Effect of vaccination route (intradermal vs. intramuscular) against porcine reproductive and respiratory syndrome using a modified live vaccine on systemic and mucosal immune response and virus transmission in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with worldwide distribution and an enormous economic impact. To control PRRS virus (PRRSV) infection, modified live vaccines (MLVs) are widely used in the field, mainly administered via an intramuscular (IM) route. Currently, some MLVs are authorized for intradermal (ID) administration, which has many practical and welfare advantages. The objectives of the study were to compare the immune responses (systemic in blood and mucosal in lungs) and vaccine efficacy in preventing challenge strain transmission after IM or needle-free ID immunization of piglets with an MLV against PRRSV-1 (MLV1). METHODS: Groups of sixteen 5-week-old specific pathogen-free piglets were vaccinated with Porcilis PRRS® (MSD) either by an IM (V+ IM) or ID route (V+ ID) using an IDAL®3G device or kept unvaccinated (V-). Four weeks after vaccination, in each group, 8 out of the 16 piglets were challenged intranasally with a PRRSV-1 field strain, and one day later, the inoculated pigs were mingled by direct contact with the remaining 8 sentinel noninoculated pigs to evaluate PRRSV transmission. Thus, after the challenge, each group (V+ IM, V+ ID or V-) included 8 inoculated and 8 contact piglets. During the postvaccination and postchallenge phases, PRRSV replication (RT-PCR), PRRSV-specific antibodies (ELISA IgG and IgA, virus neutralization tests) and cell-mediated immunity (ELISPOT Interferon gamma) were monitored in blood and bronchoalveolar lavages (BALs). RESULTS: Postvaccination, vaccine viremia was lower in V+ ID pigs than in V+ IM pigs, whereas the cell-mediated immune response was detected earlier in the V+ ID group at 2 weeks postvaccination. In the BAL fluid, a very low mucosal immune response (humoral and cellular) was detected. Postchallenge, the vaccine efficacy was similar in inoculated animals with partial control of PRRSV viremia in V+ ID and V+ IM animals. In vaccinated sentinel pigs, vaccination drastically reduced PRRSV transmission with similar estimated transmission rates and latency durations for the V+ IM and V+ ID groups. CONCLUSIONS: Our results show that the tested MLV1 induced a faster cell-mediated immune response after ID immunization two weeks after vaccination but was equally efficacious after IM or ID immunization towards a challenge four weeks later. Considering the practical and welfare benefits of ID vaccination, these data further support the use of this route for PRRS MLVs.

Classical swine fever (CSF): towards new challenges / La peste porcine classique (PPC) : vers de nouveaux défis

Classical swine fever (CSF) is a highly contagious swine-specific disease which may have a huge economic impact for porcine production. CSF is caused by a virus belonging to the Pestivirus genus, which has expanded for the past 5 years with the discovery of new species whose genetic proximity to the CSF virus could further complicate laboratory diagnosis. The various forms of the disease, and in particular the increased frequency of attenuated forms, linked to an evolution of CSF virus strains towards a reduction in their virulence, delay clinical diagnosis. Thus, a long period may elapse before an outbreak is detected, allowing the virus to circulate longer, with the risk of spreading to distant geographical areas. Efforts must be maintained in terms of surveillance and diagnostic tools development in order to detect CSF virus infection early and thus limit the spread of the disease and facilitate control measures.

La peste porcine classique (PPC) est une maladie très contagieuse, spécifique des suidés, qui continue à constituer une menace pour la production porcine malgré un statut indemne de la plupart des pays de l'Union européenne. La PPC est causée par un virus de la famille des Flaviviridae appartenant au genre Pestivirus, en extension depuis les cinq dernières années avec la découverte de nouvelles espèces, notamment chez le porc ou autres animaux de rente dont la proximité génétique avec le virus de la PPC pourrait davantage compliquer le diagnostic de laboratoire. La diversité des formes de la maladie, et notamment la fréquence accrue des formes atténuées et inapparentes liée à une évolution des souches du virus de la PPC vers une réduction de leur virulence, retarde le diagnostic clinique. Ainsi, une longue période peut s'écouler avant la détection d'un foyer, permettant au virus de la PPC de circuler plus longuement, avec le risque de diffuser vers des zones géographiques éloignées des premiers cas d'infection. Les efforts doivent être maintenus en termes de surveillance et de développement d'outils de diagnostic afin de détecter précocement une infection par le virus de la PPC et ainsi limiter la propagation de la maladie et faciliter les mesures de contrôle.

Dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype 1 PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates primarily in pulmonary alveolar macrophages (PAMs) and the resulting lung damage is influenced by strain virulence. To better understand the pathogenesis of PRRSV infection, we performed a longitudinal study of the PAM population and lung cytokines in specific pathogen-free pigs infected either with the highly pathogenic Lena strain or with the low pathogenic Finistere strain in comparison to uninfected pigs. Bronchoalveolar lavage fluid (BALF) and blood were collected to follow viral, cellular and cytokine changes in lung with respect to clinical signs and systemic events. Compared to Finistere-infected pigs, Lena-infected pigs exhibited more severe clinical signs and 10- to 100-fold higher viral loads in BALF and blood. Similarly, they showed an earlier drop in BALF cell viability and phagocytic activity along with a decrease in the macrophage count. From 8 to 15 days post-infection (dpi), monocytes increased both in BALF and blood from Lena-infected pigs. BALF and blood showed contrasting cytokine patterns, with low increase of IFN-α and TNF-α levels and high increase for IL-1α and IL-8 in BALF after Lena-infection. In contrast, in the blood, the increase was marked for IFN-α and TNF-α but limited for IL-1ß and IL-8. Down-regulation of PAM functions combined with inflammatory cytokine and monocyte recruitment may promote lung pathogenesis and virus replication in PRRSV infections with the highly pathogenic Lena strain. In contrast, the low pathogenic Finistere strain showed prolonged viral replication in lung, possibly related to the weak IFN-γ response.

Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus.

In developed countries, most of hepatitis E human cases are of zoonotic origin. Swine is a major hepatitis E virus (HEV) reservoir and foodborne transmissions after pork product consumption have been described. The risk for HEV-containing pig livers at slaughter time is related to the age at infection and to the virus shedding duration. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a virus that impairs the immune response; it is highly prevalent in pig production areas and suspected to influence HEV infection dynamics. The impact of PRRSV on the features of HEV infections was studied through an experimental HEV/PRRSV co-infection of specific-pathogen-free (SPF) pigs. The follow-up of the co-infected animals showed that HEV shedding was delayed by a factor of 1.9 in co-infected pigs compared to HEV-only infected pigs and specific immune response was delayed by a factor of 1.6. HEV shedding was significantly increased with co-infection and dramatically extended (48.6 versus 9.7 days for HEV only). The long-term HEV shedding was significantly correlated with the delayed humoral response in co-infected pigs. Direct transmission rate was estimated to be 4.7 times higher in case of co-infection than in HEV only infected pigs (0.70 and 0.15 per day respectively). HEV infection susceptibility was increased by a factor of 3.3, showing the major impact of PRRSV infection on HEV dynamics. Finally, HEV/PRRSV co-infection - frequently observed in pig herds - may lead to chronic HEV infection which may dramatically increase the risk of pig livers containing HEV at slaughter time.

CP7_E2alf oral vaccination confers partial protection against early classical swine fever virus challenge and interferes with pathogeny-related cytokine responses.

The conventional C-strain vaccine induces early protection against classical swine fever (CSF), but infected animals cannot be distinguished from vaccinated animals. The CP7_E2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for C-strain vaccine to control CSF outbreaks. In this study, single oral applications of CP7_E2alf and C-strain vaccines were compared for their efficacy to induce protection against a CSF virus (CSFV) challenge with the moderately virulent Bas-Rhin isolate, in pigs as early as two days post-immunization. This work emphasizes the powerful potential of CP7_E2alf vaccine administered orally by a rapid onset of partial protection similar to that induced by the C-strain vaccine. Furthermore, our results revealed that both vaccinations attenuated the effects induced by CSFV on production of the pig major acute phase protein (PigMAP), IFN-α, IL-12, IL-10, and TGF-ß1 cytokines. By this interference, several cytokines that may play a role in the pathogeny induced by moderately virulent CSFV strains were revealed. New hypotheses concerning the role of each of these cytokines in CSFV pathogeny are discussed. Our results also show that oral vaccination with either vaccine (CP7_E2alf or C-strain) enhanced CSFV-specific IgG2 production, compared to infection alone. Interestingly, despite the similar antibody profiles displayed by both vaccines post-challenge, the production of CSFV-specific IgG1 and neutralizing antibodies without challenge was lower with CP7_E2alf vaccination than with C-strain vaccination, suggesting a slight difference in the balance of adaptive immune responses between these vaccines.

Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that has a significant socio-economic impact. One concern associated with this disease is the ability of its etiological agent, the FMD virus (FMDV), to persist in its hosts through underlying mechanisms that remain to be elucidated. While persistence has been described in cattle and small ruminants, it is unlikely to occur in pigs. One of the factors limiting the progress in understanding FMDV persistence and, in particular, differential persistence is the lack of suitable in vitro models. A primary bovine cell model derived from the dorsal soft palate, which is the primary site of replication and persistence of FMDV in cattle, has been developed, and it seemed relevant to develop a similar porcine model. Cells from two sites of FMDV replication in pigs, namely, the dorsal soft palate and the oropharyngeal tonsils, were isolated and cultured. The epithelial character of the cells from the dorsal soft palate was then assessed by immunofluorescence. The FMDV-sensitivity of these cells was assessed after monolayer infection with FMDV O/FRA/1/2001 Clone 2.2. These cells were also grown in multilayers at the air-liquid interface to mimic a stratified epithelium susceptible to FMDV infection. Consistent with what has been shown in vivo in pigs, our study showed no evidence of persistence of FMDV in either the monolayer or multilayer model, with no infectious virus detected 28 days after infection. The development of such a model opens up new possibilities for the study and diagnosis of FMDV in porcine cells.

Challenge of Naïve and Vaccinated Pigs with a Vaccine-Derived Recombinant Porcine Reproductive and Respiratory Syndrome Virus 1 Strain (Horsens Strain).

In July 2019, a vaccine-derived recombinant Porcine reproductive and respiratory syndrome virus 1 strain (PRRSV-1) (Horsens strain) infected more than 40 Danish sow herds, resulting in severe losses. In the present study, the pathogenicity of the recombinant Horsens strain was assessed and compared to a reference PRRSV-1 strain using a well-characterized experimental model in young SPF pigs. Furthermore, the efficacies of three different PRRSV-1 MLV vaccines to protect pigs against challenge with the recombinant strain were assessed. Following challenge, the unvaccinated pigs challenged with the Horsens strain had significant increased viral load in serum compared to all other groups. No macroscopic changes were observed at necropsy, but tissue from the lungs and tonsils from almost all pigs were PRRSV-positive. The viral load in serum was lower in all vaccinated groups compared to the unvaccinated group challenged with the Horsens strain, and only small differences were seen among the vaccinated groups. The findings in the present study, combined with two other recent reports, indicate that this recombinant "Horsens" strain indeed is capable of inducing infection in growing pigs as well as in pregnant sows that is comparable to or even exceeding those induced by typical PRRSV-1, subtype 1 strains. However, absence of notable clinical signs and lack of significant macroscopic changes indicate that this strain is less virulent than previously characterized highly virulent PRRSV-1 strains.

Phenotypic and Genetic Evolutions of a Porcine Reproductive and Respiratory Syndrome Modified Live Vaccine after Limited Passages in Pigs.

Modified live vaccines (MLVs) against the porcine reproductive and respiratory syndrome virus (PRRSV) have been regularly associated with safety issues, such as reversion to virulence. In order to characterize the phenotypic and genetic evolution of the PRRSV-1 DV strain from the Porcilis® PRRS MLV after limited passages in pigs, three in vivo experiments were performed. Trial#1 aimed (i) at studying transmission of the vaccine strain from vaccinated to unvaccinated contact pigs. Trial#2 and Trial#3 were designed (ii) to assess the reproducibility of Trial#1, using another vaccine batch, and (iii) to compare the virulence levels of two DV strains isolated from vaccinated (passage one) and diseased contact pigs (passage two) from Trial#1. DV strain isolates from vaccinated and contact pigs from Trial#1 and Trial#2 were submitted to Next-Generation Sequencing (NGS) full-genome sequencing. All contact animals from Trial#1 were infected and showed significantly increased viremia compared to vaccinated pigs, whereas no such change was observed during Trial#2. In Trial#3, viremia and transmission were higher for inoculated pigs with passage two of the DV strain, compared with passage one. In this study, we showed that the re-adaptation of the DV strain to pigs is associated with faster replication and increased transmission of the vaccine strain. Punctually, a decrease of attenuation of the DV vaccine strain associated with clinical signs and increased viremia may occur after limited passages in pigs. Furthermore, we identified three mutations linked to pig re-adaptation and five other mutations as potential virulence determinants.

