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Porcine reproductive and respiratory syndrome virus (PRRSV) represents a macrophage (MØ)-tropic virus which is unable to induce interferon (IFN) type I in its target cells. Nevertheless, infected pigs show a short but prominent systemic IFN alpha (IFN-α) response. A possible explanation for this discrepancy is the ability of plasmacytoid dendritic cells (pDC) to produce IFN-α in response to free PRRSV virions, independent of infection. Here, we show that the highly pathogenic PRRSV genotype 1 strain Lena is unique in not inducing IFN-α production in pDC, contrasting with systemic IFN-α responses found in infected pigs. We also demonstrate efficient pDC stimulation by PRRSV Lena-infected MØ, resulting in a higher IFN-α production than direct stimulation of pDC by PRRSV virions. This response was strain-independent, required integrin-mediated intercellular contact, intact actin filaments in the MØ and was partially inhibited by an inhibitor of neutral sphingomyelinase. Although infected MØ-derived exosomes stimulated pDC, an efficient delivery of the stimulatory component was dependent on a tight contact between pDC and the infected cells. In conclusion, with this mechanism the immune system can efficiently sense PRRSV, resulting in production of considerable quantities of IFN-α. This is adding complexity to the immunopathogenesis of PRRSV infections, as IFN-α should alert the immune system and initiate the induction of adaptive immune responses, a process known to be inefficient during infection of pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Japanese encephalitis virus (JEV), a main cause of severe viral encephalitis in humans, has a complex ecology, composed of a cycle involving primarily waterbirds and mosquitoes, as well as a cycle involving pigs as amplifying hosts. To date, JEV transmission has been exclusively described as being mosquito-mediated. Here we demonstrate that JEV can be transmitted between pigs in the absence of arthropod vectors. Pigs shed virus in oronasal secretions and are highly susceptible to oronasal infection. Clinical symptoms, virus tropism and central nervous system histological lesions are similar in pigs infected through needle, contact or oronasal inoculation. In all cases, a particularly important site of replication are the tonsils, in which JEV is found to persist for at least 25 days despite the presence of high levels of neutralizing antibodies. Our findings could have a major impact on the ecology of JEV in temperate regions with short mosquito seasons.

Several mosquito-borne Flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV) can cause severe clinical disease. Being zoonotic, Flaviviruses infect a wide variety of terrestrial vertebrates, which dependent of the virus-host interactions, can enhance ongoing epidemics and maintain the virus in the environment for prolonged periods. Targeted species can vary from amphibians, birds to various mammals, dependent on the virus. For many mosquito-borne flaviviruses the spectrum of targeted species is incompletely understood, in particular with respect to their contribution to the maintenance of virus in certain geographical regions. Furthermore, little is known about virus and host factors contributing to species tropism. The present study utilized human and porcine monocyte-derived dendritic cells (MoDC) as a cell culture model to better understand Flavivirus species tropism and innate immune responses. MoDC were selected based on their presence in the skin and their role as an early target cell for several Flaviviruses and their role as immune sentinels. While differences in viral infectivity and replication were minor when comparing porcine with human MoDC for some of the tested Flaviviruses, a particularly strong replication in human MoDC was found with USUV, while JEV appeared to have a stronger tropism for porcine MoDC. With respect to innate immune responses we found high induction of TNF and IFN-β in both human and porcine MoDC after infection with JEV, WNV, and USUV, but not with DENV, ZIKV, and Wesselsbron virus. Spondweni virus induced these cytokine responses only in porcine MoDC. Overall, innate immune responses correlated with early infectivity and cytokine production. In conclusion, we demonstrate Flavivirus-dependent differences in the interaction with MoDC. These may play a role in pathogenesis but appear to only partially reflect the expected species tropism.

The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as and models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs. JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as and models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.

