An effective vaccine against influenza A virus based on the matrix protein 2 (M2).

The ever-increasing antigenic diversity of the hemagglutinin (HA) of influenza A virus (IAV) poses a significant challenge for effective vaccine development. Notably, the matrix protein 2 (M2) is a highly conserved 97 amino acid long transmembrane tetrameric protein present in the envelope of IAV. More than 99 % of IAV strains circulating in American swine herds share the identical pandemic (pdm) isoform of M2, making it an ideal target antigen for a vaccine that could elicit broadly protective immunity. Here, using soluble nanoscale membrane assemblies termed nanodiscs (NDs), we designed this membrane mimetic nanostructures displaying full-length M2 in its natural transmembrane configuration (M2ND). Intramuscular (IM) immunization of swine with M2ND mixed with conventional emulsion adjuvant elicited M2-specific IgG antibodies in the serum that recognized influenza virions and M2-specific interferon-γ secreting cells present in the blood. Intranasal (IN) immunization with M2ND adjuvanted with a mycobacterial extract elicited M2-specific IgA in mucosal secretions that also recognized IAV. Immunization with an influenza whole inactivated virus (WIV) vaccine supplemented with a concurrent IM injection of M2ND mixed with an emulsion adjuvant increased the level of protective immunity afforded by the former against a challenge with an antigenically distinct H3N2 IAV, as exhibited by an enhanced elimination of virus from the lung. The lone IM administration of the M2ND vaccine mixed with an emulsion adjuvant provided measurable protection as evidenced by a >10-fold reduction or complete elimination of the challenge virus from the lung, but it did not diminish the viral load in nasal secretions nor the extent of pneumonia that ensued after the virus challenge. In contrast, an improved formulation of the M2ND vaccine that incorporated synthetic CpG oligodeoxynucleotides (CpG-ODN) in the nanostructures administered alone, via the IN and IM routes combined, provided a significant level of protective immunity against IAV as evidenced by a decreased viral load in both the upper and lower respiratory tracts and fully eliminated the occurrence of pneumonia in 89 % of the pigs immunized with this biologic. Notably, to be effective, the M2 protein must be displayed in the ND assemblies, as shown by the observation that simply mixing M2 with empty NDs incorporating CpG-ODN (eND-CpG-ODN) did not provide protective immunity. This novel M2-based vaccine offers great promise to help increase the breadth of protection afforded by conventional WIV vaccines against the diversity of IAV in circulation and, plausibly, as a broadly protective stand-alone biologic.

Lateral flow paired with RT-LAMP: A speedy solution for Influenza A virus detection in swine.

Influenza A Virus in swine (IAV-S) is a zoonotic pathogen that is nearly ubiquitous in commercial swine in the USA. Swine possess sialic acid receptors that allow co-infection of human and avian viruses with the potential of pandemic reassortment. We aimed to develop a fast and robust testing method for IAV-S detection on swine farms. Two primers of the RT-LAMP assay were labeled for use in a lateral flow readout. A commercially available lateral flow kit was used to read the amplicon product. With a runtime of ∼ 45 minutes, the limit of detection for the assay is comparable with an RT-qPCR Cq less than 35, with a sensitivity of 83.5 % and a specificity of 89.6 %. This assay allows veterinarians and producers with limited access to diagnostic services to perform and detect Matrix gene amplification on-site with low equipment costs. The time from sample collection to detection is less than one hour, making this method an accessible, convenient, and affordable tool to prevent the spread of zoonotic disease.