Concomitant Swine Influenza A Virus Infection Alters PRRSV1 MLV Viremia in Piglets but Does Not Interfere with Vaccine Protection in Experimental Conditions.

Modified-live vaccines (MLVs) against porcine reproductive and respiratory syndrome viruses (PRRSVs) are usually administrated to piglets at weaning when swine influenza A virus (swIAV) infections frequently occur. SwIAV infection induces a strong interferon alpha (IFNa) response and IFNa was shown to abrogate PRRSV2 MLV replication and an inherent immune response. In this study, we evaluated the impacts of swIAV infection on the replication of a PRRSV1 MLV (MLV1), post-vaccine immune responses and post-challenge vaccine efficacy at both the systemic and pulmonary levels. Piglets were either swIAV inoculated and MLV1 vaccinated 6 h apart or singly vaccinated or mock inoculated and mock vaccinated. Four weeks after vaccination, the piglets were challenged with a PRRSV1 field strain. The results showed that swIAV infection delayed MLV1 viremia by six days and post-vaccine seroconversion by four days. After the PRRSV1 challenge, the swIAV enhanced the PRRSV1-specific cell-mediated immunity (CMI) but the PRRSV1 field strain viremia was not better controlled. High IFNa levels that were detected early after swIAV infection could have been responsible for both the inhibition of MLV1 replication and CMI enhancement. Thus, whereas swIAV infection had a negative impact on humoral responses post-vaccination, it did not interfere with the protective effectiveness of the PRRSV MLV1 in our experimental conditions.

Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (swIAV) are major pathogens of the porcine respiratory disease complex, but little is known on their interaction in super-infected pigs. In this study, we investigated clinical, virological and immunological outcomes of successive infections with PRRSV-1 and H1N2 swIAV. Twenty-four specific pathogen-free piglets were distributed into four groups and inoculated either with PRRSV at study day (SD) 0, or with swIAV at SD8, or with PRRSV and swIAV one week apart at SD0 and SD8, respectively, or mock-inoculated. In PRRSV/swIAV group, the clinical signs usually observed after swIAV infection were attenuated while higher levels of anti-swIAV antibodies were measured in lungs. Concurrently, PRRSV multiplication in lungs was significantly affected by swIAV infection, whereas the cell-mediated immune response specific to PRRSV was detected earlier in blood, as compared to PRRSV group. Moreover, levels of interferon (IFN)-α measured from SD9 in the blood of super-infected pigs were lower than those measured in the swIAV group, but higher than in the PRRSV group at the same time. Correlation analyses suggested an important role of IFN-α in the two-way interference highlighted between both viral infections.

Porcine Reproductive and Respiratory Syndrome Virus Interferes with Swine Influenza A Virus Infection of Epithelial Cells.

Respiratory infections are still a major concern in pigs. Amongst the involved viruses, the porcine reproductive and respiratory syndrome virus (PRRSV) and the swine influenza type A virus (swIAV) have a major impact. These viruses frequently encounter and dual infections are reported. We analyzed here the molecular interactions between viruses and porcine tracheal epithelial cells as well as lung tissue. PRRSV-1 species do not infect porcine respiratory epithelial cells. However, PRRSV-1, when inoculated simultaneously or shortly before swIAV, was able to inhibit swIAV H1N2 infection, modulate the interferon response and alter signaling protein phosphorylations (ERK, AKT, AMPK, and JAK2), in our conditions. SwIAV inhibition was also observed, although at a lower level, by inactivated PRRSV-1, whereas acid wash treatment inactivating non-penetrated viruses suppressed the interference effect. PRRSV-1 and swIAV may interact at several stages, before their attachment to the cells, when they attach to their receptors, and later on. In conclusion, we showed for the first time that PRRSV can alter the relation between swIAV and its main target cells, opening the doors to further studies on the interplay between viruses. Consequences of these peculiar interactions on viral infections and vaccinations using modified live vaccines require further investigations.

PCV2 co-infection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs.

Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection.

Persistent viremia and presence of hepatitis E virus RNA in pig muscle meat after experimental co-infection with porcine reproductive and respiratory syndrome virus.

Although hepatitis E virus (HEV) transmission has been demonstrated after consumption of products containing infected pig liver, human cases can be also associated with other pig meat products, such as sausages. Data on HEV viremia and dissemination in muscle meat of infected animals are still sparse, especially during long-term infection. Previously, we have shown that experimental co-infection of pigs with HEV and porcine reproductive and respiratory syndrome virus (PRRSV) lengthens HEV infection up to 49 days and increases the likelihood of the presence of HEV RNA in the liver of the pig at a later stage of infection. In the present study, we show that during experimental HEV-PRRSV co-infection, prolonged HEV viremia, up to 49 days post-inoculation (dpi), is detected. The long-term viremia observed was statistically associated with the absence of HEV seroconversion. HEV RNA was also frequently detected, at a late stage of infection (49 dpi), in the three different types of muscle tested: femoral biceps, psoas major or diaphragm pillar. The HEV RNA load could reach up to 1 ·â€¯106 genome copies per gram of muscle. Detection of HEV in muscle meat was statistically associated with high HEV loads in corresponding liver and fecal samples. The presence of HEV in pig blood, femoral biceps and major psoas, corresponding to ham and tenderloin muscles respectively, is of concern for the food industry. Hence, these results indicate new potential risks for consumers and public health regarding pork products.

Assessment of pulmonary tissue responses in pigs challenged with PRRSV Lena strain shows better protection after immunization with field than vaccine strains.

The porcine reproductive and respiratory syndrome virus (PRRSV) is plaguing porcine production. Previously piglets were immunized with a PRRSV-1 commercial modified live virus vaccine (MLV1), a PRRSV-2 MLV (MLV2) or a Western European strain (Finistere: Fini) to assess and compare the protection brought by these strains upon challenge with virulent Lena strain. Lena viremia was reduced in all the immunized groups with a slightly higher level of protection following immunization with Fini. Using lung samples collected from the same experiment, tissue response to Lena challenge was assessed at the acute and chronic stages of infection. A pre-immunization with any one of the three PRRSV strains globally exacerbated microscopic lung lesions. Ten days post-challenge (DPC), MLV1 group score was higher than unimmunized group score and 42 DPC, MLV2 group score was higher than in unimmunized group. Lowest lung Lena viral loads were measured in Fini group. Using principal component analysis, we showed 10 DPC that the lesion score was positively correlated with chemokine receptors and negatively correlated with viral load. Forty-two DPC, variables for lesion score, IL6, IL8, and CCL20 transcripts were positively correlated together and negatively correlated with CCL28, CXCL6, and CXCR4 transcripts suggesting a role for the latter ones in the tissue recovery process. In conclusions, our study shows a significant impact of the three immunizations on pulmonary tissue with the best protection against Lena challenge conferred by Fini strain. Furthermore, it gives insight into the interactions between vaccine and Fini strains and the lung upon Lena challenge.

A Field Recombinant Strain Derived from Two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) Modified Live Vaccines Shows Increased Viremia and Transmission in SPF Pigs.

In Europe, modified live vaccines (MLV) are commonly used to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, they have been associated with safety issues such as reversion to virulence induced by mutation and/or recombination. On a French pig farm, we identified a field recombinant strain derived from two PRRSV-1 MLV (MLV1). As a result, we aimed to evaluate its clinical, virological, and transmission parameters in comparison with both parental strains. Three groups with six pigs in each were inoculated with either one of the two MLV1s or with the recombinant strain; six contact pigs were then added into each inoculated group. The animals were monitored daily for 35 days post-inoculation (dpi) for clinical symptoms; blood samples and nasal swabs were collected twice a week. PRRS viral load in inoculated pigs of recombinant group was higher in serum, nasal swabs, and tonsils in comparison with both vaccine groups. The first viremic contact pig was detected as soon as 2 dpi in the recombinant group compared to 10 and 17 dpi for vaccine groups. Estimation of transmission parameters revealed fastest transmission and longest duration of infectiousness for recombinant group. Our in vivo study showed that the field recombinant strain derived from two MLV1s demonstrated high viremia, shedding and transmission capacities.

Maternally-derived neutralizing antibodies reduce vaccine efficacy against porcine reproductive and respiratory syndrome virus infection.

