Porcine Reproductive And Respiratory Syndrome Virus Antigen Research Articles

Characterization of in vitro viral neutralization targets of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in alveolar macrophage and evaluation of protection potential against HP-PRRSV challenged based on combination of HP-PRRSV-structure proteins in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global pork industry, resulting in substantial economic losses. Current control measures rely on modified live virus (MLV) vaccines with safety concerns. However, the lack of consensus on protective PRRSV antigens is impeding the development of effective and safety subunit vaccines. In this study, we conducted in vitro virus neutralization (VN) assays in MARC-145 and CRL-2843CD163/CD169 cell lines and primary porcine alveolar macrophages (PAMs) to systemically identify PRRSV structural proteins (SPs) recognized by virus-neutralizing antibodies in hyperimmune serum collected from piglets infected with highly pathogenic PRRSV (HP-PRRSV). Additionally, piglets immunized with different combinations of recombinant PRRSV-SPs were challenged with HP-PRRSV to evaluate their in vivo protection potential. Intriguingly, different in vitro VN activities of serum antibodies elicited by each PRRSV SP were observed depending on the cell type used in the VN assay. Notably, antibodies specific for GP3, GP4, and M exhibited highest in vitro VN activities in PAMs, correlating with complete protection (100% survival) against HP-PRRSV challenge in vivo after immunization of piglets with combination of GP3, GP4, M and N (GP3/GP4/M/N). Further analysis of lung pathology, weight gain, and viremia post-challenge revealed that the combination of GP3/GP4/M/N provided superior protective efficacy against severe infection. These findings underscore the potential of this SP combination to serve as an effective PRRSV subunit vaccine, marking a significant advancement in pork industry disease management.

Genetic and Pathogenic Characteristics of an Emerging Highly Virulent Recombinant Lineage Korean Clade C PRRSV Strain

A strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from lung tissue of a pig showing severe respiratory clinical signs from a farm in Gyeongsang province of South Korea. This PRRSV strain, designated as SNUVR220803, was classified within the lineage Korean clade C (LKC) based on a phylogenetic analysis of the ORF5 gene. A whole‐genome analysis was conducted on the SNUVR220803 strain, which appears to be a recombinant between the PRRSV strains K07‐2273 (part of LKC lineage) and Ingelvac MLV (part of Lineage 5). The Nsp2 amino acid sequence of this strain features a deletion of four additional amino acids, setting it apart from the typical Korean clades A, B, and C lineages. An animal inoculation experiment was conducted with 24 pigs divided into three groups: 12 pigs in the inoculated group, six in the sentinel group, and six in the negative control group. Inoculated pigs exhibited persisting hyperthermia (≥40.3°C) for 5 days, palpebral edema, and cyanosis. Subsequently, these pigs suffered from severe respiratory distress and cachexia, leading to a mortality rate of 58.3% (7 out of 12 pigs) at 14 days postinoculation (dpi). Body weight decreased post‐SNUVR220803 strain infection in both the inoculated and sentinel groups. Gross pathology revealed noncollapsed lungs and serous effusion in the pericardial and peritoneal cavities. Microscopic analysis revealed severe interstitial pneumonia, while immunohistochemistry confirmed the presence of PRRSV antigen in the lungs, lymph nodes, thymus, kidneys, and the heart. Additionally, the levels of cytokines such as tumor necrosis factor‐α (TNF‐α), interferon‐α (IFN‐α), and IL‐10 were significantly elevated in the plasma of infected pigs. These observations indicate that the LKC recombinant strain, combined with Lineage 5, possesses high virulence and infectivity as characterized by distinctive exudative lesions.

