Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log2) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7-14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection

Analysis of the genetic diversity and mRNA expression level in porcine reproductive and respiratory syndrome virus vaccinated pigs that developed short or long viremias after challenge

Porcine reproductive and respiratory syndrome virus (PRRSv) infection alters the host's cellular and humoral immune response. Immunity against PRRSv is multigenic and vary between individuals. The aim of the present study was to compare several genes that encode for molecules involved in the immune response between two groups of vaccinated pigs that experienced short or long viremic periods after PRRSv challenge. These analyses include the sequencing of four SLA Class I, two Class II allele groups, and CD163, plus the analysis by quantitative realtime qRT-PCR of the constitutive expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA and other molecules in peripheral blood mononuclear cells

Combining Laboratory and Mathematical Models to Infer Mechanisms Underlying Kinetic Changes in Macrophage Susceptibility to an RNA Virus

Background: Macrophages are essential to innate immunity against many pathogens, but some pathogens also target macrophages as routes to infection. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an RNA virus that infects porcine alveolar macrophages (PAMs) causing devastating impact on global pig production. Identifying the cellular mechanisms that mediate PAM susceptibility to the virus is crucial for developing effective interventions. Previous evidence suggests that the scavenger receptor CD163 is essential for productive infection of PAMs with PRRSV. Here we use an integrative in-vitro-in-silico modelling approach to determine whether and how PAM susceptibility to PRRSV changes over time, to assess the role of CD163 expression on such changes, and to infer other potential causative mechanisms altering cell susceptibility. Results: Our in-vitro experiment showed that PAM susceptibility to PRRSV changed considerably over incubation time. Moreover, an increasing proportion of PAMs apparently lacking CD163 were found susceptible to PRRSV at the later incubation stages, thus conflicting with current understanding that CD163 is essential for productive infection of PAMs with PRRSV. We developed process based dynamic mathematical models and fitted these to the data to assess alternative hypotheses regarding potential underlying mechanisms for the observed susceptibility and biomarker trends. The models informed by our data support the hypothesis that although CD163 may have enhanced cell susceptibility, it was not essential for productive infection in our study. Instead the models promote the existence of a reversible cellular state, such as macrophage polarization, mediated in a density dependent manner by autocrine factors, to be responsible for the observed kinetics in cell susceptibility. Conclusions: Our dynamic model-inference approach provides strong support that PAM susceptibility to the PRRS virus is transient, reversible and can be mediated by compounds produced by the target cells themselves, and that these can render PAMs lacking the CD163 receptor susceptible to PRRSV. The results have implications for the development of therapeutics aiming to boost target cell resistance and prompt future investigation of dynamic changes in macrophage susceptibility to PRRSV and other viruses

Distribución tisular de los receptores Toll-like (TLR) 3, 7 y 9 en el cerdo y efecto in vitro de la infección por el virus de síndrome respiratorio y reproductivo porcino en su regulación en macrófagos alveolares porcinos

