Article

Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus

November 2003
Journal of General Virology 84(Pt 10):2755-60

November 2003
84(Pt 10):2755-60

DOI:10.1099/vir.0.19230-0

Source
PubMed

Authors:

Sanipa Suradhat

Chulalongkorn University

Roongroje Thanawongnuwech

Chulalongkorn University

Recent studies suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system by upregulating interleukin (IL)-10 gene expression. To determine the effect of PRRSV on porcine cytokine gene expression in vivo, we infected pigs with either the European or North American strain of PRRSV and monitored cytokine gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) using a multiplex PCR assay. Our results showed that both European and North American strains of PRRSV significantly upregulated IL-10 gene expression in PBMC of infected pigs from 5 days post-infection (p.i.). In addition, upregulation of IL-10 and interferon (IFN)-gamma gene expression was observed in BALC starting from 9 days p.i. The upregulation of cytokine gene expression in BALC was observed concurrent with an increased percentage of lymphocytes in the BALC population, suggesting a role for peripheral leukocytes in cytokine production in lungs. Our results showed that PRRSV infection resulted in an upregulation of IL-10 gene expression in vivo and that both European and North American strains induced comparable levels of IL-10 gene expression in the infected pigs, despite differences in the clinical signs. Our data support the notion that induction of IL-10 production may be one of the strategies used by PRRSV to modulate the host's immune responses, and this may contribute to the unique clinical picture observed following PRRSV infection.

Effect of oxidized soybean oil on the immune response to porcine reproductive and respiratory syndrome modified live virus vaccine in nursery pigs

Article

Full-text available

Sep 2019

Effect of oxidized soybean oil on the immune response to porcine reproductive and respiratory syndrome modified live virus vaccine in nursery pigs

Research

Full-text available

Dec 2019

This study evaluated the effect of oxidized soybean oil on the immune response to porcine reproductive and respiratory syndrome modified live vaccine (PRRS-MLV), and the oxidative stress status in nursery pigs. Seventy castrated weaned pigs, from a free PRRS virus infected herd, were divided into two groups: treatment group (n=63) and control group (n=7). The treatment pigs were vaccinated with the PRRS-MLV and then divided into three groups based on different diet types containing different soybean oils: (A) 5% fresh oil, (B) 5% heated oil for 43 h, and (C) 5% heated oil for 38 h. The control pigs were not vaccinated and consumed (A) diet. Blood samples were collected for PRRS immune response and for measuring oxidative status. No significant differences were observed among groups in malondialdehyde (MDA), superoxide dismutase-1 (SOD-1), ELISA titer, and SN antibodies against PRRS virus. Notably, the percentage of interferon-gamma (IFN-) producing cell increased significantly in group A in comparison to that in the other groups on D28 and D56. In contrast, the percentage of interleukin-10 (IL-10) producing cell was highest in group C, followed by group B and group A, respectively. Moreover, group C exhibited a higher number of IL-10 producing cells compared to group A on D56 (p=0.082). Oxidized soybean oil exerts a negative effect on the cell-mediated immune response to PRRS-MLV, especially in pigs fed oil containing high peroxide value and high numbers of total polar compounds. These findings suggest that pig farmers should be more concerned with the quality of oil used in the diet for nursery pigs.

Different susceptibility to porcine reproductive and respiratory syndrome virus infection among Chinese native pig breeds