Fluorescent immunosensor based on fluorescence resonance energy transfer between CdSe/ZnS quantum dots and Au nanorods for PRRSV detection

An ultrasensitive and selective sandwich-type fluorescent immunosensor based on fluorescence resonance energy transfer (FRET) between CdSe/ZnS quantum dots (QDs) and Au nanorods (AuNRs) was developed for the rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV). Modified CdSe/ZnS QDs and AuNRs were bioconjugated with primary antibodies M and GP5, respectively. When the target antigen was present, a well-known sandwich-type form CdSe/ZnS QDs-M antibody-antigen-GP5 antibody-AuNRs was produced, and thus reducing the distance between the QDs and NRs. As a result, FRET occurred and the fluorescence intensity of CdSe/ZnS QDs decreased, and this decrease was used to determine the antigen concentration. Ultrasensitive detection of PRRSV at low concentrations in swine serum was achieved with a limit of detection of 0.55 TCID50/mL and a linear detection range of 101–3.5 ​× ​104 TCID50/mL. Therefore, the fluorescent immunosensor is selective and highly sensitive; specifically, it can detect PRRSV at low concentrations in swine serum over a large concentration range. This proposed detection method can facilitate the development of high-performance fluorescent immunosensors for the detection of PRRSV and other antigens.

Polyplex nanomicelle delivery of self-amplifying RNA vaccine

Self-amplifying RNA (SaRNA) is a burgeoning platform that exploits the replication machinery of alphaviruses such as Venezuelan equine encephalitis (VEE) virus or Sindbis virus (SIN). SaRNA has been used for development of human vaccines, but has not been evaluated for porcine vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the worldwide pork industry, but current vaccines trigger delayed neutralizing antibody response and confer only partial protection. Here we first compared two SaRNA systems based on VEE and SIN, and demonstrated that in vitro transcribed VEE-based SaRNA conferred prolonged reporter gene expression and RNA amplification in pig cells with low cytotoxicity, but SIN-based SaRNA imparted evident cytotoxicity and limited gene expression in pig cells. Transfection of VEE-based SaRNA that encodes the major PRRSV antigen dNGP5 (SaRNA-dNGP5) conferred persistent expression for at least 28 days in pig cells. We next complexed SaRNA-dNGP5 with the polyaspartamide block copolymer PEG-PAsp(TEP) to form polyplex nanomicelle with high packaging efficiency and narrow size distribution. The polyplex nanomicelle enabled sustained dNGP5 expression and secretion in vitro. Compared with the commercial PRRS vaccine, nanomicelle delivery of SaRNA-dNGP5 into animal models accelerated the induction of potent neutralizing antibodies with minimal side effects, and elicited stronger IL-4 and IFN-γ responses against homologous and heterologous PRRSV. These properties tackle the problems of current vaccines and implicate the potential of SaRNA-dNGP5 nanomicelle as an effective PRRS vaccine.

Development and use of indirect liquid-phase ELISA test system for detection of PRRS virus antigen during in-process control of raw materials intended for vaccine production

Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board.

Cell-mediated immune response and protective efficacy of porcine reproductive and respiratory syndrome virus modified-live vaccines against co-challenge with PRRSV-1 and PRRSV-2

Cell-mediated immunity (CMI), IL-10, and the protective efficacy of modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines (MLV) against co-challenge with PRRSV-1 and PRRSV-2 (HP-PRRSV) were investigated. Seventy, PRRSV-free, 3-week old, pigs were allocated into 7 groups. Six groups were intramuscularly vaccinated with MLV, including Porcilis (PRRSV-1 MLV, MSD Animal Health, The Netherlands), Amervac (PRRSV-1 MLV, Laboratorios Hipra, Spain), Fostera (PRRSV-2 MLV, Zoetis, USA), Ingelvac PRRS MLV and Ingelvac PRRS ATP (PRRSV-2, Boehringer Ingelheim, USA), and Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, The Netherlands). Unvaccinated pigs were left as control. Lymphocyte proliferative response, IL-10 and IFN-γ production were determined. At 35 days post-vaccination (DPV), all pigs were inoculated intranasally with 2 ml of each PRRSV-1 (105.4 TCID50/ml) and PRRSV-2 (105.2 TCID50/ml, HP-PRRSV). Following challenge, sera were quantitatively assayed for PRRSV RNA. Pigs were necropsied at 7 days post-challenge. Viremia, macro- and microscopic lung lesion together with PRRSV antigen presence were evaluated in lung tissues. The results demonstrated that, regardless of vaccine genotype, CMI induced by all MLVs was relatively slow. Increased production of IL-10 in all vaccinated groups was observed at 7 and 14 DPV. Pigs in Amervac, Ingelvac MLV and Ingelvac ATP groups had significantly higher levels of IL-10 compared to Porcilis, Fostera and Prime Pac groups at 7 and 14 DPV. Following challenge, regardless to vaccine genotype, vaccinated pigs had significantly lower lung lesion scores and PRRSV antigens than those in the control group. Both PRRSV-1 and PRRSV-2 RNA were significantly reduced. Prime Pac pigs had lowest PRRSV-1 and PRRSV-2 RNA in serum, and micro- and macroscopic lung lesion scores (p < 0.05) compared to other vaccinated groups. In conclusion, PRRSV MLVs, regardless of vaccine genotype, can reduce viremia and lung lesions following co-challenge with PRRSV-1 and PRRSV-2 (HP-PRRSV). The main difference between PRRSV MLV is the production of IL-10 following vaccination.