A clinically attenuated double-mutant of porcine reproductive and respiratory syndrome virus-2 that does not prompt overexpression of proinflammatory cytokines during co-infection with a secondary pathogen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to suppress the type I interferon (IFNs-α/ß) response during infection. PRRSV also activates the NF-κB signaling pathway, leading to the production of proinflammatory cytokines during infection. In swine farms, co-infections of PRRSV and other secondary bacterial pathogens are common and exacerbate the production of proinflammatory cytokines, contributing to the porcine respiratory disease complex (PRDC) which is clinically a severe disease. Previous studies identified the non-structural protein 1ß (nsp1ß) of PRRSV-2 as an IFN antagonist and the nucleocapsid (N) protein as the NF-κB activator. Further studies showed the leucine at position 126 (L126) of nsp1ß as the essential residue for IFN suppression and the region spanning the nuclear localization signal (NLS) of N as the NF-κB activation domain. In the present study, we generated a double-mutant PRRSV-2 that contained the L126A mutation in the nsp1ß gene and the NLS mutation (ΔNLS) in the N gene using reverse genetics. The immunological phenotype of this mutant PRRSV-2 was examined in porcine alveolar macrophages (PAMs) in vitro and in young pigs in vivo. In PAMs, the double-mutant virus did not suppress IFN-ß expression but decreased the NF-κB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV-2. Co-infection of PAMs with the mutant PRRSV-2 and Streptococcus suis (S. suis) also reduced the production of NF-κB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV-2 and S. suis. The double-mutant PRRSV-2 was clinically attenuated, and the expressions of proinflammatory cytokines and chemokines were significantly reduced in pigs after bacterial co-infection. Compared to the wild-type PRRSV-2 and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV-2 exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-κB activation functions of PRRSV-2 may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS-2 and PRDC during bacterial co-infection.

RT-LAMP as Diagnostic Tool for Influenza-A Virus Detection in Swine.

Point-of-care diagnostic technologies are becoming more widely available for production species. Here, we describe the application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect the matrix (M) gene of influenza A virus in swine (IAV-S). M-specific LAMP primers were designed based on M gene sequences from IAV-S isolated in the USA between 2017 and 2020. The LAMP assay was incubated at 65 °C for 30 min, with the fluorescent signal read every 20 s. The assay's limit of detection (LOD) was 20 M gene copies for direct LAMP of the matrix gene standard, and 100 M gene copies when using spiked extraction kits. The LOD was 1000 M genes when using cell culture samples. Detection in clinical samples showed a sensitivity of 94.3% and a specificity of 94.9%. These results show that the influenza M gene RT-LAMP assay can detect the presence of IAV in research laboratory conditions. With the appropriate fluorescent reader and heat block, the assay could be quickly validated as a low-cost, rapid, IAV-S screening tool for use on farms or in clinical diagnostic labs.

Bacillus-Based Direct-Fed Microbial Reduces the Pathogenic Synergy of a Coinfection with Salmonella enterica Serovar Choleraesuis and Porcine Reproductive and Respiratory Syndrome Virus.

Viral respiratory infections predispose lungs to bacterial coinfections causing a worse outcome than either infection alone. Porcine reproductive and respiratory syndrome virus (PRRSV) causes pneumonia in pigs and is often associated with bacterial coinfections. We examined the impact of providing weanling pigs a Bacillus-based direct-fed microbial (DFM) on the syndrome resulting from infection with either Salmonella enterica serotype Choleraesuis alone, or in combination with PRRSV. Nine days after the bacterial challenge, Salmonella was isolated from ileocecal lymph nodes of all challenged pigs regardless of DFM treatment. Compared to the single bacterial challenge, the dual challenge with Salmonella and PRRSV resulted in a pathogenic synergy exhibited by a higher rate of Salmonella colonization in the lung and a more extensive and severe interstitial pneumonia. Provision of DFM to dually challenged pigs reduced the rate of lung colonization by Salmonella, eliminated or reduced the presence of PRRSV in the lung, and reduced the extent and severity of gross lung pathology. Dually challenged pigs that received DFM had increased concentrations of interleukin 1 (IL-1) and IL-8 in lung lavage fluids, accompanied by increased expression in their blood cells of nucleotide-binding oligomerization domain receptor 2 (NOD2) and triggering receptor expressed in myeloid cells 1 (TREM-1) molecules. These changes in pulmonary inflammatory cytokine production and increased expression of NOD2 and TREM-1 suggest that the DFM exerted a systemic modulating effect on innate immunity. These observations are consistent with the notion that tonic stimulation by gut-derived microbial products can poise innate immunity to fight infections in the respiratory tract.

