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Le Syndrome Dysgénésique et Respiratoire Porcin (PRRS) est la maladie infectieuse endémique la plus couteuse en élevage porcin dont l'agent responsable est un Arterivirus, le PRRSV, qui présente une grande diversité génétique. L'infection par le PRRSV est fréquemment associée à l'infection par les virus influenza. La vaccination est une méthode de lutte adaptée contre ces virus. Dans le cas du PRRSV, les vaccins les plus utilisés sont des virus vivants modifiés (MLV) qui induisent une immunité protectrice peu efficace contre les variants viraux. Dans le cas du virus influenza, les vaccins inactivés utilisés présentent la même insuffisance.Dans ce travail de thèse, j'ai évalué des stratégies vaccinales visant à induire une immunité efficace contre des variants viraux, en utilisant des antigènes conservés entre souches, adressés aux cellules présentatrices d'antigènes (APC), et j'ai analysé l'effet de différentes voies et modes d'administration.Dans le cas du virus grippal, le ciblage d'antigènes conservés (HA2, M2e, NP) au CD11c a permis d'augmenter la réponse T uniquement lors d'administration par voie intramusculaire (IM) et fut sans effet sur la réponse anticorps. La vaccination par voie intradermique s'est traduit par une exacerbation de la pathologie lors d'une épreuve virale, alors que la vaccination par voie IM a réduit les symptômes, la durée d'excrétion virale en corrélation avec une meilleure réponse anticorps anti-HA2 et M2e.Dans le cas du virus PRRSV qui fut mon sujet principal d'étude, j'ai cherché à optimiser des réponses lymphocytaires T IFNγ en employant une stratégie vaccinale ADN codant des antigènes contenant des épitopes T conservés entre souches, ciblés aux APC. En effet, alors que les mutations virales conduisent à un échappement aux anticorps neutralisants, la réponse lymphocytaire T IFNγ a été proposée impliquée dans la protection croisée. J'ai montré que l'immunogénicité optimale de vaccins ADN PRRSV, conduisant à la réponse T la plus large, est obtenue par l'administration intradermique associée aux nanoparticules de PLGA (NP), suivi d'une électroporation (EP), par rapport à EP seul ou délivrance intradermique ou transcutanée avec des patches à micro-aiguilles résorbables. Cette immunogénicité optimale est associée à une bonne transfection des cellules de la peau, à une accumulation de cellules inflammatoires, et à une mobilisation des cellules dendritiques. J'ai ensuite utilisé ce mode d'administration EP+NP pour immuniser des porcs avec des plasmides codant des antigènes conservés du PRRSV adressés ou non aux APC via CD11c ou XCR1. Les porcs ont été immunisés soit avec des injections répétées d'ADN seul soit en prime-boost ADN-MLV. Le régime ADN-MLV s'est montré supérieur pour l'induction de réponse B et T à celui de l'ADN ou du MLV seuls, et le ciblage aux APC a nettement augmenté la réponse anticorps mais pas la réponse T IFNγ. Dans une expérience suivante à visée d'application sur le terrain, j'ai utilisé le régime ADN-MLV (sans NP cette fois), délivré avec EP ou avec jet sous pression (PJ). Dans ces conditions, la primo-vaccination avec ADN n'a pas significativement augmenté la réponse T IFNγ induite par le MLV, mais elle a clairement augmenté la réponse anticorps avec un bénéfice du ciblage des APC. L'immuno-potentialisation induite par la primo-vaccination ADN n'a pas conduit à l'amélioration de la protection contre une épreuve avec un virus hétérologue et a montré que cette protection n'est au final pas corrélée avec la réponse lymphocytaire T IFNγ et opère en l'absence d'anticorps neutralisants détectables. Enfin, l'ensemble de ce travail montre que l'effet du ciblage des APC chez le porc est influencé par la voie d'administration et par le régime d'administration comme le prime-boost ADN-MLV. / The