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

Revisiting the use of Live Attenuated viruses as models to study the pathogenesis and the mechanisms involved in protection against African swine fever

Trabajo presentado en el 8th EPIZONE Annual Meeting "Primed for tomorrow", celebrado en Copenhague (Dinamarca) del 23 al 25 de septiembre de 2014.The meeting is entitled: "Primed for tomorrow" and will address the latest developments aimed at monitoring and understanding the evolution, emergence, transmission and spread of epizootic viruses. The focus will remain on the EPIZONE themes aimed at improved disease control through integration and collaboration of research in diagnosis, intervention strategies, risk assessment, surveillance and epidemiology. A stimulating scientific programme will be provided by invited speakers and selected poster and oral presentations describing recent research on epizootic diseases of cattle, pigs, poultry, sheep, goats, fish and horses. This meeting builds on previous highly successful EPIZONE meetings and will provide extensive opportunities for networking, scientific exchange and fostering collaboration

DNA Vaccination Partially Protects against African Swine Fever Virus Lethal Challenge in the Absence of Antibodies

The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8+ T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8+ T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease

Sheep experimental model for rift valley fever virus Infection for the study of immunopathogenesis, pathology and vaccinology

El virus de la Fiebre del Valle del Rift (RVFV) es un flebovirus zoonótico transmitido por mosquitos que afecta a los rumiantes causando abortos y hepatitis aguda con necrosis multifocal. Los síntomas de la RVF varían desde el síndrome gripal hasta la retinitis y la encefalitis. En esta tesis, mostramos la susceptibilidad leve-subaguda de las ovejas Ripollesas de 9-10 semanas de edad a la infección por RVFV. Cuatro aislamientos virales diferentes de brotes de campo se replicaron de forma eficiente in vivo tras la inoculación subcutánea experimental, detectándose viremia y diseminacion viral dependientes del aislado de RVFV. Se demostró la transmisión horizontal a un cordero centinela no infectado. La infección en corderos únicamente causó pirexia transitoria, aunque se observó opacidad corneal ("ojo azul") en 3 de 16 ovejas inoculadas subcutáneamente. En una aproximación preliminar, se investigó el tejido formolado y parafinado procedente de estos animales afectados por la opacidad corneal mediante histopatología y qRT-PCR, para su caracterización. En cuatro corderos infectados se diagnosticó uveítis anterior con endotelitis linfoplasmocítica. Para evaluar la protección conferida por una única dosis subcutánea de una vacuna de virus Ankara modificado (MVA) que codifica las glicoproteínas Gn y Gc de RVFV en corderos, se inmunizaron 3 grupos de 6 a 7 corderos de 5-7 semanas de edad de la siguiente manera: Un grupo vacunal (denominado rMVA-GnGc), un segundo grupo control vectorial (vector MVA) y un tercer grupo control no vacunado (solución salina). Catorce días más tarde, todos los animales se sometieron a un desafío subcutáneo con 105 TCID50 del aislamiento virulento RVFV 56/74 y se evaluó la eficacia de la vacuna usando puntos finales estándar. Dos corderos (uno del grupo vacunado y uno del control vectorial) sucumbieron al desafío, mostrando lesiones hepáticas características. Los corderos de los grupos control vectorial y control no vacunado fueron febriles de los días 2 a 5 después del desafío (pc) mientras que los del grupo rMVA-GnGc mostraron un único pico de pirexia al día 3 pc. Se detectó ARN de RVFV en ambos hisopados nasal y oral de los días 3 a 7 pc en algunos corderos de los grupos control vectorial y no vacunado, pero no se pudo detectar diseminación viral en los corderos supervivientes vacunados. Caracterizamos patológicamente la nueva lesión ocular detectada en un abordaje secundario. Se seleccionaron 2 grupos de 5 corderos por grupo (n = 10) de la base de datos histórica de infecciones experimentales de RVFV realizadas en el Centro de Investigación en Sanidad Animal (España, NBS3), en base a sus datos clínicos, viremia y diagnóstico de lesiones ocular y hepática. Se caracterizó la uveítis anterior diagnosticada (8 de 10) con endotelitis linfoplasmocítica (2 de 10). CD3, CD20 e infiltrados inflamatorios mononucleares positivos a la lisozima se observaron en ojos parafinados RVFV-positivos. El marcaje positivo de CD20 sólo se observó en los infiltrados en uvea anterior. También se diagnosticó una nueva retinitis T dependiente (CD3 positivo) en 5 de 10 corderos infectados con RVFV. Se desarrolló un protocolo de inmunoquímica contra RVFV basado en un anticuerpo monoclonal murino. En conclusión, la raza Ripollesa se infecta fácilmente con RVFV sin manifestaciones clínicas evidentes. Un modelo de desafío con corderos de 5 a 10 semanas de edad ha demostrado ser eficaz para testar vacunas. Se sugiere que una sola dosis de la vacuna rMVA-GnGc puede ser suficiente para reducir la diseminación de RVFV y la duración de la viremia, pero no proporciona inmunidad estéril ni protección contra la enfermedad. Hasta donde sabemos, esta es la primera descripción patológica de uveítis anterior con lesión retiniana en un modelo ovino, muy parecida a las lesiones oculares humanas.Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic phlebovirus that primarily affects ruminants by causing abortions and acute hepatitis with multifocal necrosis as major findings. Human RVF symptoms range from flu-like syndrome to retinitis and encephalitis. The increasing interest in RVFV deserve revisiting experimental sheep infection. In this thesis, we show the susceptibility of 9–10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates from field outbreaks efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and RVFV-isolate dependent virus shedding were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in old lambs and no other clinical symptoms were observed, although corneal opacity (“blue eye”) was found in 3 out of 16 subcutaneously inoculated sheep. To better characterize this corneal opacity, in a preliminary approach, formalin-fixed paraffin wax-embedded tissue from these ocular condition-affected animals was investigated by histopathology and quantitative real time reverse transcriptase polymerase chain reaction. Anterior uveitis with lymphoplasmacytic endotheliitis was diagnosed in these four RVFV-infected lambs. To evaluate the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the RVFV glycoproteins Gn and Gc in lambs, 3 groups of 6 to 7 Ripollesa lambs of 5–7 weeks old were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 105 TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. We characterize pathologically the new ocular detected condition in a secondary approach. Two groups of five lambs per group were selected (n=10) from the historical database of RVFV experimental infections performed in Center of Research in Animal Health (Spain, NBS3 facility) in the basis of their clinical data, viremia and diagnosed ocular and hepatic lesions (two previous experiments). The previously diagnosed anterior uveitis (8 out of 10) with lymphoplasmacytic endotheliitis (2 out of 10) was characterized. CD3, CD20 and lysozyme-positive mononuclear inflammatory infiltrates were observed in RVFV-positive paraffin-embedded eyes. CD20 labelling was only observed in infiltrates in anterior uvea. A novel T-cell dependent retinitis was also diagnosed in 5 out of 10 RVFV-infected lambs based on CD3-positive labelling. An immunochemistry protocol based on a murine monoclonal antibody was developed at CReSA BLS2 facility. In conclusion, Ripollesa sheep are readily infected with RVFV without apparent clinical manifestations. A 5-10 weeks old Ripollesa breed challenge model has proven to be effective in vaccine testing because of its susceptibility to virus. It is suggested that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. To our knowledge, this is the first pathological description of RVFV-related anterior uveitis with retinal injury in a RVFV-challenge sheep model, resembling ocular human lesions

Sheep experimental model for rift valley fever virus Infection for the study of immunopathogenesis, pathology and vaccinology /

