam amplo espaço para se exercitarem, e no abrigo #3 os animais eram separados em grupos e alojados em gaiolas. No abrigo #2 foram detectadas 8% de amostras positivas para o cPIV e 6% para o CaHV-1; e no abrigo #3, 8% de amostras positivas para o CAdV-2 e 1% para o CDV. Das amostras obtidas em clínicas de Santa Maria, 40% (10/25) foram positivas para um dos vírus pesquisados, sendo 28% (7/25) para o cPIV, e 4% (1/25) para cada um dos outros vírus. Assim, os resultados obtidos demonstram que infecções e coinfecções por vírus respiratórios são comuns em cães de abrigos no estado do RS, estando relacionadas com a densidade populacional, condições sanitárias e nutricionais e estação do ano. Estes vírus também circulam em cães domésticos em Santa Maria, estando associados com doença respiratória. Este estudo reforça a importância de medidas de prevenção, tendo em vista que a vacinação e boas condições ambientais podem reduzir e/ou prevenir as infecções causadas por vírus respiratórios em cães.