Modified live virus (MLV) vaccines are commonly used to reduce the impact of porcine reproductive and respiratory syndrome (PRRS) but limited efficacy is achieved in field conditions. Here, we evaluated the impact of maternally-derived neutralizing antibodies (MDNAs) on vaccine efficacy after PRRS virus (PRRSV) challenge. Piglets with low (A-) or high (A+) MDNA levels derived from a commercial pig herd were moved to experimental facilities to be vaccinated (V+) or not (V-) with a PRRSV-1 MLV vaccine at 3 weeks of age (woa). Because of unexpectedly low vaccine detection in A-V+ piglets post-vaccination (pv), all V+ piglets received a second vaccination at 4 woa. Five weeks (W5) pv, piglets were inoculated with a PRRSV-1 field strain to evaluate vaccine protection, and were mingled 24 h later with non-inoculated piglets of similar immune status to assess viral transmission. Vaccine strain was detected at W2 pv in 69% and 6% of A-V+ and A+V+ piglets, and at W5 pv in 50% and 25% of A-V+ and A+V+ piglets, respectively. At W5 pv, 94% of A-V+ and 44% of A+V+ piglets seroconverted, with a significant IFNg response induction in the A-V+ group only. After challenge, compared to the V- inoculated group, viremia was 100-fold lower at 10 days post-infection in A-V+ whereas viremia was not significantly reduced in A+V+ piglets. A lower transmission rate was estimated for the A-V+ group: 0.15 [0.07-0.29] versus 0.44 [0.18-1.76] and 0.32 [0.14-0.68] for the A+V+ and V- groups, respectively. Investigations about the low vaccine strain detection after the first vaccination suggested a relationship between IFNa levels and vaccine strain detection in A-V+ piglets. We showed that MDNAs impair vaccine efficacy against PRRSV both in inoculated and contact piglets, probably by reducing vaccine replication. IFNa may also interfere with PRRSV vaccination. These new data could help improving vaccination protocols.

The miRNA-targeted transcriptome of porcine alveolar macrophages upon infection with Porcine Reproductive and Respiratory Syndrome Virus.

Host miRNAs are known to modulate the cell response to virus infections. We characterized the miRNA-targeted transcriptome of porcine alveolar macrophages (PAMs) at early times after infection with a subtype 1.1 strain of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). We performed the immunoprecipitation of RISC (RNA-induced Silencing Complex) followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip) to evaluate the relative enrichment or depletion of expressed genes in RISC. The miRNA-mediated regulation occurred early after PRRSV infection and decreased fast (1,241 and 141 RISC-bound genes at 7 h and 10 h post-infection, respectively); it affected several cell functions with evidence of miRNA buffering of upregulated interferon-related genes. Eight miRNAs were highly enriched in RISC of both control and infected cells with no evidence of differential expression. Although miR-335-5p was the miRNA with most predicted targets among enriched RISC-bound genes, no effects on surface markers, cytokine expression and PRRSV replication were detected upon miR-335-5p mimics of primary PAMs. Our results do not point to specific miRNA-driven mechanisms regulating the early response to infection with this PRRSV 1.1 strain and indicate that the miRNome expressed by steady-state PAMs reacts promptly to counterbalance PRRSV infection by a pervasive modulation of host functions.

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus.

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.

Estimating Parameters Related to the Lifespan of Passively Transferred and Vaccine-Induced Porcine Reproductive and Respiratory Syndrome Virus Type I Antibodies by Modeling Field Data.

The outputs of epidemiological models are strongly related to the structure of the model and input parameters. The latter are defined by fitting theoretical concepts to actual data derived from field or experimental studies. However, some parameters may remain difficult to estimate and are subject to uncertainty or sensitivity analyses to determine their variation range and their global impact on model outcomes. As such, the evaluation of immunity duration is often a puzzling issue requiring long-term follow-up data that are, most of time, not available. The present analysis aims at characterizing the kinetics of antibodies against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) from longitudinal data sets. The first data set consisted in the serological follow-up of 22 vaccinated gilts during 21 weeks post-vaccination (PV). The second one gathered the maternally derived antibodies (MDAs) kinetics in piglets from three different farms up to 14 weeks of age. The peak of the PV serological response against PRRSv was reached 6.9 weeks PV on average with an average duration of antibodies persistence of 26.5 weeks. In the monitored cohort of piglets, the duration of passive immunity was found relatively short, with an average duration of 4.8 weeks. The level of PRRSv-MDAs was found correlated with the dams' antibody titer at birth, and the antibody persistence was strongly related to the initial MDAs titers in piglets. These results evidenced the importance of PRRSv vaccination schedule in sows, to optimize the delivery of antibodies to suckling piglets. These estimates of the duration of active and passive immunity could be further used as input parameters of epidemiological models to analyze their impact on the persistence of PRRSv within farms.

Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.