Departament responsable de la tesi: Departament de Sanitat i d'Anatomia Animals.El virus de la Fiebre del Valle del Rift (RVFV) es un flebovirus zoonótico transmitido por mosquitos que afecta a los rumiantes causando abortos y hepatitis aguda con necrosis multifocal. Los síntomas de la RVF varían desde el síndrome gripal hasta la retinitis y la encefalitis. En esta tesis, mostramos la susceptibilidad leve-subaguda de las ovejas Ripollesas de 9-10 semanas de edad a la infección por RVFV. Cuatro aislamientos virales diferentes de brotes de campo se replicaron de forma eficiente in vivo tras la inoculación subcutánea experimental, detectándose viremia y diseminacion viral dependientes del aislado de RVFV. Se demostró la transmisión horizontal a un cordero centinela no infectado. La infección en corderos únicamente causó pirexia transitoria, aunque se observó opacidad corneal ("ojo azul") en 3 de 16 ovejas inoculadas subcutáneamente. En una aproximación preliminar, se investigó el tejido formolado y parafinado procedente de estos animales afectados por la opacidad corneal mediante histopatología y qRT-PCR, para su caracterización. En cuatro corderos infectados se diagnosticó uveítis anterior con endotelitis linfoplasmocítica. Para evaluar la protección conferida por una única dosis subcutánea de una vacuna de virus Ankara modificado (MVA) que codifica las glicoproteínas Gn y Gc de RVFV en corderos, se inmunizaron 3 grupos de 6 a 7 corderos de 5-7 semanas de edad de la siguiente manera: Un grupo vacunal (denominado rMVA-GnGc), un segundo grupo control vectorial (vector MVA) y un tercer grupo control no vacunado (solución salina). Catorce días más tarde, todos los animales se sometieron a un desafío subcutáneo con 105 TCID50 del aislamiento virulento RVFV 56/74 y se evaluó la eficacia de la vacuna usando puntos finales estándar. Dos corderos (uno del grupo vacunado y uno del control vectorial) sucumbieron al desafío, mostrando lesiones hepáticas características. Los corderos de los grupos control vectorial y control no vacunado fueron febriles de los días 2 a 5 después del desafío (pc) mientras que los del grupo rMVA-GnGc mostraron un único pico de pirexia al día 3 pc. Se detectó ARN de RVFV en ambos hisopados nasal y oral de los días 3 a 7 pc en algunos corderos de los grupos control vectorial y no vacunado, pero no se pudo detectar diseminación viral en los corderos supervivientes vacunados. Caracterizamos patológicamente la nueva lesión ocular detectada en un abordaje secundario. Se seleccionaron 2 grupos de 5 corderos por grupo (n = 10) de la base de datos histórica de infecciones experimentales de RVFV realizadas en el Centro de Investigación en Sanidad Animal (España, NBS3), en base a sus datos clínicos, viremia y diagnóstico de lesiones ocular y hepática. Se caracterizó la uveítis anterior diagnosticada (8 de 10) con endotelitis linfoplasmocítica (2 de 10). CD3, CD20 e infiltrados inflamatorios mononucleares positivos a la lisozima se observaron en ojos parafinados RVFV-positivos. El marcaje positivo de CD20 sólo se observó en los infiltrados en uvea anterior. También se diagnosticó una nueva retinitis T dependiente (CD3 positivo) en 5 de 10 corderos infectados con RVFV. Se desarrolló un protocolo de inmunoquímica contra RVFV basado en un anticuerpo monoclonal murino. En conclusión, la raza Ripollesa se infecta fácilmente con RVFV sin manifestaciones clínicas evidentes. Un modelo de desafío con corderos de 5 a 10 semanas de edad ha demostrado ser eficaz para testar vacunas. Se sugiere que una sola dosis de la vacuna rMVA-GnGc puede ser suficiente para reducir la diseminación de RVFV y la duración de la viremia, pero no proporciona inmunidad estéril ni protección contra la enfermedad. Hasta donde sabemos, esta es la primera descripción patológica de uveítis anterior con lesión retiniana en un modelo ovino, muy parecida a las lesiones oculares humanas.Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic phlebovirus that primarily affects ruminants by causing abortions and acute hepatitis with multifocal necrosis as major findings. Human RVF symptoms range from flu-like syndrome to retinitis and encephalitis. The increasing interest in RVFV deserve revisiting experimental sheep infection. In this thesis, we show the susceptibility of 9-10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates from field outbreaks efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and RVFV-isolate dependent virus shedding were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in old lambs and no other clinical symptoms were observed, although corneal opacity ("blue eye") was found in 3 out of 16 subcutaneously inoculated sheep. To better characterize this corneal opacity, in a preliminary approach, formalin-fixed paraffin wax-embedded tissue from these ocular condition-affected animals was investigated by histopathology and quantitative real time reverse transcriptase polymerase chain reaction. Anterior uveitis with lymphoplasmacytic endotheliitis was diagnosed in these four RVFV-infected lambs. To evaluate the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the RVFV glycoproteins Gn and Gc in lambs, 3 groups of 6 to 7 Ripollesa lambs of 5-7 weeks old were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 105 TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. We characterize pathologically the new ocular detected condition in a secondary approach. Two groups of five lambs per group were selected (n=10) from the historical database of RVFV experimental infections performed in Center of Research in Animal Health (Spain, NBS3 facility) in the basis of their clinical data, viremia and diagnosed ocular and hepatic lesions (two previous experiments). The previously diagnosed anterior uveitis (8 out of 10) with lymphoplasmacytic endotheliitis (2 out of 10) was characterized. CD3, CD20 and lysozyme-positive mononuclear inflammatory infiltrates were observed in RVFV-positive paraffin-embedded eyes. CD20 labelling was only observed in infiltrates in anterior uvea. A novel T-cell dependent retinitis was also diagnosed in 5 out of 10 RVFV-infected lambs based on CD3-positive labelling. An immunochemistry protocol based on a murine monoclonal antibody was developed at CReSA BLS2 facility. In conclusion, Ripollesa sheep are readily infected with RVFV without apparent clinical manifestations. A 5-10 weeks old Ripollesa breed challenge model has proven to be effective in vaccine testing because of its susceptibility to virus. It is suggested that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. To our knowledge, this is the first pathological description of RVFV-related anterior uveitis with retinal injury in a RVFV-challenge sheep model, resembling ocular human lesions

First-described recently discovered non-toxic vegetal-derived furocoumarin preclinical efficacy against SARS-CoV-2: a promising antiviral herbal drug

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiology of coronavirus disease 2019 (COVID19) pandemic. ICEP4 purified compound (ICEP4) is a recently discovered furocoumarin-related purified compound coming from roots and seeds of Angelica archangelica (herbal drug). ICEP4-related herbal preparations have been extensively used as active herbal ingredient in traditional medicine treatments in several European countries. Extraction method of patent pending ICEP4 (patent application no. GB2017123.7) has previously shown strong manufacturing robustness, long-lasting stability, and repeated chemical consistency. Here we show that ICEP4 presents a significant in vitro cytoprotective effect in highly virulent-SARS-CoV-2 challenged Vero E6 cellular cultures, using doses of 34.5 and 69 μM. No dose-related ICEP4 toxicity was observed in Vero E6 cells, M0 macrophages, B, CD4+ T and CD8+ T lymphocytes, Natural Killer (NK) or Natural Killer T (NKT) cells. No dose-related ICEP4 inflammatory response was observed in M0 macrophages quantified by IL6 and TNFα release in cell supernatant. No decrease in survival rate was observed after either 24 hr acute or 21-day chronic exposure in in vivo toxicity studies performed in C. elegans. Therefore, ICEP4 toxicological profile has demonstrated marked differences compared to others vegetal furocoumarins. Successful ICEP4 doses against SARS-CoV-2-challenged cells are within the maximum threshold of toxicity concern (TTC) of furocoumarins as herbal preparation, stated by European Medicines Agency (EMA). The characteristic chemical compounding of ICEP4, along with its safe TTC, allow us to assume that the first-observation of a natural antiviral compound has occurred. The potential druggability of a new synthetic ICEP4-related compound remains to be elucidated. However, well-established historical use of ICEP4-related compounds as herbal preparations may point towards an already-safe, widely extended remedy, which may be ready-to-go for large-scale clinical trials under the EMA emergency regulatory pathway. To the best of the authors' knowledge, ICEP4-related herbal drug can be postulated as a promising therapeutic treatment for COVID19.Iratxe Uranga-Murillo is supported by a predoctoral contract from Aragón Government. ii Dr. Maykel Arias is supported by a Juan de la Cierva postdoctoral contract. iii Dr. Julian Pardo´s laboratory is funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón -Group B29_17R-, Ministerio de Ciencia, Innovación e Universidades - MCNU-, Agencia Estatal de Investigación -SAF2017‐83120‐C2‐1‐R-), Instituto de Salud Carlos III, Fundación Inocente Inocente, ASPANOA and Carrera de la Mujer de Monzón.N

Sow vaccination with a protein fragment against virulent glaesserella (Haemophilus) parasuis modulates immunity traits in their offspring

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer's disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-β both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains

Live attenuated African swine fever viruses as ideal tools to dissect the mechanisms involved in viral pathogenesis and immune protection

African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8 + T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.This work has been funded by the Spanish Government (project reference numbers AGL201022229 and AGL201348998C21R). Anna Lacasta and Paula López-Monteagudo were financially supported by an FPU fellowship and an FPI fellowship, respectively, both from the Spanish Government

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