Article

Full-text available

Aug 2018
ARCH VIROL

China is rich in native pig breeds, yet information regarding the susceptibility/resistance of local breeds to porcine reproductive and respiratory syndrome virus (PRRSV) infection is lacking. In the present study, an in vitro method based on assessing PRRSV replication in porcine alveolar macrophages (PAMs) was established to evaluate PRRSV susceptibility/resistance in a commercial pig breed (Landrace) and five native pig breeds from Jiangsu and Anhui provinces in China. Expression levels of cytokines (IL-8, IL-10, TNF-α and IFN-γ), Toll-like receptor 3 (TLR3), CD163 (PRRSV receptor), and sialoadhesin (Sn, PRRSV receptor) in infected pigs were determined using real-time PCR, and the association between PRRSV susceptibility/resistance and the abundance of the cytokines and receptors was investigated. The viral replication rate and titer at 0, 6, 12 18, 24 and 36 hours postinfection (hpi) were determined to assess the proliferation dynamics of PRRSV NJGC in PAMs. Based on the PRRSV proliferation dynamics, the results indicated that Dingyuan pigs were the most susceptible to PRRSV infection, whereas Jiangquhai pigs were the least susceptible to PRRSV infection among the six pig breeds tested, as indicated by measuring PRRSV replication and the viral load in PAMs. The different levels of susceptibility to PRRSV infection in PAMs may be associated with differences in the abundance of CD163 (PRRSV receptor), cytokines IL-8, IFN-γ, and TNF-α in Jiangquhai and Dingyuan pig breeds after viral inoculation.

Síndrome reproductivo y respiratorio del cerdo(PRRS): Revisión

Article

Full-text available

Mar 2015

The porcine reproductive and respiratory syndrome (PRRS), is a viral disease that is manifested in severe reproductive failure in pregnant sows, lower levels of semen quality in boars, and respiratory problems in pigs of all ages but mainly piglets. Economically it is one of the most important diseases worldwide and is present in the majority of porcine producing countries, where, in most of them, it is still endemic. The PRRS virus (PRRSV) has a high mutation rate, reason why there is great genetic diversity in the North American linage strains (NA PRRSV), and between the NA PRRSV and the European (EU PRRSV) lineages. This affects strain homogeneity and gives few or null cross antigenicity among strains, which is important for a successful vaccine. The modified virus vaccine, is the only commercially available vaccine that provides some level of immunity confidence, but it has shown the possibility to revert to pathogenicity by mutation or recombination with field strains. The commercially available vaccines are only used to diminish the level of impact of the disease. The virus shows the capacity of immune suppression and immune regulation, which allows the virus to extend the viremia period in infected animals. The virus spreads through saliva, trans placental and mammal secretions and with a high probability feces, been the main transmission by direct contact and secondary by infected objects; also shows a posterior selectivity toward a limited lymphoid tissue, this enables it to remain unnoticed until favorable conditions allow the disease to develop into an outbreak or pandemic.

Immunoregulatory signal FoxP3, cytokine gene expression and IFN-γ cell responsiveness upon porcine reproductive and respiratory syndrome virus (PRRSV) natural infection

Article

Sep 2015
RES VET SCI

The study aims at evaluating gene expression of pro-inflammatory (IL-1β, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age. Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks. In the early phase of infection, pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT. The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS). Review

Article

Full-text available

Jan 2015

Comparison of Host Immune Responses to Homologous and Heterologous Type II Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Challenge in Vaccinated and Unvaccinated Pigs

Article

Full-text available

Feb 2014
BMRI

Porcine reproductive and respiratory syndrome (PRRS) is a high-consequence animal disease with current vaccines providing limited protection from infection due to the high degree of genetic variation of field PRRS virus. Therefore, understanding host immune responses elicited by different PRRSV strains will facilitate the development of more effective vaccines. Using IngelVac modified live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06-72109 strain (a Kansas isolate of PRRSV), we compared immune responses induced by vaccination and/or PRRSV infection. Our results showed that MLV can provide complete protection from homologous virus (VR-2332) and partial protection from heterologous (KS-06) challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge, with vaccinated pigs having higher titers to VR-2332 compared to KS-06 strain. Challenge strain did not alter the cytokine expression profiles in the serum of vaccinated pigs or subpopulations of T cells. However, higher frequencies of IFN- γ -secreting PBMCs were generated from pigs challenged with heterologous PRRSV in a recall response when PBMCs were re-stimulated with PRRSV. Thus, this study indicates that serum neutralizing antibody titers are associated with PRRSV vaccination-induced protection against homologous and heterologous challenge.