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus.

The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.

The NC229 multi-station research consortium on emerging viral diseases of swine: Solving stakeholder problems through innovative science and research.

The NC229 research consortium was created in 1999 in response to the emergence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral agent responsible for devastating economic losses to the swine industry. The project follows the traditional "consortium" approach for Multistate Agricultural Research driven through the US State Agricultural Experiment Stations (SAES), wherein stakeholder-driven needs to combat swine infectious diseases are identified and scientific solutions pursued by combining funds from federal, state, commodity groups, and the animal health industry. The NC229 consortium was the main driving force in successfully competing for a USDA multi-station Coordinated Agricultural Project (PRRS CAP-I) in 2004-2008, immediately followed by a renewal for 2010-2014 (PRRS CAP-II)-, resulting in an overall record achievement of almost $10 million dollars. The CAP funding was not only useful for quality research, extension, and education in PRRS and related diseases, but also instrumental in enabling the group to leverage swine industry funding of more than $34 million dollars, distributed between creative research and extension on PRRS during the last 20 years. The North American/International PRRS Symposium, now recognized by the community as a highly effective platform for the exchange of basic research findings and fundamental translational technology, is directly derived from the NC229 consortium. Other significant offshoots from NC229 include the PHGC (PRRS Host Genomic Consortium), a platform for discoveries on the role of host genetics during PRRSV infection, since 2007. Since 2009, the NC229 consortium has expanded its collective research interests beyond PRRSV to include nine other emerging viral diseases of swine. In the current project (2019-2024), African Swine Fever Virus (ASFV) retains a central focus, with the goal of harnessing the group's expertise in promoting preparedness for the global control of ASFV.

Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AMϕ), causing dysregulated alpha interferon (IFN-α) and tumor necrosis factor alpha (TNF-α) production through a mechanism(s) yet to be resolved. Here, we show that AMϕ infected with PRRSV secreted a reduced quantity of IFN-α following exposure of the cell to synthetic double-stranded RNA (dsRNA). This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and that were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2α (eIF2α) and the appearance of stress granules. Notably, the typical rapid production of TNF-α by AMϕ exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV, depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h postinfection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2α by the stress sensor PERK (protein kinase RNA [PKR]-like ER kinase) occurred, inhibition of TNF-α production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2α phosphorylation, a synergistic response was observed due to the earlier NF-κB activation via the stress sensor IRE1α (inositol-requiring kinase 1α). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely, IRE1α, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-α production, respectively.IMPORTANCE The activation of AMϕ is controlled by the microenvironment to deter excessive proinflammatory cytokine responses to microbes that could impair lung function. However, viral pneumonias frequently become complicated by secondary bacterial infections, triggering severe inflammation, lung dysfunction, and death. Although dysregulated cytokine production is considered an integral component of the exacerbated inflammatory response in viral-bacterial coinfections, the mechanism responsible for this event is unknown. Here, we show that PRRSV replication in porcine AMϕ triggers activation of the IRE1α branch of the UPR, which causes a synergistic TNF-α response to LPS exposure. Thus, the severe pneumonias typically observed in pigs afflicted with PRRSV-bacterial coinfections could result from dysregulated, overly robust TNF-α production in response to opportunistic pathogens that is not commensurate with the typical restrained reaction by uninfected AMϕ. This notion could help in the design of therapies to mitigate the severity of viral and bacterial coinfections.

Veterinary Curriculum Transformation at the University of Illinois, 2006-2016.