Porcine Reproductive and Respiratory Syndrome (PRRS) is the most damaging infectious disease in pigs worldwide. The etiologic agent is an Arterivirus, the PRRSV, which presents a large genetic diversity. PRRSV infection is frequently associated with influenza virus co-infection. Vaccination is a highly suitable way to control these viruses. In the case of PRRSV, the most effective commercial vaccines are modified live vaccines (MLV) which induce only a partial protection against heterologous strains. In the case of the influenza virus, the available inactivated vaccines show the same weakness.With the goal to control emerging influenza and PRRSV variants, I evaluated vaccine strategies involving conserved viral antigens between strains which were targeted to antigen-presenting cells (APC) and delivered by different routes and methods.In the case of influenza virus, the targeting of conserved antigens (HA2, M2e and NP) to CD11c led to increased IFNγ T cell responses only when vaccines were delivered by the intramuscular (IM) route and had no effect on the humoral response. The intradermal route exacerbated disease following challenge whereas the IM route reduced the symptoms, the duration of viral excretion in correlation with higher anti-HA2 and anti-M2e antibody responses.In the case of PRRSV, which was my main subject, I sought to optimize the IFNγ T cell responses by using DNA vaccines encoding antigens with conserved T-epitopes between strains, and targeted to APC. Indeed, whereas viral mutants escape neutralizing antibodies, it has been proposed that the IFNγ T cell responses are instrumental for cross-protection. I showed that the broadest T cell responses were induced by DNA vaccines combined to nanoparticles PLGA (NP) injected by the intradermal route, followed by electroporation (EP) compared with EP-only, intradermal route-only or transcutaneous dissolvable microneedles. This optimal immunogenicity was associated with a high transfection level of skin cells, an accumulation of inflammatory cells, and dendritic cells mobilisation. Next I used the EP+NP method to immunize pigs with plasmids encoding conserved PRRSV antigens targeted or not to APC via CD11c or XCR1. Pigs were immunized either with repeated injections of DNA alone or with a prime-boost DNA-MLV. The DNA-MLV regimen induced improved humoral and IFNγ T cell responses compared to DNA alone or MLV alone and the APC-targeting significantly increased the humoral response but not the IFNγ T cell response. Finally, I evaluated the DNA-MLV regimen efficacy, with an applied perspective, using naked DNA without NP and delivered by EP or by a convenient needle free injection technology (PJ). In these conditions, the DNA prime did not significantly increase the IFNγ T cell response induced by the MLV, but clearly increased the humoral response with a benefit of the APC-targeting. However, the immune potentiation induced by the DNA prime did not lead to an improved protection following a heterologous challenge. The heterologous protection was not correlated to the measured humoral and IFNγ T cell responses, and neutralizing antibodies were undetectable. Thus cross-protective effectors have not been sufficiently activated by our DNA-MLV strategy and the immune correlates of protection against heterologous PRRSV are still to be identified to develop cross-protective vaccines. Finally, this work shows that the effect of APC-targeting in pigs is influenced by delivery routes and methods and by vaccine regimen such as the prime-boost DNA-MLV.

Background: In this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the sameherd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs. Results: At the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N. Conclusions: Only one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated.