Differential Responses of Disease-Resistant and Disease-Susceptible Primate Macrophages and Myeloid Dendritic Cells to Simian Hemorrhagic Fever Virus Infection

Article

Full-text available

Jan 2014
J VIROL

Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but asymptomatic, persistent infections in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (MΦs) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque MΦs but in only ∼10% of baboon MΦs. Although SHFV infected ∼50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12/23(p40), TNF-α and MIP-1α, in response to SHFV infection were observed in macaque but not baboon cultures suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor, and also SOCS3, a negative regulator of pro-inflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1β and MIP-1α but not TNF-α suggesting a role for IL-10 in suppressing SHFV induced pro-inflammatory cytokine production in macaques.

Dual infection of a Thai isolate HP-PRRSV and the pdmH1N1 2009 SIV in weanling pigs

Article

Mar 2019

Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

Article

Full-text available

May 2023

Porcine alveolar macrophages (PAMs) are widely used for in vitro studies of porcine respiratory viruses. Gene expression in these cells is altered by viral infection and cellular immune response. Real-time reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for analyzing these changes. In order to obtain reliable quantitative RT-qPCR data and come to sound conclusions, stable reference genes are needed for normalization of target gene expression. In the present study, we evaluated the expression stability of nine reference genes in PAMs during cultivation and upon porcine reproductive and respiratory syndrome virus (PRRSV) inoculation. Using geNorm and NormFinder algorithms, we identified PSAP and GAPDH as the most stable reference genes under all experimental conditions. The selected reference genes were used for the normalization of CD163 expression under different conditions. This study demonstrates that selection of appropriate reference genes is essential for normalization and validation of RT-qPCR data across all experimental conditions. This study provides a new set of stable reference genes for future studies with porcine respiratory viruses in PAMs.

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2

Article

Full-text available

Apr 2023
PLOS ONE

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Immune Molecules’ mRNA Expression in Porcine Alveolar Macrophages Co-Infected with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

Article

Full-text available

Mar 2023

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2–PRRSV co-infected group (PCV2–PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV–PCV2 co-infected group (PRRSV–PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1β, IL-10 and TGF-β) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.

Respiratory viral infections drive different lung cytokine profiles in pigs

Article

Full-text available

Jan 2021
BMC Vet Res

Background Swine influenza A virus (IAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are considered key viral pathogens involved in the porcine respiratory disease complex. Concerning the effect of one virus on another with respect to local immune response is still very limited. Determination of presence and quantity of cytokines in the lung tissue and its relation to the lung pathology can lead to a better understanding of the host inflammatory response and its influence on the lung pathology during single or multi-virus infection. The aim of the present study was to explore and compare the patterns of lung cytokine protein response in pigs after single or dual infection with swine IAV and/or PRRSV. Results Inoculation with IAV alone causes an increase in lung concentration of IFN-α, IFN-ɣ, TNF-α, IL-6, IL-8 and IL-10, especially at 2 and 4 DPI. In PRRSV group, beyond early IFN-α, IFN-ɣ, IL-6, IL-8 and IL-10 induction, elevated levels of cytokines at 10 and 21 DPI have been found. In IAV+PRRSV inoculated pigs the lung concentrations of all cytokines were higher than in control pigs. Conclusions Current results indicate that experimental infection of pigs with IAV or PRRSV alone and co-infection with both pathogens induce different kinetics of local cytokine response. Due to strong positive correlation between local TNF-α and IL-10 concentration and lung pathology, we hypothesize that these cytokines are involved in the induction of lung lesions during investigates infection. Nevertheless, no apparent increase in lung cytokine response was seen in pigs co-inoculated simultaneously with both pathogens compared to single inoculated groups. It may also explain no significant effect of co-infection on the lung pathology and pathogen load, compared to single infections. Strong correlation between local concentration of TNF-α, IFN-ɣ, IL-8 and SwH1N1 load in the lung, as well as TNF-α, IL-8 and PRRSV lung titres suggested that local replication of both viruses also influenced the local cytokine response during infection.

Novel lineage 1 recombinants of porcine reproductive and respiratory syndrome virus isolated from vaccinated herds: genome sequences and cytokine production profiles

Article

Full-text available

Oct 2020
ARCH VIROL

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.