Susceptibility of porcine pulmonary microvascular endothelial cells to porcine reproductive and respiratory syndrome virus.

Microvascular endothelial cells possess versatile functions and their roles in a variety of viral infections have been documented. Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces severe lung inflammatory lesions in piglets, which is manifested as pulmonary endothelial dysfunction. However, the underlying mechanism of PRRSV affecting porcine pulmonary microvascular endothelial cells (PMECs) remains unknown. This study aimed to evaluate the susceptibility of PMECs to PRRSV. Primary PMECs were isolated and purified from piglet lungs, and the expression of three PRRSV receptors was characterized using immunofluorescence. Overt cytopathic effects of the PRRSV strain HN in PMECs were observed at day five post-infection, and PRRSV antigens in PMECs were determined at both RNA and protein levels using immunofluorescence and quantitative RT-PCR assays. The viral antigen significantly increased at 96 hr post-infection, and infectious virus was recovered from the supernatant of the infected PMECs. The results show that PMECs can be infected with the PRRSV strain HN, and that their receptor expression pattern is different from that of alveolar macrophages. The results of this study shed light on the potential roles of PMECs in PRRSV infection and provide a comprehensive understanding of the pathogenesis underlying its severe manifestation.

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1β, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2posCD172Apos CD163int and CD163neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.

A DNA Prime Immuno-Potentiates a Modified Live Vaccine against the Porcine Reproductive and Respiratory Syndrome Virus but Does Not Improve Heterologous Protection.

The porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus inducing abortion in sows and respiratory disease in young pigs, is a leading infectious cause of economic losses in the swine industry. Modified live vaccines (MLVs) help in controlling the disease, but their efficacy is often compromised by the high genetic diversity of circulating viruses, leading to vaccine escape variants in the field. In this study, we hypothesized that a DNA prime with naked plasmids encoding PRRSV antigens containing conserved T-cell epitopes may improve the protection of MLV against a heterologous challenge. Plasmids were delivered with surface electroporation or needle-free jet injection and European strain-derived PRRSV antigens were targeted or not to the dendritic cell receptor XCR1. Compared to MLV-alone, the DNA-MLV prime- boost regimen slightly improved the IFNγ T-cell response, and substantially increased the antibody response against envelope motives and the nucleoprotein N. The XCR1-targeting of N significantly improved the anti-N specific antibody response. Despite this immuno-potentiation, the DNA-MLV regimen did not further decrease the serum viral load or the nasal viral shedding of the challenge strain over MLV-alone. Finally, the heterologous protection, achieved in absence of detectable effective neutralizing antibodies, was not correlated to the measured antibody or to the IFNγ T-cell response. Therefore, immune correlates of protection remain to be identified and represent an important gap of knowledge in PRRSV vaccinology. This study importantly shows that a naked DNA prime immuno-potentiates an MLV, more on the B than on the IFNγ T-cell response side, and has to be further improved to reach cross-protection.

