RESUMEN
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.
Asunto(s)
Coinfección , Interferón Tipo I , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Citocinas/genética , Citocinas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Macrófagos Alveolares/metabolismo , Interferón Tipo I/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/metabolismoRESUMEN
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.
Asunto(s)
Bacillus , Coinfección , Neumonía , Virus del Síndrome Respiratorio y Reproductivo Porcino , Salmonella enterica , Animales , Salmonella , Serogrupo , Porcinos , Receptor Activador Expresado en Células Mieloides 1RESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AMÏ), 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 AMÏ 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 AMÏ 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 AMÏ 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 AMÏ 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 AMÏ. This notion could help in the design of therapies to mitigate the severity of viral and bacterial coinfections.
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Interferón-alfa/genética , Macrófagos Alveolares/virología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Respuesta de Proteína Desplegada , Animales , Línea Celular , Factor 2 Eucariótico de Iniciación/metabolismo , Genotipo , Interferón-alfa/metabolismo , Lipopolisacáridos/efectos adversos , Macrófagos Alveolares/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Factor de Necrosis Tumoral alfa/metabolismo , Replicación Viral , eIF-2 Quinasa/metabolismoRESUMEN
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.
Asunto(s)
Prácticas Clínicas , Curriculum/tendencias , Educación en Veterinaria/organización & administración , Facultades de Medicina Veterinaria/organización & administración , Acreditación , Educación en Veterinaria/normas , Humanos , Illinois , Innovación Organizacional , Facultades de Medicina Veterinaria/normasRESUMEN
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 â¼ 45â¯minutes, the limit of detection for the assay is comparable with an RT-qPCR Cq less than 35, with a sensitivity of 83.5â¯% and a specificity of 89.6â¯%. 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.
Asunto(s)
Virus de la Influenza A , Técnicas de Amplificación de Ácido Nucleico , Infecciones por Orthomyxoviridae , Sensibilidad y Especificidad , Enfermedades de los Porcinos , Animales , Porcinos , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/diagnóstico , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/veterinariaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Interferón Tipo I/antagonistas & inhibidores , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Animales , Regulación hacia Abajo , Expresión Génica , Factor 7 Regulador del Interferón/biosíntesis , América del Norte , Oligodesoxirribonucleótidos/inmunología , ARN Mensajero/biosíntesis , Porcinos , Virus de la Gastroenteritis Transmisible/inmunologíaRESUMEN
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.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Técnicas de Cultivo de Célula/métodos , Macrófagos/citología , Vacunas Atenuadas/farmacología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Células de la Médula Ósea/virología , Línea Celular , Proliferación Celular , Macrófagos/virología , Pase Seriado , Porcinos , Vacunas Atenuadas/inmunología , Replicación ViralRESUMEN
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.
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Enfermedades Transmisibles Emergentes/veterinaria , Enfermedades Transmisibles Emergentes/virología , Investigación/organización & administración , Virosis/veterinaria , Animales , Congresos como Asunto , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Investigación/economía , Participación de los Interesados , Porcinos , Estados Unidos , Virosis/prevención & controlRESUMEN
OBJECTIVE: To determine whether 6.5-week-old gilts that have not previously been exposed to porcine reproductive and respiratory syndrome (PRRS) virus can be acclimatized to an endemic strain of the virus by commingling with age-matched gilts inoculated with the endemic PRRS virus strain and whether 10.5-week-old gilts can be acclimatized by commingling with age-matched inoculated or contact-exposed animals. DESIGN: Randomized controlled longitudinal study. ANIMALS: 80 gilts seronegative for PRRS on a farm in the Midwestern United States with a history of PRRS. PROCEDURES: 20 gilts were inoculated with the endemic PRRS virus strain at 6.5 weeks of age (group 1) and were commingled with 20 gilts that were not inoculated (group 2). Four weeks later, the remaining 40 gilts (group 3) were commingled with gilts in groups 1 and 2. Presence of viral RNA in the tonsils, seroconversion rate, serum neutralizing antibody titers, interferon-gamma-mediated cellular immunity, and reproductive outcomes were analyzed. RESULTS: Acclimatization of PRRS virus-naïve pigs was achieved by means of contact exposure at both 6.5 and 10.5 weeks of age. No differences were observed among the 3 groups with respect to development of anti-PRRS virus-specific immune responses or reproductive outcomes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that contact exposure of 6.5- to 10.5-week-old pigs that had not previously been exposed to PRRS virus to pigs inoculated with endemic PRRS virus may be an efficient acclimatization strategy for controlling outbreaks on commercial farms on which PRRS is endemic.