Isolation and characterization of a new population of nasal surface macrophages and their susceptibility to PRRSV-1 subtype 1 (LV) and subtype 3 (Lena)

Article

Full-text available

Dec 2020
VET RES

Sialoadhesin (Sn) and CD163 have been recognized as two important mediators for porcine reproductive and respiratory syndrome virus (PRRSV) in host macrophages. Recently, it has been demonstrated that the highly virulent Lena strain has a wider macrophage tropism than the low virulent LV strain in the nasal mucosa. Not only CD163+Sn+ macrophages are infected by Lena but also CD163+Sn- macrophages. This suggests that an alternative receptor exists for binding and internalization of PRRSV Lena in the CD163+Sn- macrophages. Further investigation to find the new entry receptor was hampered by the difficulty of isolating these macrophages from the nasal mucosa. In the present study, a new population of CD163+Sn- cells has been identified that is specifically localized in the nasal lamina propria and can be isolated by an intranasal digestion approach. Isolated nasal cells were characterized using specific cell markers and their susceptibility to two different PRRSV-1 strains (LV and Lena) was tested. Upon digestion, 3.2% (flow cytometry)-6.4% (confocal microscopy) of the nasal cells were identified as CD163+ and all (99.7%) of these CD163+ cells were Sn-. These CD163+Sn- cells, designated as "nasal surface macrophages", showed a 4.9 times higher susceptibility to the Lena strain than to the LV strain. Furthermore, the Lena-inoculated cell cultures showed an upregulation of CD163. These results showed that our new cell isolation system is ideal for the further functional and phenotypical analysis of the new population of nasal surface macrophages and further research on the molecular pathogenesis of PRRSV in the nose.

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

Article

Full-text available

Feb 2020

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

Molecular serotyping of Haemophilus parasuis isolated from diseased pigs and the relationship between serovars and pathological patterns in Taiwan

Article

Full-text available

Nov 2018

Background Haemophilus parasuis is the etiological agent of Glässer’s disease, and causes severe economic losses in the swine industry. Serovar classification is intended as an indicator of virulence and pathotype and is also crucial for vaccination programs and vaccine development. According to a polysaccharide biosynthesis locus analysis, H. parasuis isolates could be classified by a molecular serotyping assay except serovars 5 and 12 detected by the same primer pair. The aim of this study was to identify H. parasuis isolates from diseased pigs in Taiwan by using a molecular serotyping assay and to analyze the relationship between serovars and pathological patterns. Methods From August 2013 to February 2017, a total of 133 isolates from 277 lesions on 155 diseased animals from 124 infected herds serotyped by multiplex PCR and analyzed with pathological data. Results The dominant serovars of H. parasuis in Taiwan were serovars 5/12 (37.6%), 4 (27.8%) and 13 (15%) followed by molecular serotyping non-typable (MSNT) isolates (13.5%). Nevertheless, the serovar-specific amplicons were not precisely the same sizes as previously indicated in the original publication, and MSNT isolates appeared with unexpected amplicons or lacked serovar-specific amplicons. Most H. parasuis isolates were isolated from nursery pigs infected with porcine reproductive and respiratory syndrome virus. The percentage of lung lesions (30.4%) showing H. parasuis infection was significantly higher than that of serosal lesions. Discussion Collectively, the distribution of serovars in Taiwan is similar to that found in other countries, but MSNT isolates remain due to genetic variations. Furthermore, pulmonary lesions may be optimum sites for H. parasuis isolation, the diagnosis of Glässer’s disease, and may also serve as points of origin for systemic H. parasuis infections in hosts.

Induction of porcine reproductive and respiratory syndrome virus (PRRSV)-specific regulatory T lymphocytes (Treg) in the lungs and tracheobronchial lymph nodes of PRRSV-infected pigs

Article

Mar 2018
VET MICROBIOL

Regulatory T lymphocytes (Treg) residing within the tissues, are known to possess immunosuppressive properties which contribute to immunomodulation within the organs. PRRSV infection usually weakens lung defense mechanisms, leading to porcine respiratory disease complex (PRDC). Induction of circulatory Treg is one of the reported mechanisms involved in PRRSV-induced immunomodulation. However, whether PRRSV can induce tissue-infiltrating Treg in the lungs and lymph nodes is still unclear. To investigate the effect of PRRSV on induction of porcine Treg in the tissues, we isolated mononuclear cells from the lungs and tracheobronchial lymph nodes, and identified the existence of Treg by flow cytometry. The results demonstrated that PRRSV could induce Treg proliferation in the cultured mononuclear cells derived from lungs and tracheobronchial lymph nodes, regardless of the pig's PRRSV infective status. Furthermore, PRRSV-infected pigs exhibited higher numbers of total tissue-infiltrating Treg and PRRSV-specific Treg in the lungs and tracheobronchial lymph nodes than the PRRSV-negative pigs. To determine if the lung Treg could produce an inhibitory cytokine, the numbers of IL-10-producing Treg were determined. Significantly higher numbers of IL-10-producing Treg in the lungs of PRRSV-infected pigs were observed. Altogether, our findings indicate the potent effect of PRRSV on induction of Treg in the lungs and tracheobronchial lymph nodes of the infected pigs. The findings expand our understanding in PRRSV immunopathogenesis within the target organs.

Cross-reactive Immunity to Porcine Reproductive and Respiratory Syndrome Virus and its Contribution to Protection

Thesis

Sep 2017

Ignacio Correas

Due to the vast geographical distribution and significant economic losses generated, porcine reproductive and respiratory syndrome virus (PRRSV) can be considered the most important swine pathogen of contemporary times. Current control and eradication strategies against PRRSV have difficulty succeeding because of their complex nature and the absence of an effective vaccine. A major obstacle for PRRSV vaccine development is the broad heterogeneity of the virus, both at the genetic and antigenic level, its rapid evolution, and an incomplete knowledge of the immune responses responsible for clearing the virus from the host. Specifically, how known correlates of protection against PRRSV—neutralizing antibodies and T cells—cross-react with heterologous isolates and mediate cross-protection is inadequately understood. The objectives of this dissertation were (i) to determine the extent of cross-reactivity of immune responses against PRRSV, and (ii) to ascertain how cross-reactive immune responses mediate protection against heterologous isolates. T cell responses were found to be cross-reactive among PRRSV-2 isolates, but extremely variable among individual animals, while the neutralizing antibody response induced by a single infection with PRRSV was deemed to be solely self-neutralizing. Sequential exposure to heterologous PRRSV-2 isolates elicited neutralizing antibodies to the isolates used for infection and challenge, as well as other heterologous PRRSV-2 isolates. Furthermore, prior exposure to PRRSV afforded cross-protection against heterologous challenge, with reduction in viremia, tissue viral load and the extent of microscopic lung lesions; however, protection was still suboptimal. T cell cross-reactivity between PRRSV-1 and PRRSV-2 was evaluated at the structural protein level and was deemed to be feeble or absent. Prior exposure to PRRSV-1 did not prime the T cell response against the PRRSV-2 structural proteins after PRRSV-2 challenge. Collectively, the results in this dissertation contribute to furthering the understanding of immune responses against PRRSV and may be used in the development of a better vaccine. Advisor: Fernando A. Osorio

Generation of murine macrophage-derived cell lines expressing porcine CD163 that support porcine reproductive and respiratory syndrome virus infection

Article

Full-text available

Nov 2017
BMC BIOTECHNOL

Background Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits a highly restricted tropism for cells of the monocyte-macrophage lineage, utilizing porcine CD163 (pCD163) as an indispensable cellular receptor for infection. Transfection the gene of pCD163 into several non-permissive cell lines followed by protein expression confers susceptibility to PRRSV. A lack of specialized porcine antibody tools for use with existing porcine-derived primary cells and cell lines has hampered studies of both PRRSV pathogenesis and virus triggering of immune response cascades. Therefore, we constructed PRRSV-susceptible murine alveolar macrophage-derived MH-S and peritoneal macrophage-like RAW264.7 cell lines by achieving pCD163 cell surface expression in these cells. We then evaluated PRRSV susceptibility and cytokine expression patterns induced upon PRRSV infection of these pCD163-expressing cell lines. Results Growth of MH-SCD163 and RAW264.7CD163 cells was indistinguishable from growth of un-transfected parental cell lines. Meanwhile, various stages of the PRRSV replication cycle, including viral particle attachment, internalization, disassembly and infection were confirmed in both pCD163-transfected cell lines. Analysis of PRRSV replication using immunofluorescence staining of virus and viral titration of cell lysates demonstrated that both MH-SCD163 and RAW264.7CD163 cells supported replication of various genotype 2 PRRSV isolates. Moreover, PRRSV replication in MH-SCD163 cells was similar to that observed in porcine alveolar macrophages (PAMs) and was more efficient than in RAW264.7CD163 cells. However, peak virus titers in MH-SCD163 cells were attained at 60 h post-infection (pi) versus 48 hpi in PAMs. Analysis of cytokine expression showed that post-PRRSV infection, mRNA expression patterns of anti-inflammatory cytokines (IL-4 and IL-10) and pro-inflammatory cytokines (TNF-α and IFN-γ) in MH-SCD163 cells were more similar to those observed in PAMs versus levels in RAW264.7CD163 cells. Conclusions MH-S and RAW264.7 cells were not susceptible to PRRSV infection until transfection and subsequent expression of pCD163 were achieved in these cell lines. The PRRSV-susceptible MH-SCD163 cell line efficiently supported viral replication of various genotype 2 PRRSV isolates and exhibited similar cytokine expression patterns as observed in PAMs. In conclusion, this work describes the development of new tools to further understand PRRSV pathogenesis and immune response mechanisms to PRRSV infection. Electronic supplementary material The online version of this article (10.1186/s12896-017-0399-5) contains supplementary material, which is available to authorized users.

Mechanisms of Adaptive Immunity to Porcine Reproductive and Respiratory Syndrome Virus

Article

Full-text available

Jun 2017

The adaptive immune response is necessary for the development of protective immunity against infectious diseases. Porcine reproductive and respiratory syndrome virus (PRRSV), a genetically heterogeneous and rapidly evolving RNA virus, is the most burdensome pathogen of swine health and wellbeing worldwide. Viral infection induces antigen-specific immunity that ultimately clears the infection. However, the resulting immune memory, induced by virulent or attenuated vaccine viruses, is inconsistently protective against diverse viral strains. The immunological mechanisms by which primary and memory protection are generated and used are not well understood. Here, we summarize current knowledge regarding cellular and humoral components of the adaptive immune response to PRRSV infection that mediate primary and memory immune protection against viruses.

Antagonizing Cytokine-Mediated JAK-STAT Signaling by Porcine Reproductive and Respiratory Syndrome Virus

Article

Dec 2016

Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by myriad cytokines, which are involved in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of its significance in immune response, JAK-STAT pathway is often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One of the possible reasons for the weak protective immune response is that PRRSV interferes with cytokine-mediated JAK-STAT signaling. PRRSV inhibits interferon-activated JAK-STAT signaling by blocking nuclear translocation of STAT1 and STAT2. The mechanism is that PRRSV non-structural protein 1β (nsp1β) induces degradation of karyopherin α1 (KPNA1), a critical adaptor in nucleo-cytoplasmic transport. PRRSV also antagonizes IL6-activated JAK-STAT3 signaling via inducing degradation of STAT3. In this review, we briefly introduce JAK-STAT signaling, summarize the PRRSV interference with it, and provide perspective on the perturbation in the context of PRRSV-elicited immune response.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via Nsp5 by Inducing STAT3 Degradation

Article

Full-text available

Jan 2017
J VIROL

Significance: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low level of virus neutralizing antibodies as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of the protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Modulates Interferon-β Expression Mainly Through Attenuating Interferon-Regulatory Factor 3 Phosphorylation

Article

Jun 2016

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that emerged from classic PRRSV causes more severe damage to the swine industry. The earlier reports indicating inhibition of interferon-β (IFN-β) expression by PRRSV through total blockage of IFN-regulatory factor 3 (IRF3) nuclear translocation made us investigate the mechanism of IFN-β expression in HP-PRRSV infection. For this purpose, the IRF3 nuclear translocation in the control group [Poly (I:C)] and test group [Poly (I:C)+HP-PRRSV] was detected by immunofluorescence, and the results showed that IRF3 nuclear translocation in cells with PRRSV was weaker than cells without PRRSV, which was different from the previous study. In addition, the IFN-β mRNA and protein expression was observed to be inhibited by HP-PRRSV along with decreased IRF3 mRNA and total protein, and IRF3 nuclear translocation of test group was suppressed in MARC-145 and porcine alveolar macrophage cells in comparison with the control group. The quantity of phosphorylated IRF3 protein was also reduced after HP-PRRSV infection. However, CREB-binding protein (CBP) expression did not change between the control and test group. These results indicate that the inhibition of IFN-β expression is mainly due to the quantitative change in the amount of phosphorylated IRF3 in the cytoplasm, but not dependent on the complete blockage of IRF3 nuclear translocation or the restraining of CBP expression in the nucleus by HP-PRRSV.

Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1

Article

Full-text available

May 2016

Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection.

Cloning and expression of porcine CD163: Its use for characterization of monoclonal antibodies to porcine CD163 and development of an ELISA to measure soluble CD163 in biological fluids

Article

Feb 2008

CD163 is a member of the family of proteins with scavenger receptor cysteine-rich domains, whose expression is restricted to monocytes and macrophages. It functions as a receptor for haemoglobin/haptoglobin complexes and it has also been implicated in the regulation of inflammatory processes. Treatment of monocytes/macrophages with phorbol esters, LPS or cross-linking of Fc gamma receptors, causes shedding of CD163 from cell surface by a protease-dependent mechanism. The level of soluble CD163 (sCD163) in serum and other body fluids has been considered a useful marker of macrophage activation. In addition, porcine CD163 has been shown to play a role in the infection of monocytes/macrophages by the African swine fever virus. The porcine CD163 cDNA has been cloned, and expressed in transfected CHO cells. Clones of CHO cells stably expressing porcine CD163 have been used for the characterization of three new mAbs against porcine CD163. Using two of these mAbs, that bind to different epitopes on CD163 molecule, a sandwich enzyme-linked immunoassay (ELISA) has been developed to measure levels of sCD163 in porcine sera and biological fluids. The assay was calibrated using lysates of CD163 transfectants, showing a sensitivity of 105 cells mL-1, that allowed to detect sCD163 in sera and in culture supernatants of activated alveolar macrophages. This assay can be a useful method of monitoring the degree of activation of macrophages in a variety of inflammatory and infectious diseases of swine.

Montanide<sup>TM</sup> Gel01 ST Adjuvant Enhances PRRS Modified Live Vaccine Efficacy by Regulating Porcine Humoral and Cellular Immune Responses

Article

Full-text available

Jan 2013

Xiangdong Li

Synergistic effects of sequential infection with highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Article

Full-text available

Aug 2013
VIROL J

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Coinfection with highly pathogenic PRRSV (HP-PRRSV) and PCV2 in the field has recently become extensive in some Asian countries. A synergistic pathogenicity between PRRSV and PCV2 infections has previously been reported. However, the consequences of the sequential infection of pigs with these two viruses are unknown. Thirty 35-day-old piglets were randomly divided into six groups (n = 5 each): HP-PRRSV/PCV2 (group 1, inoculated with HP-PRRSV, then inoculated with PCV2 one week later), PCV2/HP-PRRSV (group 2, inoculated with PCV2, then inoculated with HP-PRRSV one week later), HP-PRRSV+PCV2 (group 3, inoculated with HP-PRRSV and PCV2 concurrently), HP-PRRSV (group 4, inoculated with HP-PRRSV), PCV2 (group 5, inoculated with PCV2), and the control (group 6, uninfected). This experiment lasted 28 days. Clinical symptoms and rectal temperatures were recorded each day after inoculation, body weight was recorded weekly, and serum samples were obtained for viral nucleic acid quantification and antibody titration. Variations in CD3+, CD4+ CD8--, CD3+, CD4--, and CD8+ cells, natural killer (NK) cells, and mononuclear cells were determined by flow cytometry. The serum concentrations of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), and macrophage granulocyte-colony stimulating factor (GM-CSF) were determined. Pathological changes in different tissues from the experimentally infected pigs were recorded. The piglets in group 1 had the highest viral loads, the lowest antibody titers, the most-severe clinical signs, and the highest mortality (3/5, 60%; the mortality in the other groups was 0%), and interstitial pneumonia was more severe in this group compare to the other HP-PRRSV infected groups. The serum levels of IFN-gamma, TNF-alpha, IL-10, and GM-CSF varied (increased or decreased) most widely in group 1, as did each immunocyte subgroup. HP-PRRSV infection followed by PCV2 infection enhanced the replication of both viruses in the experimental piglets and led to more-severe clinical signs and lesions, indicating greater synergistic effects during the sequential infection of piglets with HP-PRRSV and then PCV2.