Los receptores Toll-like (TLRs), en particular los que se encuentran en vesículas intracelulares de origen endosomal (TLR3, TLR7 y TLR9), están involucrados en la respuesta innata antivírica. La unión a sus respectivos ligandos conduce a la activación de cascadas intracelulares que resultan en una producción de citoquinas pro-inflamatorias (TNF-α) y antivirales (interferones de tipo I). Existe muy poca información sobre la distribución celular y tisular de estos receptores en la especie porcina así como su regulación en condiciones normales o de infección. En el primer estudio de la presente tesis se valoró la distribución tisular de los TLRs endosomales en pulmón y tejidos linfoides primarios y secundarios de cerdos sanos de distintas edades. El marcaje del TLR9 se realizó con un anticuerpo comercial con reactividad específica para el porcino. En cambio, en el caso de TLR3 y TLR7 los anticuerpos se dirigían a las moléculas humanas pero, supuestamente, poseían reactividad cruzada con las moléculas de origen porcino. Los resultados obtenidos permitieron valorar la distribución del TLR9 en los distintos tejidos examinados, pero no la de los TLR3 y TLR7 ya que el uso de los anticuerpos dirigidos frente a estos dos últimos receptores no produjo resultados satisfactorios. Así, el marcaje obtenido con el anticuerpo anti-TLR3 fue muy variable en función del tejido utilizado; es decir, en algunos órganos como el pulmón, tonsila o linfonodos parecía ser específico, pero en otros como en el hígado, producía un intenso color de fondo inespecífico. Por el contrario, sí hubo un marcaje específico para el TLR9 que reveló una expresión constitutiva de dicho receptor en células de la periferia de los folículos linfoides de los linfonodos, la tonsila y las placas de Peyer, (células de tipo epitelial, dendríticas, macrófagos o linfocitos) un hecho que sugería que este receptor probablemente puede jugar un papel importante en la activación el sistema inmunitario de los cerdos a partir de las 3 semanas de vida. El segundo estudio de esta tesis consistió en determinar la variación de la expresión de TLR3, TLR7 y TLR9 a lo largo del tiempo en una población de células presentadoras de antígeno. La población de estudio elegida fue la de macrófagos alveolares porcinos (PAMs). Los resultados de la cinética de expresión mediante citometría de flujo nos mostró que estas células presentaban una expresión basal elevada de TLR3 y TLR9 pero no de TLR7. Una de las explicaciones posibles para este marcaje basal señalaría a la necesaria manipulación de estas células antes de ser congeladas como responsable de esta expresión. Por otra parte, es difícil conocer con exactitud el ambiente en el cual se encontraban los PAMs en el pulmón antes de ser recogidos (concentración de interleuquinas, quimioquinas, otras moléculas, etc). Dado que los animales donantes estaban sanos, no presentaban ningún tipo de lesión pulmonar y eran libres de los patógenos víricos comunes del cerdo (circovirus porcino de tipo 2, influenza A y virus del PRRS entre otros) la causa de esta elevada expresión no está clara. En cuanto a la expresión de TLR7, apenas se pudo detectar una expresión basal en los PAMs utilizados. Para el tercer estudio de esta tesis se buscó un modelo de infección con un virus RNA que pudiera influir en la regulación de estos TLRs y además pudiera añadir nuevos conocimientos respecto a la inmunopatogenia de dicha infección. En el campo de las enfermedades infecciosas del porcino, una de las mayores incógnitas inmunológicas del momento la encontramos en la infección con el virus del PRRS. Los resultados de este estudio demostraron que dos cepas del mismo genotipo del virus del PRRS causaban una regulación diferente del TLR3 y del patrón de citoquinas pro-inflamatorias. En concreto, en los estudios de citometría de flujo, la cepa 3262 al inducir la expresión de TLR3 en PAMs, sobre todo a dosis elevadas (m.o.i=1), activaría la producción de TNF-α+; en cambio, la cepa 3267 o la cepa vacunal DV activaron TLR3 con menor intensidad y no inducirían TNF-α; sugiriendo en definitiva, que la regulación del patrón de citoquinas antivirales o pro-inflamatorias en los macrófagos dependería del tipo de cepa utilizada. Resulta interesante señalar que a pesar de las diferencias observadas en la citometría de flujo respecto el porcentaje de células que expresaban TLR3 y en la intensidad de su expresión según el tipo de virus utilizado, la expresión relativa del mRNA no parecía modificarse. Estas diferencias resultan interesantes y apuntan a que distintas cepas de campo de genotipo europeo podrían ejercer un efecto regulador de diferente intensidad sobre moléculas inhibitorias de la cascada de señalización de los TLRs. Además esta regulación parece depender de diferentes factores tales