The organization and delivery of a curriculum is the responsibility of the faculty in educational institutions. Curricular revision is often a hotly debated topic in any college faculty. At the University of Illinois, a 2006 mandate for curriculum modernization from the American Veterinary Medical Association Council on Education provided impetus for a long-discussed curricular revision. After two iterations and a lengthy development process, a new curriculum was gradually implemented at Illinois with the August 2009 matriculation of the Class of 2013. The goals of the revision included earlier clinical exposure for veterinary students through introductions to clinical rotations in years 1 to 3 and an integrated body systems approach in lecture/laboratory courses. A new Clinical Skills Learning Center facilitates development of clinical skills earlier in the curriculum and promotes the development of those skills throughout all 4 years of the curriculum. New outcomes assessments include comprehensive written examinations and Objective Structured Clinical Examinations (OSCEs) in years 2 and 3. Curriculum management, including grading of clinical rotations in all 4 years, is achieved through a commercially available software package. For the past 5 years, when candidates were asked why they chose to apply to Illinois, the new curriculum (27.4%) was the most common answer given during interviews. The Illinois revision has resulted in measurably increased veterinary student self-confidence (p<.001) at graduation.

Development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a leading cause of economic burden to the pork industry worldwide. The routinely used modified live PRRS virus vaccine (PRRS-MLV) induces clinical protection, but it has safety concerns. Therefore, in an attempt to develop a safe and protective inactivated PRRSV vaccine, we generated PRRS-virus-like-particles (PRRS-VLPs) containing the viral surface proteins GP5-GP4-GP3-GP2a-M or GP5-M using a novel baculovirus expression system. Our in vitro results indicated that the desired PRRSV proteins were incorporated in both the VLPs preparations based on their reactivity in immunogold electron microscopy and ELISA. To boost their immunogenicity in pigs, we entrapped the PRRS-VLPs in PLGA nanoparticles and coadministered them intranasally with a potent adjuvant. We then evaluated their efficacy in pigs against a viral challenge using a virulent heterologous field isolate. Our results indicated that PRRS-VLPs induced an anamnestic immune response, since we observed boosted IgG and IFN-γ production in vaccinated and virus-challenged animals, but not during the pre-challenge period. Importantly, a two-log reduction in the lung viral load was detected in PRRS-VLP-vaccinated animals. In conclusion, we generated PRRS-VLPs containing up to five viral surface proteins and demonstrated their immunogenicity in pigs, but further studies are required to improve its immunogenicity and efficacy as a vaccine candidate.

Innate and adaptive immunity against Porcine Reproductive and Respiratory Syndrome Virus.

Many highly effective vaccines have been produced against viruses whose virulent infection elicits strong and durable protective immunity. In these cases, characterization of immune effector mechanisms and identification of protective epitopes/immunogens has been informative for the development of successful vaccine programs. Diseases in which the immune system does not rapidly clear the acute infection and/or convalescent immunity does not provide highly effective protection against secondary challenge pose a major hurdle for clinicians and scientists. Porcine reproductive and respiratory syndrome virus (PRRSV) falls primarily into this category, though not entirely. PRRSV causes a prolonged infection, though the host eventually clears the virus. Neutralizing antibodies can provide passive protection when present prior to challenge, though infection can be controlled in the absence of detectable neutralizing antibodies. In addition, primed pigs (through natural exposure or vaccination with a modified-live vaccine) show some protection against secondary challenge. While peripheral PRRSV-specific T cell responses have been examined, their direct contribution to antibody-mediated immunity and viral clearance have not been fully elucidated. The innate immune response following PRRSV infection, particularly the antiviral type I interferon response, is meager, but when provided exogenously, IFN-α enhances PRRSV immunity and viral control. Overall, the quality of immunity induced by natural PRRSV infection is not ideal for informing vaccine development programs. The epitopes necessary for protection may be identified through natural exposure or modified-live vaccines and subsequently applied to vaccine delivery platforms to accelerate induction of protective immunity following vaccination. Collectively, further work to identify protective B and T cell epitopes and mechanisms by which PRRSV eludes innate immunity will enhance our ability to develop more effective methods to control and eliminate PRRS disease.

Differences in immune cell function between tuberculosis positive and negative Asian elephants.