Flaviviruses replicate in a wide variety of species and have a broad cellular tropism. They are isolated from various body fluids, and Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) RNAs have been detected in nasopharyngeal swabs. Consequently, we evaluated the cellular tropism and host responses upon ZIKV, JEV, WNV, and Usutu virus (USUV) infection using a relevant model of the human upper respiratory tract epithelium based on primary human nasal epithelial cells (NECs) cultured at the air-liquid interface. NECs were susceptible to all the viruses tested, and confocal analysis showed evidence of infection of ciliated and non-ciliated cells. Each flavivirus productively infected NECs, leading to apical and basolateral live virus shedding with particularly high basal release for JEV and WNV. As demonstrated by a paracellular permeability assay, the integrity of the epithelium was not affected by flavivirus infection, suggesting an active release of live virus through the basolateral surface. Also, we detected a significant secretion of interferon type III and the pro-inflammatory cytokine IP-10/CXCL10 upon infection with JEV. Taken together, our data suggest that the human upper respiratory tract epithelium is a target for flaviviruses and could potentially play a role in the spread of infection to other body compartments through basolateral virus release. Undoubtedly, further work is required to evaluate the risks and define the adapted measures to protect individuals exposed to flavivirus-contaminated body fluids.

Pigs are considered to be the main amplifying host for Japanese encephalitis virus (JEV), and their infection can correlate with human cases of disease. Despite their importance in the ecology of the virus as it relates to human cases of encephalitis, the pathogenesis of JEV in pigs remains obscure. In the present study, the localization and kinetics of virus replication were investigated in various tissues after experimental intravenous infection of pigs. The data demonstrate a rapid and broad spreading of the virus to the central nervous system (CNS) and various other organs. A particular tropism of JEV in pigs not only to the CNS but also for secondary lymphoid tissue, in particular the tonsils with the overall highest viral loads, was observed. In this organ, even 11 days post infection, the latest time point of the experiment, no apparent decrease in viral RNA loads and live virus was found despite the presence of a neutralizing antibody response. This was also well beyond the clinical and viremic phase. These results are of significance for the pathogenesis of JEV, and call for further experimental studies focusing on the cellular source and duration of virus replication in pigs.

We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against Mycoplasma hyopneumoniae. Seven groups of six M. hyopneumoniae-free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce M. hyopneumoniae-specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.

While Zika virus (ZIKV) circulated for decades (African lineage strains) without report of outbreaks and severe complications, its emergence in French Polynesia and subsequently in the Americas (Asian lineage strains) was associated with description of severe neurological defects in newborns/neonates and adults. With the aim to identify virus lineage-dependent factors, we compared cell susceptibility, virus replication, cell death and innate immune responses following infection with two African and three contemporary Asian lineage strains of ZIKV. To this end, we used green monkey Vero and Aedes albopictus C6/36 cells and human monocyte-derived dendritic cells (DCs). The latter are involved in the pathogenesis of several mosquito-borne Flavivirus infections. In Vero and C6/36 cells, we observed strain- but not lineage-dependent differences in infection profiles. Nevertheless, in human DCs, no significant differences in susceptibility and virus replication were found between lineages and strains. ZIKV induced antiviral interferon type I/III in a limited fashion, with the exception of one African strain. None of the strains induced cell death or DC maturation in terms of MHC II, CD40, CD80/86 or CCR7 expression. Taken together, our data suggest that a large collection of virus isolates needs to be investigated before conclusions on lineage differences can be made.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus responsible for a widespread contagious disease of domestic pigs with high economic impact. Switzerland is one of the rare PRRSV-free countries in Europe, although sporadic outbreaks have occurred in the past. The PRRSV isolate IVI-1173 from the short outbreak in Switzerland in 2012 was entirely sequenced, and a functional full-length cDNA clone was constructed. Genetic and antigenic characterization of IVI-1173 revealed the importance of amino acid 90 of the nucleocapsid protein N as part of a conformational epitope. IVI-1173 was not detected by SDOW17, a monoclonal antibody against N widely used to detect PRRSV-infected cells. Substitution of alanine at position 90 of N [N(A90)] with a threonine [N(T90)] restored reactivity of vIVI1173-N(T90) to SDOW17 completely. The relevance of this amino acid for the conformational SDOW17 epitope of PRRSV N was further confirmed by the opposite substitution in a functional cDNA clone of the genotype 2 isolate RVB-581. Finally, N proteins from ten genotype 1 strains differing from threonine at position 90 were analysed for reactivity with SDOW17. N(A90) totally disrupted or severely affected the epitope in 7 out of 8 strains tested. Based on these findings, 225 genotype 1 strains were screened for the prevalence of N(A90). N(A90) is rare in classical subtype 1 and in subtype 3 strains, but is frequent in Russian subtype 1 (70%) and in subtype 2 (45%) isolates. In conclusion, this study highlights the variable antigenic properties of N among genotype 1 PRRSV strains.