como: la cepa vírica, el tiempo de infección y la dosis infectiva inicial. Nuestros resultados pueden ser útiles para abrir y conducir una nueva línea de investigaciones orientadas hacia el área de la inmunidad innata frente al virus del PRRS.Toll-like receptors (TLRs), particularly those found within intracellular vesicles of endosomal origin (TLR3, TLR7 and TLR9), are involved in the innate antiviral responses. Binding of those receptors to their respective ligands leads to the activation of intracellular cascades resulting in the release of pro-inflammatory cytokines (TNF-α) and antiviral (type I) interferons. The knowledge on the distribution of those receptors in porcine organs, tissues and cells and its regulation in physiological states or in infection is scarce. In the first study of the present thesis the distribution of endosomal TLRs in lung and primary and secondary lymphoid tissues of healthy pigs of different ages was assessed. Labeling of TLR9 was performed using a commercial antibody with specific reactivity for the porcine TLR9. For TLR3 and TLR7 the antibodies used in the study were directed to human molecules but they were supposed to cross-react with the porcine counterpart molecules. The results allowed the assessment of the distribution of TLR9 in the different tissues examined, but not that of TLR3 and TLR7 since the use of antibodies directed against the latter two receptors did not yield satisfactory results. Thus, labeling obtained with the anti-TLR3 antibody was highly variable depending on the tissue examined, that is, in some organs such as lungs, tonsils or lymph nodes labeling was apparently specific but in others, as in the liver, the se of that antibody resulted in an intense non-specific background. By contrast, TLR9 labeling was specific and revealed a constitutive expression of this receptor in cells of the periphery of lymphoid follicles of lymph nodes, tonsils and Peyer's patches (epithelial cells, dendritic cells, macrophages or lymphocytes) a fact suggesting that this receptor can probably play an important role in activating the immune system of pigs of 3 week-old piglets. The second study of this thesis was aimed to determine the variation of the expression of TLR3, TLR7 and TLR9 over time in a population of antigen-presenting cells. Porcine alveolar macrophages (PAMs) were used for this purpose. The results of the kinetics of expression as assessed by flow cytometry showed that PAMs had a high basal expression of TLR3 and TLR9 but not of TLR7. A possible explanation for this basal labeling could point to the unavoidable manipulation of PAMs needed for their collection. Moreover, it is difficult to know precisely the environmental conditions in which PAMs were in the lungs before being collected (concentration of interleukins, chemokines, presence of other molecules, etc.). Since PAM donors were healthy, showed no lung lesions and were demonstrated to be free of common viral pathogens of pigs (porcine circovirus type 2, influenza A and PRRS virus among others) the cause of this elevated expression remains unclear. As for TLR7, basal expression in the PAMs used was low or nil. The third study of the present thesis aimed to a model of infection with an RNA virus that might influence the regulation of these TLRs and also could add new knowledge regarding the pathogenesis of the infection. In the field of infectious diseases of swine, one of most interesting models of RNA virus infections is PRRS virus for which immunopathogenesis is largely understood. The results of this study showed that two strains of the same genotype of PRRS virus resulted in a different regulation of TLR3 and in a different pattern of pro-inflammatory cytokines. Specifically, in flow cytometry experiments, strain 3262, induced the expression of TLR3 in PAMs, particularly at high multiplicities of infection (m.o.i = 1) and triggered the production of TNF-α+ whereas strain 3267 or the vaccine strain DV resulted in lower TLR3 expression and did not induce TNF-α, suggesting ultimately that the regulation of the antiviral or pro-inflammatory cytokine patterns in macrophages depends on the strain used. Interestingly, despite the differences observed in flow cytometry for TLR3, the relative mRNA expression did not apparently change under different circumstances. This was an interesting observation that suggests that different field strains of genotype I PRRSV might exert a regulatory effect of different intensity on inhibitory molecules of the signaling cascade of TLRs. Furthermore, this regulation seems to depend on various factors such as the viral strain, the time of infection and the multiplicity of infection. Our results may be useful as a basis for further studies in the area of innate immunity against PRRS virus