Asunto(s)
Anticuerpos Antivirales/sangre , Síndrome Respiratorio y de la Reproducción Porcina/transmisión , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunación/veterinaria , Adaptación Fisiológica/inmunología , Factores de Edad , Animales , Animales Recién Nacidos , Exposición a Riesgos Ambientales , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Estudios Longitudinales , Pruebas de Neutralización/métodos , Pruebas de Neutralización/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Distribución Aleatoria , Porcinos/crecimiento & desarrollo , Aumento de PesoRESUMEN
The efficacy of two different types of commercial vaccines against PRRSV (Euro-type) was evaluated based on clinical parameters upon challenge as well as post-challenge virological profiles (viremia and viral load in tissues upon necropsy, measured in both cases by quantitative real time PCR). In an attempt to establish correlates of protective immunity, two commonly proposed parameters predictive of immunity were measured: (1) serologic responses (ELISA and neutralizing antibodies), (2) frequency of gamma interferon-producing cells in peripheral blood mononuclear cell fraction. The vaccines compared consisted of two commercially available products that are regularly marketed in Spain: one modified live virus and one killed vaccine. The efficacy assay was carried out by vaccinating twice 3 weeks apart groups of 5 and-a-half month-old female swine and then challenging them with a European type 1 PRRSV strain (Lelystad). The results obtained indicate that the modified live virus vaccine was the only type of vaccine capable of establishing protective immunity, as measured by viral load in blood and tissues. The killed vaccine, in spite of this product evoking a spontaneous interferon-gamma response and post-challenge titers of virus-neutralizing antibody, evoked no measurable protective immunity. In the case of the modified live vaccine, the protection exhibited did not appear to be based on humoral but rather on cell-mediated immunity.
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Interferón gamma/biosíntesis , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Química Farmacéutica , Femenino , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Pruebas Serológicas , Organismos Libres de Patógenos Específicos , PorcinosRESUMEN
OBJECTIVE: To compare immunologic responses and reproductive outcomes in sows housed under field conditions following controlled exposure to a wild-type strain of porcine reproductive and respiratory syndrome virus (PRRSV strain WTV) or vaccination with a modified-live virus (MLV) vaccine. DESIGN: Randomized controlled trial. ANIMALS: 30 PRRSV-naïve 10-week-old female pigs. PROCEDURE: Humoral and cell-mediated immune responses were monitored while pigs were held in isolation for 84 days after inoculation with the WTV strain (n = 10), inoculation with the WTV strain and 42 days later vaccination with a killed-virus vaccine (10), or vaccination with an MLV vaccine (10). Reproductive outcomes were measured after pigs were released into the farm herd. RESULTS: Inoculation with the WTV strain, regardless of whether a killed-virus vaccine was subsequently administered, elicited faster and more substantial production of strain-specific neutralizing antibodies, as well as a more rapid generation of interferon-gamma secreting cells, than did vaccination with the MLV vaccine. Despite the enhanced immune responses in pigs inoculated with the WTV strain, animals vaccinated with the MLV vaccine produced a mean of 2.45 more pigs than did sows exposed to the WTV strain, mainly because of a lower rate for failure to conceive. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that current assays of immunity to PRRSV correlate only imperfectly with degree of clinical protection and that the practice of controlled exposure of sows to a circulating PRRSV strain should be reconsidered in light of negative clinical outcomes.
Asunto(s)
Anticuerpos Antivirales/sangre , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Reproducción , Vacunas Virales/inmunología , Animales , Femenino , Tamaño de la Camada , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Embarazo , Índice de Embarazo , Porcinos , Vacunas Atenuadas , Vacunas de Productos Inactivados , Vacunas Virales/administración & dosificaciónRESUMEN
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.
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Aborto Veterinario/virología , Brotes de Enfermedades/veterinaria , Muerte Fetal/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Animales , Femenino , Muerte Fetal/virología , Inmunidad Celular , Modelos Logísticos , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Embarazo , Factores de Riesgo , PorcinosRESUMEN
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.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Inmunidad Adaptativa , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Antígenos Virales , Reacciones Cruzadas , Epítopos , Interacciones Huésped-Patógeno/inmunología , Evasión Inmune , Inmunidad Celular , Inmunidad Innata , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Sus scrofa , Porcinos , Linfocitos T/inmunología , Vacunas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Interferón-alfa/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Animales , Citocinas/inmunología , Inmunidad Celular , Interferón-alfa/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Plásmidos/genética , Plásmidos/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Análisis de Regresión , Factores de Tiempo , Regulación hacia Arriba/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
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.
Asunto(s)
Interferón-alfa/inmunología , Interleucina-12/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , ARN Bicatenario/inmunología , Porcinos/inmunología , Células TH1/inmunología , Animales , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Factores de Tiempo , Vacunas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunas Virales/farmacología , Viremia/veterinaria , Animales , Peso Corporal/inmunología , Líquido del Lavado Bronquioalveolar/virología , Inmunización/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Síndrome Respiratorio y de la Reproducción Porcina/virología , ARN Viral/química , ARN Viral/genética , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Organismos Libres de Patógenos Específicos , Porcinos , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/farmacología , Carga Viral/veterinaria , Vacunas Virales/inmunología , Viremia/inmunología , Viremia/prevención & control , Viremia/virologíaRESUMEN
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.
Asunto(s)
Epítopos de Linfocito T/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , ARN Viral/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Animales , Proliferación Celular , Células Cultivadas , Secuencia Conservada , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , PorcinosRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Hipersensibilidad/dietoterapia , Lacticaseibacillus rhamnosus/fisiología , Porcinos , Alimentación Animal , Animales , Ascaris suum/inmunología , Pruebas de Provocación Bronquial/veterinaria , Suplementos Dietéticos , Humanos , Hipersensibilidad/complicaciones , Hipersensibilidad/patología , Hipersensibilidad/prevención & control , Enfermedades Pulmonares/dietoterapia , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/prevención & control , Probióticos/administración & dosificación , Probióticos/uso terapéutico , Organismos Libres de Patógenos EspecíficosRESUMEN
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.