Dendritic cell-targeted porcine reproductive and respiratory syndrome virus (PRRSV) antigens adjuvanted with polyinosinic-polycytidylic acid (poly (I:C)) induced non-protective immune responses against heterologous type 2 PRRSV challenge in pigs

Porcine Reproductive and Respiratory Syndrome (PRRS) is an economically important swine viral disease worldwide. Current modified live-attenuated vaccines are ineffective against heterologous strains of PRRS virus (PRRSV) circulating in the field. In this study, we evaluated three dendritic cell (DC)-targeted vaccine candidates for their protective efficacy against heterologous PRRSV challenge. Ectodomain regions of DNA-shuffled structural proteins GP3, GP4, GP5 and M of PRRSV were fused together to form the vaccine antigen which was in turn fused with one of three recombinant antibodies each specific to a DC receptor: DC-SIGN, Langerin, and DEC205. The recombinant antibody-fused vaccine antigens were co-administered with polyinosinic-polycytidylic acid (poly (I:C)) adjuvant and subsequently challenged with a heterologous type 2 PRRSV strain (NADC20) in pigs. Our results demonstrate that pigs in DC-SIGN- and DEC205-targeted, but not Langerin- and non-targeted, vaccine groups showed significant IFN-γ- and IL-4-specific CD4T cell immune responses against the vaccine antigen in 7days post-challenge. Pigs in DC-SIGN- and Langerin-targeted vaccine groups showed greatly reduced IgG responses as compared to the DEC205- and non-targeted vaccine groups. The immune responses induced by DC-targeted vaccines did not reduce viremia and lung pathological lesions in type 2 PRRSV-challenged pigs. In contrast, pigs in Langerin-targeted vaccine group showed significantly increased serum viral titers and viral antigen in lung tissues at 7 and 14days post-challenge respectively. In conclusion, specific targeting of PRRSV antigen through DC-SIGN or DEC205 or Langerin-specific antibodies in the presence of poly (I:C) adjuvant induced immune responses that failed to protect pigs against heterologous type 2 PRRSV challenge.

Type 2 porcine reproductive and respiratory syndrome virus infection increases apoptosis at the maternal-fetal interface in late gestation pregnant gilts.

The pathogenesis of fetal death associated with porcine reproductive and respiratory syndrome (PRRS) is hypothesized to be a consequence of PRRS virus-induced apoptosis at the maternal-fetal interface (MFI). The objectives of this study were to evaluate distribution and degree of apoptosis in the uterine and fetal placental tissues during the experimental type 2 PRRS virus (PRRSV) infection and determine associations between apoptosis at the MFI, PRRSV RNA concentration and antigen staining intensity, PRRSV-induced microscopic lesions, and fetal preservation status. A total of 114 naïve, high-health pregnant gilts were inoculated with type 2 PRRSV on gestation day 85±1 with euthanasia 21 days later; 19 sham-inoculated gilts served as controls. Two hundred and fifty samples of uterine tissue with fetal placenta were selected based on negative, low PRRSV RNA, and high PRRSV RNA concentration (0, < or > 2.7 log10 copies/mg, respectively). TUNEL assay was used to detect apoptosis in the endometrium and at the MFI. PRRSV RNA concentration and numbers of PRRSV immunopositive cells in uterine and placental tissue were positively associated with the severity of apoptosis in the endometrium and the MFI (P<0.001, P<0.05 and P<0.001, respectively). The number of TUNEL positive cells at the MFI was also positively associated with the severity (P<0.001) of vasculitis, but not total numbers of inflammatory cells in the endometrium. Increased numbers of TUNEL positive cells at the MFI were associated with PRRSV load in the fetal thymus, and greater odds of meconium staining of the fetus at 21 days post infection (P<0.001 for both). These findings suggest an important role of apoptosis in the pathogenesis of uterine epithelial and trophoblastic cell death at the MFI. Moreover, apoptosis at the MFI is significantly associated with fetal demise during in utero type 2 PRRSV infection.