Distribución tisular de los receptores Toll-like (TLR) 3, 7 y 9 en el cerdo y efecto in vitro de la infección por el virus de síndrome respiratorio y reproductivo porcino en su regulación en macrófagos alveolares porcinos

Los receptores Toll-like (TLRs), en particular los que se encuentran en vesículas intracelulares de origen endosomal (TLR3, TLR7 y TLR9), están involucrados en la respuesta innata antivírica. La unión a sus respectivos ligandos conduce a la activación de cascadas intracelulares que resultan en una producción de citoquinas pro-inflamatorias (TNF-α) y antivirales (interferones de tipo I). Existe muy poca información sobre la distribución celular y tisular de estos receptores en la especie porcina así como su regulación en condiciones normales o de infección. En el primer estudio de la presente tesis se valoró la distribución tisular de los TLRs endosomales en pulmón y tejidos linfoides primarios y secundarios de cerdos sanos de distintas edades. El marcaje del TLR9 se realizó con un anticuerpo comercial con reactividad específica para el porcino. En cambio, en el caso de TLR3 y TLR7 los anticuerpos se dirigían a las moléculas humanas pero, supuestamente, poseían reactividad cruzada con las moléculas de origen porcino. Los resultados obtenidos permitieron valorar la distribución del TLR9 en los distintos tejidos examinados, pero no la de los TLR3 y TLR7 ya que el uso de los anticuerpos dirigidos frente a estos dos últimos receptores no produjo resultados satisfactorios. Así, el marcaje obtenido con el anticuerpo anti-TLR3 fue muy variable en función del tejido utilizado; es decir, en algunos órganos como el pulmón, tonsila o linfonodos parecía ser específico, pero en otros como en el hígado, producía un intenso color de fondo inespecífico. Por el contrario, sí hubo un marcaje específico para el TLR9 que reveló una expresión constitutiva de dicho receptor en células de la periferia de los folículos linfoides de los linfonodos, la tonsila y las placas de Peyer, (células de tipo epitelial, dendríticas, macrófagos o linfocitos) un hecho que sugería que este receptor probablemente puede jugar un papel importante en la activación el sistema inmunitario de los cerdos a partir de las 3 semanas de vida. El segundo estudio de esta tesis consistió en determinar la variación de la expresión de TLR3, TLR7 y TLR9 a lo largo del tiempo en una población de células presentadoras de antígeno. La población de estudio elegida fue la de macrófagos alveolares porcinos (PAMs). Los resultados de la cinética de expresión mediante citometría de flujo nos mostró que estas células presentaban una expresión basal elevada de TLR3 y TLR9 pero no de TLR7. Una de las explicaciones posibles para este marcaje basal señalaría a la necesaria manipulación de estas células antes de ser congeladas como responsable de esta expresión. Por otra parte, es difícil conocer con exactitud el ambiente en el cual se encontraban los PAMs en el pulmón antes de ser recogidos (concentración de interleuquinas, quimioquinas, otras moléculas, etc). Dado que los animales donantes estaban sanos, no presentaban ningún tipo de lesión pulmonar y eran libres de los patógenos víricos comunes del cerdo (circovirus porcino de tipo 2, influenza A y virus del PRRS entre otros) la causa de esta elevada expresión no está clara. En cuanto a la expresión de TLR7, apenas se pudo detectar una expresión basal en los PAMs utilizados. Para el tercer estudio de esta tesis se buscó un modelo de infección con un virus RNA que pudiera influir en la regulación de estos TLRs y además pudiera añadir nuevos conocimientos respecto a la inmunopatogenia de dicha infección. En el campo de las enfermedades infecciosas del porcino, una de las mayores incógnitas inmunológicas del momento la encontramos en la infección con el virus del PRRS. Los resultados de este estudio demostraron que dos cepas del mismo genotipo del virus del PRRS causaban una regulación diferente del TLR3 y del patrón de citoquinas pro-inflamatorias. En concreto, en los estudios de citometría de flujo, la cepa 3262 al inducir la expresión de TLR3 en PAMs, sobre todo a dosis elevadas (m.o.i=1), activaría la producción de TNF-α+; en cambio, la cepa 3267 o la cepa vacunal DV activaron TLR3 con menor intensidad y no inducirían TNF-α; sugiriendo en definitiva, que la regulación del patrón de citoquinas antivirales o pro-inflamatorias en los macrófagos dependería del tipo de cepa utilizada. Resulta interesante señalar que a pesar de las diferencias observadas en la citometría de flujo respecto el porcentaje de células que expresaban TLR3 y en la intensidad de su expresión según el tipo de virus utilizado, la expresión relativa del mRNA no parecía modificarse. Estas diferencias resultan interesantes y apuntan a que distintas cepas de campo de genotipo europeo podrían ejercer un efecto regulador de diferente intensidad sobre moléculas inhibitorias de la cascada de señalización de los TLRs. Además esta regulación parece depender de