An investigation of the distribution of porcine reproduction and respiratory syndrome virus (PRRSV) and factors associated with the presence of PRRSV in Ontario from 2004 – 2007 was conducted. Surveys on the presence of clinical signs associated with PRRS, management practices, animal suppliers, and herd location were administered to the managers of 458 PRRSV positive herds and 61 PRRSV negative herds. Open reading frame (ORF) 5 of the PRRSV genome was sequenced from herds with PRRSV. PRRSV positive herds were compared to PRRSV negative herds. Management practices associated with being PRRSV positive were: not washing animal- and feed-delivery vehicles, feed-delivery and animal-transport vehicles visiting multiple herds at one time, allowing a truck driver to enter the barn, not requiring visitors to shower prior to farm entry, and not utilizing all-in all-out flow in gilt and finisher barns. Specific PRRSV restriction fragment length polymorphism (RFLP) genotypes of the ORF5 gene were compared with clinical signs. Herds with RFLP type ‘1-undetermined-4’, ‘1-undetermined-2’ and 1-3-4 were associated with clinical signs in sows and 2-6-2 was associated with finisher mortality compared to herds with vaccine virus. Additionally, genotypes 1-3-4 and 1-8-4 increased in frequency during this study. The between-herd PRRSV similarity of genome and clinical signs were compared. Abortions and stillbirths were associated with similarity in genetic sequences between herds. This relationship did not extend to those herds where vaccine virus was identified. Patterns in space and time of herds with different RFLP types of PRRSV were investigated after accounting for ownership. There was weak evidence to suggest local spread the genotype 1-3-4. The association between genetic similarity and proximity in space, time, ownership, animal, and semen suppliers was tested. Significant correlation was detected for distances up to 30 km. After controlling for ownership, only small associations between breeding stock and semen suppliers and genetic similarity of PRRSV were found. The spread of PRRSV among herds in Ontario cannot be attributed to any one factor. However, similarity in ownership between herds was a key variable indicating that movement of animals, personnel, and vehicles among herds must be measured in future investigations of PRRSV dynamics. / Ontario Pork and the Canada-Ontario Research and Development (CORD) Program and the OMAFRA/University of Guelph agreement

Respiratorische Krankheitsbilder, an denen mehr als ein Pathogen ursächlich beteiligt ist, gewinnen in der Schweinepopulation zunehmend an Bedeutung. Diagnostische Methoden zum simultanen Nachweis mehrerer Erreger sind Bestandteil einer schnellen und effizienten Therapie und tragen zum ökonomischen Bestandsmanagement bei. Im Rahmen dieser Arbeit sollten Methoden für den Multiplex-Nachweis von Antikörpern und Nukleinsäuren viraler Erreger von respiratorischen Krankheitsgeschehen des Schweins entwickelt werden. Die Methode für den Multiplex-Nachweis von Antikörpern sollte auf Basis der xMAP® Flüssigchip-Technologie (Luminex Corporation, Austin, T, USA) an der LiquiChip®- Workstation (Qiagen, Hilden, D) etabliert werden. Da es sich um eine für den Antikörpernachweis im veterinärmedizinischen Bereich bislang nicht genutzte Methode handelte, erfolgte die Prüfung der Machbarkeit zunächst im Einfach-Format am Beispiel des Porzinen Circovirus Typ 2. Im Laufe der Arbeit wurde deutlich, daß die Kopplung des PCV2 ORF2-Proteins als Capture-Molekül sowie die Erstellung der Versuchsansätze mit akzeptablem Aufwand ohne Spezialtechniken durchführbar war. Aufgrund der Anordnung der Proben auf Platten im 96-well-Format und der vollautomatischen Messung war ein hoher Probendurchsatz möglich. Nach der Einführung von Waschschritten in die Versuchsansätze konnten hohe Fluoreszenzsignale erzeugt werden. Im Laufe der Optimierungsversuche wurde allerdings die fehlende Korrelation dieser Fluoreszenzsignale mit den Ergebnissen der Referenzmethode deutlich. Aufgrund der unbekannten Testeigenschaften sowie fehlender Kontrollmöglichkeiten wurden diese nicht sogleich als unspezifische bzw. falsch positive Signale erkannt. Erst durch die Testung von positiven und negativen Feldseren an verschiedensten Bead-Arten wurde ersichtlich, daß die Fluoreszenzen nicht ausschließlich durch die spezifische Bindung der PCV2-Antikörper an das Capture-Protein entstanden. Im Ausschlussverfahren konnte die Ursache eingegrenzt werden. Bestandteile aus dem Schweineserum führten vermutlich durch unspezifische Bindungen an die LiquiChip®-Beads zu einem Fluoreszenzereignis. Durch Vorinkubation der Beads in Pferdeserum und der Feldseren mit einem Block-Puffer wurde versucht, diese Serumbestandteile abzusättigen und so eine Bindung an die Beads zu verhindern. Die Inkubationsvarianten führten weder zu einer Minimierung der unspezifischen Bindung noch zu einer verbesserten Differenzierung PCV2-positiver und negativer Seren. Die in der vorliegenden Arbeit verwendeten Bead-Arten sind für den Nachweis von Antikörpern gegen das PCV2 ORF2-Protein nicht geeignet. Alternative Bead-Arten für einen vergleichbaren Versuchsansatz stehen derzeit nicht zur Verfügung. Ein weiteres Ziel der Arbeit bestand darin, eine bereits in der Diagnostik von Schweineviren etablierte Methode, die PCR, zu einer Multiplex-PCR zu erweitern. Als zu detektierende Parameter wurden die derzeit bedeutendsten viralen Erreger von respiratorischen Erkrankungen des Schweins, das PRRS-Virus (Typ 1 und Typ 2), das Porzine Influenzavirus mit den Subtypen H1N1, H3N2 und H1N2 und PCV2 gewählt. Es wurden die Primersequenzen von bereits etablierten Einfach-PCRs an die besonderen Ansprüche einer Multiplex-PCR angepasst und die Methode zunächst im Einzelansatz auf Funktionsfähigkeit überprüft. Im Anschluss wurden die Parameter zu einer Multiplex-PCR zusammengeführt, die Methode optimiert und auf Spezifität, Sensitivität und Verhalten in der Routinediagnostik überprüft. Aufgrund der im Gegensatz zur Einfach-PCR zum Teil herabgesetzten Sensitivität ist diese Methode für Ausschlussuntersuchungen weniger geeignet. Für die Untersuchung von Probenmaterial klinisch erkrankter Tiere ist sie jedoch gut geeignet und bietet die Möglichkeit einen schnellen Überblick über das Erregerspektrum zu erhalten. Es muss jedoch berücksichtigt werden, daß bestimmte Parameter, z.B. PCV2, die Sensitivität des Nachweises der anderen Parameter sehr deutlich herabsetzen kann. Dies ist insbesondere von Bedeutung, da PCV2-DNA in Probenmaterial von klinisch erkrankten Tieren in sehr hohen Mengen vorhanden ist und dadurch die weiteren Parameter noch zusätzlich beeinflusst werden können.

Le virus du syndrome dysgénésique et respiratoire porcin (SDRPv) est un pathogène à l’origine de problèmes respiratoires et de reproduction. La réponse immunitaire face au SDRPv est caractérisée par une virémie persistante et un retard dans la mise en place des anticorps neutralisants. Les macrophages alvéolaires (AM) sont la cible principale du virus mais plusieurs études in vitro suggèrent une infection des cellules dendritiques (DC). Dans ce manuscrit, de nouvelles cibles cellulaires du virus ont été découvertes dans le poumon et dans les ganglions trachéo-bronchiques. Les cellules du parenchyme pulmonaire AM-like sont infectés in vivo par le SDRPv de Type 1 alors que les cDC1, cDC2 et moDC du poumon ne sont pas infectées. La souche Lena Type 1.3 se distingue des autres souches par sa capacité à induire une polarisation vers une réponse Th1 in vitro. Une expérience de transfert de lymphocytes T mémoires in vivo suggèrent une réponse cellulaire accrue et délétère lors de l’infection par la souche Lena. Trois populations de macrophages ont été identifiées dans les ganglions et nommées : macrophages périfolliculaires (PFMacro), macrophages des cordons lymphatiques (cordMacro) et macrophages efférents (effMacro). Les effMacro et les PFMacro sont infectés par les souches européennes. Quant à la réponse humorale, 5 stades de différenciation des LB dans les ganglions ont été identifiés et une étude de l’impact de l’infection sur la différenciation des lymphocytes B est en cours. / Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded RNA virus that causes reproductive failure and respiratory problems in swine. Immune response against PRRSV is characterized by a persistent viremia and a delay in neutralizing antibodies’ production. Main targets of PRRSV are Alveolar Macrophages (AM) but in vitro studies suggest that PRRSV could replicate in dendritic cells (DC). In this manuscript, new cellular targets of PRRSV in the lung and the tracheo-bronchial lymph nodes have been depicted. This work revealed that pulmonary parenchymal AM-like are susceptible to PRRSV in vivo. Moreover, study of DC infection in the lung reveals that cDC1, cDC2 and moDC are not infected by Type 1 PRRSV. In tracheo-bronchial lymph nodes, 3 populations of pig macrophages (Macro) have been identified and called perifollicular macrophages (PFMacro), lymphatic cord macrophages (cordMacro) and efferent macrophages (effMacro). In vivo infection revealed that PFMacro and effMacro are susceptible to Type 1 PRRSV infection. In order to understand the delay in humoral response establishment, 5 populations of B cells have been identified in the lymph nodes. The interaction of SDRPv with these populations is under study.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important swine pathogen. As the emergences of novel virulent strains of PRRSV continue to occur worldwide, rapid vaccine development is the key for effective control of ongoing PRRSV outbreaks. With the availability of the PRRSV reverse genetics systems, rapid vaccine development against PRRSV through the manipulation of the reverse genetics becomes feasible. To facilitate the vaccine development effort and study of PRRSV genes, we first established a DNA-launched infectious clone of the passage 14 PRRSV strain VR2385, pIR-VR2385-CA, and identified a spontaneous 435-bp deletion in the nsp2 gene. To characterize the biological and pathological significance of this nsp2 deletion, we restored deleted nsp2 sequence back to pIR-VR2385-CA and constructed another clone pIR-VR2385-R. VR2385-CA and VR2385-R were successfully rescued in vitro. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no significant effect on the pathogenicity of the virus. With the availability of the DNA-launched infectious clone of PRRSV, we successfully applied the molecular breeding approach to rapidly attenuate PRRSV. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred. DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were successfully rescued in vitro and shown to be attenuated both in vitro and in vivo. Furthermore, DS722, but not DS5M3, still elicit similar protection against PRRSV challenge as its parental virus. This study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positive-strand RNA virus. We subsequently utilized the novel synthetic attenuated virus engineering (SAVE) approach to attenuate PRRSV. The GP5 and nsp9 genes of PRRSV were codon-pair deoptimized with the aid of a computer algorithm. SAVE5 and SAVE9 with deoptimized GP5 gene and SAVE9 with deoptimized nsp9 gene, were successfully rescued and shown to be attenuated in vitro. An in vivo pathogenicity study indicated the attenuation of SAVE5 virus in vivo. The results have important implications for rapid vaccine development against PRRSV and other important viruses. / Ph. D.

Since emerging in Europe and the US, PRRS has spread globally and become the most significant infectious disease currently devastating the swine industry. In the US alone, the economic losses caused by this disease amount to more than 560 million US dollars every year. Modified-live PRRSV vaccines (MLV) are the most effective option currently available for the control of the disease. MLVs can confer solid protection against homologous re-infection and have significant effects in reducing viral shedding. But the vaccine efficacy varies upon heterologous challenge. None of the current vaccines are able to completely prevent respiratory infection, transplacental transmission, as well as pig-to-pig transmission of the virus. More importantly, the intrinsic risk of MLV vaccine to revert to virulent virus under farm conditions poses a great safety concern. The unsatisfactory efficacy and safety of current PRRSV vaccines drives the continuous efforts of developing a new generation of vaccines. The strategy we focus on for novel PRRSV vaccine development is subunit vaccine. The reasons for choosing this strategy are: 1) subunit vaccines only contain the immunogenic fragments of a pathogen. Administration of such pathogen fragments eliminates the risk of pathogens reverting back to their virulent form as in the case of modified live vaccines. 2) Subunit vaccines have advantages in terms of vaccine production since a well-defined pathogen fragment can more easily be produced consistently. To achieve of our goal of developing safe and efficacious subunit vaccines against PRRSV, three projects were completed. First, a scalable process for purification of PRRSV particles from cell culture was developed. This process produced purified viral particles for ELISA and cell-based assays used in vaccine development. Second, a plant-made oral subunit vaccine against PRRSV was developed. Administration of the plant-made vaccine, the vaccinated animals produced virus-specific serum and intestine mucosal antibodies with neutralization activity, as well as cellular immune responses with a preference of virus-specific IFN-γ production. Since neutralization antibodies and virus-specific IFN-γ response are the crucial factors contributing to protection against PRRSV infection, the plant-made oral subunit vaccine strategy is an attractive strategy for developing a new generation of the vaccine to control PRRS disease. Third, a chimeric protein consisting of the ectodomains of viral M and GP5 proteins was expressed and purified. The protein product showed a single band on a silver-stained gel and contained an endotoxin level of less than 10 EU/mg protein. In addition, the purified protein showed expected bioactivities. It was antigenic, could bind to a cellular receptor for the virus (heparan sulfate), and could block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV. / Ph. D.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated for the first time in 1991 and since then it has been associated with significant economic losses to the pig industry worldwide. Although vaccination against PRRSV is widely used, an important advance would be the development of marker vaccines allowing serologic discrimination between vaccinated and naturally infected animals. The present study aimed to identify immunogenic and conserved regions dispensable to viral replication in different PRRSV proteins, which could be used as negative serologic markers in a new generation of liveattenuated vaccines. A fine mapping of B-cell linear epitopes in different PRRSV proteins by Pepscan is presented in the first part of this thesis. The results indicated the presence of several B-cell linear epitopes in the non-structural protein 2 (Nsp2) and in all structural proteins encoded by PRRSV, which were consistently recognized by antibodies raised in pigs experimentally infected with a North American strain of the virus (NVSL97-7895). The Nsp2 was found to harbor the highest frequency of immunodominant epitopes (n=18) when compared to structural proteins. In the structural proteins, epitopes consistently recognized by immune sera were located in all studied proteins. Overall, the highest degree of immunogenicity and conservation was exhibited by two epitopes identified in the C-terminal end of the M protein (ORF6). The antibodies recognizing the immunodominant epitopes of each protein were detected as early as days 7 to 15 post-infection (p.i.) and remained detectable until the end of the experiment (day 90 p.i). Based on their immunodominance and level of amino acid (aa) conservation, two target epitopes were selected to serve as serological marker candidates in each of the following PRRSV proteins: Nsp2, GP3 and M. These epitopes were deleted in the wild-type cDNA infectious clone (FL-12) by site-directed mutagenesis. The results of this study are presented in the second part of this thesis. A Nsp2 mutant virus (FLdNsp2/44) was successfully rescued following RNA transfection in MARC 145 cells. This epitope deletion mutant fulfilled the requirements for a differential vaccine virus such as efficient growth in vitro and in vivo and induction of active seroconversion as measured by a commercial ELISA kit associated with the absence of a marker-specific peptide-ELISA response in 100% (n=15) of the vaccinated animals. In vitro and in vivo characterization of the mutant virus clearly showed that removal of a 15-mer Nsp2 epitope had no effect on the immunogenicity, growth properties or virulence when compared to the wild type virus. On the other hand, deletions of previously identified peptide marker candidates within GP3 and M genes were shown to be lethal for virus viability in vitro. Alternatively, by substitution of 5aa at a time within a M peptide marker candidate, a viable mutant virus could be recovered although it still resulted in a positive marker virus. In summary, our results provide proof of concept that PRRSV marker vaccines can be developed using such methodology. Taken together, these data indicate that the combination of a mutant virus carrying a deletion of an immunodominant epitope and the corresponding peptide ELISA represents an attractive approach for the development of PRRSV differential modified-live vaccines. / O vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV) foi isolado pela primeira vez em 1991 e, desde então, tem sido associado a perdas significativas para a suinocultura mundial. Apesar da vacinação contra o PRRSV ser amplamente utilizada, um grande avanço seria alcançado com a elaboração de vacinas diferenciais que permitam a discriminação sorológica entre animais vacinados e naturalmente infectados. O presente estudo teve como objetivo a identificação de regiões imunogênicas, conservadas e dispensáveis a replicação viral, em diferentes proteínas do PRRSV, que pudessem ser utilizadas como marcadores sorológicos negativos em uma nova geração de vacinas atenuadas. Na primeira parte desta tese estão apresentados os resultados de um mapeamento de epítopos lineares de linfócitos B em diferentes proteínas do PRRSV, pelo uso da tecnologia de Pepscan. Os resultados indicam a presença de diversas regiões imunodominantes na proteína não estrutural 2 (Nsp2) e em todas as proteínas estruturais do vírus. Essas regiões foram consistentemente reconhecidas pelo soro de suínos experimentalmente infectados com uma cepa norte-americana do PRRSV (NVSL97-7895). A maior freqüência de epítopos imunodominantes foi identificada na Nsp2 (n=18) e o mais alto grau de imunogenicidade e nível de conservação de aminoácidos foi observado em dois epítopos identificados na extremidade carboxi-terminal da proteína M (ORF6). Anticorpos reagentes com epítopos imunodominantes de cada proteína foram detectados inicialmente entre os dias 7-15 pós-infecção (pi), permanecendo em altos títulos até o final do experimento (dia 90 pi). Com base na imunodominância e nível de conservação de amino ácidos (aa) das seqüências mapeadas, dois epítopos alvos foram selecionados como candidatos a marcadores sorológicos negativos em cada uma das proteínas Nsp2, Gp3 e M. Esses epítopos foram então deletados em um clone infeccioso de cDNA (FL12) por mutagênese sítio-direcionada. Os resultados desses experimentos encontram-se descritos na segunda parte da tese. Um vírus mutante carreando a deleção de um epítopo imunodominante da Nsp2 (FLdNsp2/44) foi obtido após transfeccção de RNA viral em células MARC145. A caracterização in vitro e in vivo do vírus mutante demonstrou que a remoção dos 15 aa da Nsp2 não produziu efeito sobre a imunogenicidade, replicação ou virulência quando comparado ao vírus parental. Além disso, observou-se indução de soroconversão contra o PRRSV em animais infectados, detectada pelo uso de um teste ELISA comercial. Por outro lado, não foi detectada resposta humoral específica contra a região deletada nos animais imunizados com o FLdNsp2/44, conforme resultados de um teste ELISA contendo como antígeno um peptídeo sintético correspondente a seqüência removida. Por outro lado, deleções dos epítopos previamente identificados na Gp3 e proteína M foram letais à viabilidade viral in vitro. Alternativamente, um outro vírus mutante foi gerado pela substituição de 5 aa do epítopo identificado na proteína M, embora a alteração de resíduos não tenha sido suficiente para eliminar a imunogenicidade da região. Em resumo, os resultados do presente estudo se constituem em uma prova de conceito no sentido do desenvolvimento de vacinas diferenciais contra o PRRSV. A utilização de um vírus mutante carreando a deleção de um epítopo imunodominante, associado com um teste de ELISA baseado no peptídeo sintético correspondente a região deletada, representam uma alternativa para o desenvolvimento de vacinas diferenciais atenuadas contra o PRRSV.

Les récoltes de céréales sont souvent contaminées par des moisissures qui se développent pendant la récolte et l’entreposage et produisent des métabolites secondaires appelés mycotoxines. Le porc est reconnu pour être sensible au déoxynivalénol (DON). L’infection virale la plus importante chez le porc est causée par le virus du syndrome reproducteur et respiratoire porcin (VSRRP). Celui-ci provoque un syndrome grippal et des troubles de reproduction. L’objectif du présent projet était de déterminer l'effet in vitro de DON sur la réplication du VSRRP dans de lignées cellulaires permissives, MARC-145 et PAM, et déterminer in vivo l'impact de DON dans des aliments naturellement contaminés sur l’infection au VSRRP chez le porcelet. Tout d’abord, les cellules ont été incubées avec des doses croissantes de DON et ont été infectées avec du VSRRP pour évaluer la viabilité et la mortalité cellulaire, la réplication virale et l’expression de cytokines. Les résultats ont montré que les concentrations de DON de 560ng/ml et plus affectaient significativement la survie des cellules MARC-145 et PAM infectées par le VSRRP. En revanche, il y avait une augmentation significative de la viabilité et une réduction de la mortalité cellulaire à des concentrations de DON de 140 à 280 ng/ml pour les cellules PAM et de 70 à 280 ng/ml pour les cellules MARC-145 avec une réduction de l'effet cytopathique provoqué parle VSRRP. Au niveau in vivo, 30 porcelets divisés en 3 groupes de 10 porcelets et nourris pendant 2 semaines avec 3 différentes diètes naturellement ont été contaminées avec DON (0; 2,5 et 3,5 mg/kg). Les porcelets ont été subdivisés en 6 groupes, 3 groupes de 6 porcelets et ont été exposés au DON pendant 2 semaines et infectés par voie intratrachéale et intramusculaire avec le virus. Les 3 autres groupes de 4 porcelets servaient de contrôle non infectés. Les signes cliniques ont été enregistrés pendant 21 jours. La virémie a été évaluée par PCR. À la fin de l’expérimentation, les porcelets ont été euthanasiés et les lésions pulmonaires ont été évaluées. Les résultats ont montré que l’ingestion de DON à 3,5 mg/kg a augmenté l’effet du VSRRP sur la sévérité des signes cliniques, les lésions pulmonaires et la mortalité. L’ingestion de DON à 2,5 mg/kg a entrainé une augmentation de la virémie au jour 3 après l’infection mais sans impact sur les signes cliniques et les lésions pulmonaires. Mot clés: DON, VSRRP, MARC-145, PAM, effet cytopathique, cytokines, PCR / Cereal crops are often contaminated with moulds that grow during harvest and storage and produce secondary metabolites called mycotoxins. Pig is known to be sensitive to deoxynivalenol (DON). On the other hand, infection by porcine reproductive and respiratory syndrome virus (PRRSV) causes a flu-like syndrome and reproductive disorders. The objectives of this project were to determine the in vitro effect of DON on the replication of PRRSV in permissive cell lines, MARC-145 and PAM and the in vivo impact of DON-naturally contaminated feed on PRRSV infection in piglets. Firstly, cells were incubated with gradually increasing doses of DON and were infected with PRRSV to evaluate cytopathic effect and to assess cell viability, virus replication and cytokine mRNA expression on infected and uninfected cells. Results showed that DON concentrations of 560 ng/ml and higher were significantly detrimental to the survival of MARC-145 cells infected with PRRSV. In contrast, there was a significant increase of cell viability and decreased of cell mortality at DON concentrations within 140 to 280 ng/ml for PAM cells and 70 to 280 ng/ml ranges for MARC-145 showing a reduced cytopathic effect (CPE) caused by PRRSV. In vivo study was carried out on 30 piglets divided into 3 groups of 10 piglets fed naturally contaminated diets with different levels of DON; 0, 2.5 and 3.5 mg/kg. After 2 weeks, pigs were further divided into 6 subgroups, 3 subgroups of 6 piglets were infected intra tracheally and intramuscularly with PRRSV. The other 3 subgroups of 4 piglets were used as uninfected controls. Clinical signs were recorded for 21 days post-infection (p.i.). Sera were evaluated for viremia by PCR. At the end of the experiment, piglets were euthanized and pulmonary lesions were evaluated. Results showed that ingestion of diet highly contaminated with DON at 3.5 mg/kg increased the effect of PRRSV infection on the severity of clinical signs, weight loss, lung lesions and mortality. Diet with DON at 2.5 mg/kg showed an increase of viremia at day 3 but had not significant impact on clinical signs and lung lesions. Keywords: DON, PRRSV, MARC-145, PAM, cytopathic effect, cytokines, PCR