Fluorescence Microsphere Immunoassay for Detection of Antibodies to Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus type 2 using Protein A, Protein G, and Protein A/G

The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) specific IgG antibodies by incorporation of non-species-specific conjugates Protein A, G, and A/G in place of the secondary antibody. A total of 205 serum samples obtained from pigs experimentally infected with PRRSV and/or PCV2 were tested. For the production of recombinant antigens, PRRSV nucleoprotein (N) and PCV2 capsid protein (CP) were expressed in Escherichia coli and purified on a nickel affinity column. The purified proteins were covalently coupled to carboxylated microsphere beads. Four target antigens were assembled into a single multiplex and tested against sera from swine infected or co-infected with PRRSV or PCV2. Protein A, G, and A/G have been tested in place of secondary conjugate porcine IgG. All conjugates were capable of detecting antibody and the detection of IgG responses against PRRSV and PCV2 antigen targets were varied with A> A/G> G.In the absence of species specific reagents the incorporation of Protein A, G, and A/G provide a suitable substitutes.

A porcine reproductive and respiratory syndrome virus candidate vaccine based on the synthetic attenuated virus engineering approach is attenuated and effective in protecting against homologous virus challenge

Current porcine reproductive and respiratory syndrome virus (PRRSV) vaccines sometimes fail to provide adequate immunity to protect pigs from PRRSV-induced disease. This may be due to antigenic differences among PRRSV strains. Rapid production of attenuated farm-specific homologous vaccines is a feasible alternative to commercial vaccines. In this study, attenuation and efficacy of a codon-pair de-optimized candidate vaccine generated by synthetic attenuated virus engineering approach (SAVE5) were tested in a conventional growing pig model. Forty pigs were vaccinated intranasally or intramuscularly with SAVE5 at day 0 (D0). The remaining 28 pigs were sham-vaccinated with saline. At D42, 30 vaccinated and 19 sham-vaccinated pigs were challenged with the homologous PRRSV strain VR2385. The experiment was terminated at D54. The SAVE5 virus was effectively attenuated as evidenced by a low magnitude of SAVE5 viremia for 1–5 consecutive weeks in 35.9% (14/39) of the vaccinated pigs, lack of detectable nasal SAVE5 shedding and failure to transmit the vaccine virus from pig to pig. By D42, all vaccinated pigs with detectable SAVE5 viremia also had detectable anti-PRRSV IgG. Anti-IgG positive vaccinated pigs were protected from subsequent VR2385 challenge as evidenced by lack of VR2385 viremia and nasal shedding, significantly reduced macroscopic and microscopic lung lesions and significantly reduced amount of PRRSV antigen in lungs compared to the non-vaccinated VR2385-challenged positive control pigs. The nasal vaccination route appeared to be more effective in inducing protective immunity in a larger number of pigs compared to the intramuscular route. Vaccinated pigs without detectable SAVE5 viremia did not seroconvert and were fully susceptible to VR2385 challenge. Under the study conditions, the SAVE approach was successful in attenuating PRRSV strain VR2385 and protected against homologous virus challenge. Virus dosage likely needs to be adjusted to induce replication and protection in a higher percentage of vaccinated pigs.

Evaluation of a 20 year old porcine reproductive and respiratory syndrome (PRRS) modified live vaccine (Ingelvac® PRRS MLV) against two recent type 2 PRRS virus isolates in South Korea

Type 2 porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) was first isolated in Korea in 1994. The commercial PRRS modified live vaccine (Ingelvac® PRRS MLV, Boehringer Ingelheim Vetmedica Inc., St. Joseph, Missouri, USA) based on type 2 PRRSV, was first licensed for use in 3- to 18-week-old pigs in Korea in 1996. The objective of the present study was to evaluate the efficacy of this 20year old commercial PRRS modified live vaccine (MLV) against two recent PRRSV isolates. Two genetically distant type 2 PRRSV strains (SNUVR150004 for lineage 1 and SNUVR150324 for lineage 5), isolated in 2015, were used as challenge virus. Regardless of the challenge virus, vaccination of pigs effectively reduced the level of viremia, the lung lesions, and of the PRRSV antigen within the lung lesions. The induction of virus-specific interferon-γ secreting cells by the PRRS vaccine produced a protective immune response, leading to the reduction of PRRSV viremia. There were no significant differences in efficacy against the two recently isolated viruses by the PRRS MLV based on virological results, immunological responses, and pathological outcomes. This study demonstrates that the PRRS MLV used in this study is still effective against recently isolated heterologous type 2 PRRSV strains even after 20 years of use in over 35 million pigs

Synthetic Toll-like receptor 7 ligand inhibits porcine reproductive and respiratory syndrome virus infection in primary porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV), a common viral pathogen, causes huge annual economic losses to the swine industry worldwide. After triggering by specific ligands, the Toll-like receptor 7 (TLR7), a type of pattern-recognition receptor (PRR), induces antiviral cytokines production. Previously, we synthesized an adenine analog, designated SZU101, a TLR7-specific ligand. In this study, we assessed the inhibitory effect of SZU101 on PRRSV infection in vitro. SZU101 significantly suppressed PRRSV infection in primary porcine alveolar macrophages (PAMs) in a dose-dependent manner. Moreover, SZU101-induced inhibition involved NF-κB pathway activation in PAMs to initiate expression of TLR7-mediated cytokines and induce expression of downstream signaling IFN-stimulated genes (ISGs). Chloroquine, a TLR7 inhibitor, and BAY 11-7082, an NF-κB inhibitor, reversed both the SZU101-induced antiviral effect and induction of cytokine genes and ISGs expression. Therefore, SZU101 antiviral effects depend at least in part on TLR7-NF-κB signaling pathway. Additionally, administration of SZU101 enhanced the humoral and cell-mediated immune responses against PRRSV antigens in mice. Given these results, SZU101 holds promise as an antiviral agent and a vaccine adjuvant to prevent PRRSV infection in pigs.

Thymic depletion of lymphocytes is associated with the virulence of PRRSV-1 strains

Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3+ thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p≤0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1α, TNF-α and IL-10 were predominantly detected in the medulla (p≤0.01). An increase in the number of TNF-α and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

Cross-protection of a new type 2 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine (Fostera PRRS) against heterologous type 1 PRRSV challenge in growing pigs

The objective of the present study was to determine the cross-protection of a new type 2 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine against heterologous type 1 PRRSV challenge in growing pigs. The mean rectal temperature and respiratory score was significantly (P<0.05) lower in vaccinated challenged pigs than in unvaccinated challenged pigs. Vaccination of pigs with type 2 PRRSV reduced the levels of type 1 PRRSV viremia after challenge with type 1 PRRSV. Vaccinated challenged pigs had significantly (P<0.05) higher frequency of interferon-γ secreting cells and lower levels of interleukin-10 compared to unvaccinated challenged pigs. Vaccination of pigs with the type 2 PRRSV effectively reduced the macroscopic and microscopic lung lesion and the type 1 PRRSV antigens within lung lesions in vaccinated challenged pigs. This study demonstrates partial cross-protection of a new type 2 PRRSV modified live vaccine against heterologous type 1 PRRSV challenge in growing pigs.

Prevalence of porcine reproductive and respiratory syndrome virus detection in aborted fetuses, mummified fetuses and stillborn piglets using quantitative polymerase chain reaction.

The objective of the present study was to investigate the prevalence of porcine reproductive and respiratory syndrome (PRRS) virus detection in aborted fetuses (n=32), mummified fetuses (n=30) and stillborn piglets (n=27) from 10 swine herds in Thailand using quantitative polymerase chain reaction (qPCR). Pooled organs and umbilical cord from each fetus/piglet were homogenized and subjected to RNA extraction and cDNA synthesis. The qPCR was carried out on the ORF7 of the PRRS viral genome using fluorogenic probes for amplified product detection. The results revealed that 67.4% (60/89) of the specimens contained PRRS virus. The virus was found in 65.6% (21/32) of aborted fetuses, 63.3% (19/30) of mummified fetuses and 74.1% (20/27) of stillborn piglets (P=0.664). Genotype 1, genotype 2 and mixed genotypes of PRRS virus were detected in 19.1% (17/89), 25.8% (23/89) and 22.5% (20/89) of the specimens, respectively (P=0.316). PRRS virus antigen was retrieved from both non-PRRS-vaccinated herds (68.2%, 45/66) and PRRS-vaccinated herds (65.2%, 15/23) (P=0.794). These findings indicated that these specimens are important sources of the PRRS viral load and the viral shedding within the herd. Thus, intensive care on the routine management of dead fetuses and stillborn piglets in PRRS virus-positive herds should be emphasized.

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.