diferentes factores tales como: la cepa vírica, el tiempo de infección y la dosis infectiva inicial. Nuestros resultados pueden ser útiles para abrir y conducir una nueva línea de investigaciones orientadas hacia el área de la inmunidad innata frente al virus del PRRS.Toll-like receptors (TLRs), particularly those found within intracellular vesicles of endosomal origin (TLR3, TLR7 and TLR9), are involved in the innate antiviral responses. Binding of those receptors to their respective ligands leads to the activation of intracellular cascades resulting in the release of pro-inflammatory cytokines (TNF-α) and antiviral (type I) interferons. The knowledge on the distribution of those receptors in porcine organs, tissues and cells and its regulation in physiological states or in infection is scarce. In the first study of the present thesis the distribution of endosomal TLRs in lung and primary and secondary lymphoid tissues of healthy pigs of different ages was assessed. Labeling of TLR9 was performed using a commercial antibody with specific reactivity for the porcine TLR9. For TLR3 and TLR7 the antibodies used in the study were directed to human molecules but they were supposed to cross-react with the porcine counterpart molecules. The results allowed the assessment of the distribution of TLR9 in the different tissues examined, but not that of TLR3 and TLR7 since the use of antibodies directed against the latter two receptors did not yield satisfactory results. Thus, labeling obtained with the anti-TLR3 antibody was highly variable depending on the tissue examined, that is, in some organs such as lungs, tonsils or lymph nodes labeling was apparently specific but in others, as in the liver, the se of that antibody resulted in an intense non-specific background. By contrast, TLR9 labeling was specific and revealed a constitutive expression of this receptor in cells of the periphery of lymphoid follicles of lymph nodes, tonsils and Peyer's patches (epithelial cells, dendritic cells, macrophages or lymphocytes) a fact suggesting that this receptor can probably play an important role in activating the immune system of pigs of 3 week-old piglets. The second study of this thesis was aimed to determine the variation of the expression of TLR3, TLR7 and TLR9 over time in a population of antigen-presenting cells. Porcine alveolar macrophages (PAMs) were used for this purpose. The results of the kinetics of expression as assessed by flow cytometry showed that PAMs had a high basal expression of TLR3 and TLR9 but not of TLR7. A possible explanation for this basal labeling could point to the unavoidable manipulation of PAMs needed for their collection. Moreover, it is difficult to know precisely the environmental conditions in which PAMs were in the lungs before being collected (concentration of interleukins, chemokines, presence of other molecules, etc.). Since PAM donors were healthy, showed no lung lesions and were demonstrated to be free of common viral pathogens of pigs (porcine circovirus type 2, influenza A and PRRS virus among others) the cause of this elevated expression remains unclear. As for TLR7, basal expression in the PAMs used was low or nil. The third study of the present thesis aimed to a model of infection with an RNA virus that might influence the regulation of these TLRs and also could add new knowledge regarding the pathogenesis of the infection. In the field of infectious diseases of swine, one of most interesting models of RNA virus infections is PRRS virus for which immunopathogenesis is largely understood. The results of this study showed that two strains of the same genotype of PRRS virus resulted in a different regulation of TLR3 and in a different pattern of pro-inflammatory cytokines. Specifically, in flow cytometry experiments, strain 3262, induced the expression of TLR3 in PAMs, particularly at high multiplicities of infection (m.o.i = 1) and triggered the production of TNF-α+ whereas strain 3267 or the vaccine strain DV resulted in lower TLR3 expression and did not induce TNF-α, suggesting ultimately that the regulation of the antiviral or pro-inflammatory cytokine patterns in macrophages depends on the strain used. Interestingly, despite the differences observed in flow cytometry for TLR3, the relative mRNA expression did not apparently change under different circumstances. This was an interesting observation that suggests that different field strains of genotype I PRRSV might exert a regulatory effect of different intensity on inhibitory molecules of the signaling cascade of TLRs. Furthermore, this regulation seems to depend on various factors such as the viral strain, the time of infection and the multiplicity of infection. Our results may be useful as a basis for further studies in the area of innate immunity against PRRS virus

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log2) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7-14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection