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.

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.

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.

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.

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.

Correlation of cell-mediated immunity against porcine reproductive and respiratory syndrome virus with protection against reproductive failure in sows during outbreaks of porcine reproductive and respiratory syndrome in commercial herds.

OBJECTIVE: To determine whether cell-mediated immunity against porcine reproductive and respiratory syndrome (PRRS) virus is correlated with protection against reproductive failure in sows during clinical outbreaks of PRRS in commercial herds. DESIGN: Outbreak investigation in 4 swine breeding herds. ANIMALS: 97 sows. PROCEDURES: On each farm, blood samples were collected from sows with clinical signs (abortion or increased fetal death; case sows) and from clinically normal sows (control sows). The intensity of the cell-mediated immune (CMI) response was determined by use of an interferon-gamma enzyme-linked immunospot (ELISPOT) assay. Multiple logistic regression analyses and t tests were used to compare ELISPOT assay values between case and control sows. Multiple linear regression was used to investigate associations between cell-mediated immunity and the magnitude of clinical signs. RESULTS: In 2 farms, case sows had lower ELISPOT assay values than control sows. A negative association between the intensity of the CMI response and the number of pigs born dead per litter was detected on 1 farm. In 1 farm, no association was detected between the intensity of the CMI response and protection against reproductive failure. CONCLUSIONS AND CLINICAL RELEVANCE: Evidence that a strong CMI response was correlated with protection against clinical PRRS was detected in 3 of 4 farms. However, farms and sows within farms varied considerably in their immune responsiveness and in the degree to which they were protected clinically. Increasing cell-mediated immunity within infected herds has the potential to decrease clinical reproductive disease, but only if the sources of intra- and interfarm variation in the intensity of cell-mediated immunity to PRRS virus can be identified.

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination.

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus.

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.

Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination.

Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately 3 months. In contrast, the initial onset of virus-specific interferon (IFN)-gamma-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-gamma SC (comprising predominantly CD4/CD8 alpha double positive memory T cells with a minority being typical CD4(-)/CD8 alpha beta(+) T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-gamma response and preferentially stimulates the synthesis of antibodies incapable of neutralization.