Tuberculosis is an important health concern for Asian elephant (Elephas maximus) populations worldwide, however, mechanisms underlying susceptibility to Mycobacterium tuberculosis are unknown. Proliferative responses assessed via brominated uridine incorporation and cytokine expression measured by real-time RT-PCR were evaluated in peripheral blood mononuclear cell (PBMC) cultures from 8 tuberculosis negative and 8 positive Asian elephants. Cultures were stimulated with Mycobacterium bovis purified protein derivative (PPD-B), M. tuberculosis culture filtrate protein (CFP)-10, and Mycobacterium avium PPD (PPD-A). Following stimulation with PPD-B, proliferation was higher (α = 0.005) in positive samples; no significant differences were detected following CFP-10 or PPD-A stimulation. Tumor necrosis factor (TNF)-α, interleukin (IL)-12, and interferon (IFN)-γ expression was greater in samples from positive elephants following stimulation with PPD-B (α = 0.025) and CFP-10 (α = 0.025 TNF-α and IL-12; α = 0.005 IFN-γ). Stimulation with PPD-A also produced enhanced IL-12 expression in positive samples (α = 0.025). Findings suggested that differences in immune cell function exist between tuberculosis positive and negative elephants. Proliferative responses and expression of TNF-α, IL-12, and IFN-γ in response to stimulation with PPD-B and CFP-10 differ between tuberculosis positive and negative elephants, suggesting these parameters may be important to tuberculosis immunopathogenesis in this species.

Location of T-cell epitopes in nonstructural proteins 9 and 10 of type-II porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant swine pathogen which exhibits considerable sequence diversity. In an attempt to identify highly conserved T-cell epitopes contained in proteins of this virus, we examined heptadecamer peptides spanning the sequence of the PRRSV nonstructural proteins (NSPs) 9 and 10, both of which are highly conserved, for their ability to elicit a recall proliferative and interferon-gamma response in peripheral blood mononuclear cells obtained from pigs immunized against the type-II PRRSV strain FL-12. These studies led to the identification of four peptides, two from each NSP9 and NSP10 that appear to contain T-cell epitopes. Comparison of the amino acid sequence of these four peptide sequences to the analogous sequences from a diverse sample of type-II PRRSV strains indicated that these sequences are highly conserved and thus contain highly conserved T-cell epitopes. The identified epitopes may be important in the formulation of immunogens to provide broad cross-protection against diverse PRRSV strains.

Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus.

The abilities of the modified-live Prime Pac (PP) strain of porcine reproductive and respiratory syndrome virus (PRRSV), propagated in either traditional simian cells (MARC-145) or in a novel porcine alveolar macrophage cell line (ZMAC), to confer pigs protection against subsequent PRRSV challenge were compared. Eight week-old pigs were injected with PP virus grown in one of the two cell types and then exposed 4 weeks later to the "atypical" PRRSV isolate NADC-20. Control animals were similarly challenged or remained PRRSV-naïve. While the average adjusted body weight (aabw) of the strict control group increased 22% by 10 days post challenge (pc), this value for the non-vaccinated, challenged group dropped 4%. In contrast, prior immunization with PP virus, regardless of its host cell source, ameliorated this effect by affording a >9% rise in aabw. Likewise, nearly equivalent protection was extended to both groups of vaccinates in regards to the temporal elimination of their pc clinical distress and viremia. However, the PP virus propagated in ZMAC cells appeared to be more efficacious since four of the six pigs receiving this biologic cleared the challenge virus from the their lungs by 10 days pc as compared to only one member of the other vaccinated group. Notably, the predominant quasispecies in the ZMAC cell-prepared PP virus stock contained a highly conserved N-glycosylation site at position 184 in its glycoprotein 2 while this entity was underrepresented in the MARC-145 cell grown biologic. Since glycoprotein 2 is involved in infectivity, such additional glycosylation may enhance virus replication in porcine alveolar macrophages.

Lactobacillus rhamnosus HN001 attenuates allergy development in a pig model.

BACKGROUND: Probiotics have been studied as immunomodulatory agents of allergy. Several human probiotic trials tracking the development of eczema and other forms of allergy have yielded inconsistent results. A recent infant study demonstrated that pre and postnatal Lactobacillus rhamnosus HN001 (HN001) supplementation decreased the prevalence of eczema and IgE associated eczema. However, the influence of HN001 on the incidence of wheeze, asthma, and/or other allergic manifestations has yet to be reported. OBJECTIVE: This study was conducted to determine the effects of the probiotic HN001 on the development of allergic lung disease in a pig model. METHODS: Allergy was induced by a series of subcutaneous and intratracheal sensitizations with Ascaris suum allergen (ASA) during a six week time frame in post-weanling pigs supplemented daily with HN001, or without supplementation. One week following final sensitization intradermal skin tests and respiratory challenges were conducted. RESULTS: In response to intradermal and respiratory challenges, ASA-sensitized pigs fed HN001 had less severe skin flare reactions, smaller increases in pleural pressure, and trends towards lower changes in arterial oxygen and carbon dioxide partial pressure levels compared to control pigs. The frequency of ASA-specific IFN-γ-secreting peripheral blood mononuclear cells, as well as the amount of IL-10 produced by ASA-specific cells, was of greater magnitude in probiotic-fed pigs compared to control animals. These observations suggest that differences in clinical responses to the allergen challenges may be related to probiotic-induced modulation of Th1 (IFN-γ) and regulatory (IL-10) cytokine expression. CONCLUSIONS: Probiotic supplementation decreased the severity of allergic skin and lung responses in allergen-sensitized pigs with a corresponding increase in IFN-γ expression. A similar correlation between certain allergic responses and increased IFN-γ expression has been reported in human clinical studies of allergy; this pig model of allergy may be indicative of potential probiotic modulation of allergic lung disease in humans.

North American porcine reproductive and respiratory syndrome viruses inhibit type I interferon production by plasmacytoid dendritic cells.

Although enveloped viruses typically trigger the prodigious secretion of alpha interferon (IFN-α) by plasmacytoid dendritic cells (pDC), porcine pDC remain quiescent when exposed to porcine reproductive and respiratory syndrome virus (PRRSV). This inactivity is likely due to virus-mediated interference since the typical IFN-α response by either purified or nonsorted porcine pDC to transmissible gastroenteritis virus (TGEV) or the Toll-like receptor 9 agonist, oligodeoxynucleotide (ODN) D19, was markedly reduced in the presence of PRRSV. Suppression occurred independently of virus viability and acidification of pDC early endosomes but correlated with diminished levels of IFN-α mRNA. This change was attributed to an abrogation of transcription resulting from a decrease in the otherwise enhanced amounts of the requisite interferon regulatory factor 7 (IRF-7), whose gene expression in turn was limited as a consequence of a lessened availability of nuclear-localized signal transducer and activator of transcription 1 (STAT1). While PRRSV also inhibited tumor necrosis factor alpha (TNF-α) synthesis by pDC responding to either agent, only the interleukin-2 (IL-2) and IL-6 production instigated by ODN D19 exposure was blocked. Likewise, PRRSV did not impact a specific TGEV-associated enhancement of IL-8 expression. Moreover, an augmented phosphorylation of NF-κB seen in activated pDC was not only unaffected by PRRSV but actually occurred in its presence. Thus, as supported by a demonstrated resilience of pDC to PRRSV infection, this pathogen may interact with a cell surface protein(s) to selectively impede the completion of cascades involved in cytokine production by stimulated pDC.

Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination.

A Luminex (Luminex Corp., Austin, TX) multiplex swine cytokine assay was developed to measure 8 cytokines simultaneously in pig serum for use in assessment of vaccine candidates. The fluorescent microsphere immunoassay (FMIA) was tested on archived sera in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This FMIA simultaneously detects innate (IL-1 beta, IL-8, IFN-alpha, TNF-alpha, IL-12), regulatory (IL-10), Th1 (IFN-gamma) and Th2 (IL-4) cytokines. These proteins were measured to evaluate serum cytokine levels associated with vaccination strategies that provided for different levels of protective immunity against PRRSV. Pigs were vaccinated with a modified-live virus (MLV) vaccine and subsequently challenged with a non-identical PRRSV isolate (93% identity in the glycoprotein (GP5) gene). Protection (as defined by no serum viremia) was observed in the MLV vaccinated pigs after PRRSV challenge but not those vaccinated with killed virus vaccine with adjuvant (KV/ADJ) (99% identity in the GP5 gene to the challenge strain) or non-vaccinates. Significantly elevated levels of IL-12 were observed in the KV/ADJ group compared to MLV vaccinated and control groups. However, this significant increase in serum IL-12 did not correlate with protection against PRRSV viremia. Additional studies using this assay to measure the local cytokine tissue responses may help in defining a protective cytokine response and would be useful for the targeted design of efficacious vaccines, not only for PRRSV, but also for other swine pathogens.

Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists.

Plausible representatives of plasmacytoid dendritic cells (pDCs) in pigs have been characterized as being CD4(hi)CD172(lo). Due to their paucity in blood, we utilized novel fluorescent-activated cell sorting procedures to isolate them from PBMC. The resultant subset was greater than 98% homogeneous in regards to the selected phenotype and contained the preponderance of individuals secreting IFN-alpha after exposure to a known stimulant, transmissible gastroenteritis virus (TGEV). In addition to being a potent source of IFN-alpha, other properties of these porcine CD4(hi)CD172(lo) cells including their morphological transition from a plasma cell-like shape during quiescence to one resembling a dendritic cell (DC) after activation by TGEV and their relatively strong constitutive expression of interferon regulatory factor-7 (IRF-7) conformed to the expectations of genuine pDCs. While a substantial IFN-alpha response was also elicited from the porcine pDCs by pseudorabies virus (PrV), swine influenza virus (SIV), and TLR7 and 9 agonists, there was an agent-dependent induction of varying amounts of IL-2, IL-6, IL-8, IL-12, IFN-gamma, and TNF-alpha. Notably, porcine reproductive and respiratory syndrome virus (PRRSV) failed to provoke the pDCs to secrete any of the measured cytokines except IL-2. Moreover, whereas pDCs exposed to TGEV or the TLR9 agonist rapidly increased IRF-7 production and morphed into DCs with enhanced CD80/86 expression, similar alterations were not observed during incubation with PRRSV. This atypical response of pDCs to PRRSV may contribute to its pathogenesis, which unlike that associated with PrV, SIV or TGEV includes persistent infection and limited development of protective immunity.

Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth.

The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is known to possess the properties of an ion-channel protein, and in the present study we show that the PRRSV E protein is N-terminal myristoylated. The PRRSV E protein contains the consensus motif of (1)MGxxxS(6) for myristoylation, and in the presence of 2-hydroxymyristic acid, the virus titer decreased by 2.5 log TCID(50) and the level of viral RNA was reduced significantly. When the glycine at position 2 was mutated to alanine (G2A) using an infectious cDNA clone, a viable virus was recoverable and a mutant PRRSV was obtained. The titers of G2A mutant virus were 2.0 x 10(4) and 1.0 x 10(6)TCID(50)/ml for 'passage-2' and 'passage-3' viruses, respectively, in PAM cells, and these titers were significantly lower than those of wild-type PRRSV. When treated with the myristoylation inhibitor, the G2A mutant virus was resistant to the drug. The data show that the PRRSV E protein myristoylation is non-essential for PRRSV infectivity but promotes the growth of the virus.

Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus.

Ninety-six pentadecapeptides spanning glycoprotein 5 (GP5) of porcine reproductive and respiratory virus (PRRSV) were screened for their ability to elicit a recall interferon-gamma response from peripheral blood mononuclear cells isolated from 22 pigs infected with up to two genetically divergent PRRSV strains. Two distinct regions (amino acid residues 117-131, LAALICFVIRLAKNC, and 149-163, KGRLYRWRSPVII/VEK) appeared to contain immunodominant T-cell epitopes based on their ability to stimulate above average numbers of interferon-gamma secreting cells as compared to other GP5 peptides. A survey of PRRSV isolates indicated that these two sites are relatively conserved with at most a two amino acid variation and thus should be considered for incorporation into a multi-valent vaccine against PRRS.