SEMIQUANTITATIVE ANALYSIS OF IL-10 PRODUCTION IN BRONCHOALVEOLAR LAVAGE LEUKOCYTES OF PRRSV INFECTED PIGS

Article

Mar 2004

Evasion strategies of porcine reproductive and respiratory syndrome virus

Article

Full-text available

Mar 2023

During the co-evolution of viruses and their hosts, viruses have developed various strategies for overcoming host immunological defenses so that they can proliferate efficiently. Porcine reproductive and respiratory syndrome virus (PRRSV), a significant virus to the swine industry across the world, typically establishes prolonged infection via diverse and complicated mechanisms, which is one of the biggest obstacles for controlling the associated disease, porcine reproductive and respiratory syndrome (PRRS). In this review, we summarize the latest research on how PRRSV circumvents host antiviral responses from both the innate and adaptive immune systems and how this virus utilizes other evasion mechanisms, such as the manipulation of host apoptosis and microRNA. A thorough understanding of the exact mechanisms of PRRSV immune evasion will help with the development of novel antiviral strategies against PRRSV.

Effects of β-alanine on intestinal development and immune performance of weaned piglets

Article

Full-text available

Dec 2022

Beta-alanine is an important amino acid involved in several metabolic reactions in the body. The study aimed to investigate the effect of β-alanine supplementation on intestinal development and the immune performance of weaned piglets. Thirty-two 21-day-old healthy weaned piglets (half female and half male; Duroc × Landrace × Yorkshire) with an initial body weight of 8.11 ± 0.21 kg were randomly divided into 4 groups with 8 replicates of 1 pig each. The control group was fed a basal diet and the three experimental treatment groups were fed diets supplemented with 300, 600 and 1,200 mg/kg β-alanine, respectively. The trial lasted 28 days and the diets fed were divided into 2 phases: the late lactation period (day 1 to 14) and the nursery period (day 15 to 28), during which the weaned piglets had free access to food and water. The regulatory effects of β-alanine were further investigated in vitro using organoids obtained from the jejunum of piglets. In vivo, the addition of β-alanine to the diet had no significant effect on the growth performance of weaned piglets (P > 0.05), but significantly reduced serum levels of immunoglobulin G (IgG) (P

Tylvalosin administration in pregnant sows attenuates the enlargement and bluish coloration of inguinal lymph nodes in newborn piglets

Article

Jun 2019
RES VET SCI

In the porcine industry, some piglets show slightly enlarged and bluish inguinal lymph nodes. However, the causative factors for these signs and prevention of these signs remain unclear. Tylvalosin is a broad-spectrum antibiotic with an immunomodulatory function. This study was aimed at evaluating the effect of tylvalosin on the abovementioned signs. Thus, fifteen 90-day pregnant sows were divided into an untreated control group and 0.1 and 0.2 g/kg feed tylvalosin-treated groups until delivery. Forty-five piglets on day 2 after birth (15 each group) were blooded, then oxidative stress, serum cytokine levels, routine blood analysis, and effect of sera on macrophage phagocytic activity were examined. Fifteen piglets on day 2 after birth (5 in each group) were euthanized and pathological changes in the inguinal lymph nodes were observed. The untreated piglets showed hemorrhage, hemosiderin accumulation, and increased macrophages in the inguinal lymph nodes. However, tylvalosin administration in sows alleviated these signs in their piglets; increased total antioxidant capacity and serum glutathione levels; decreased serum IL-1β, TNF-α, and IL-10 levels; improved the percentages of neutrophils and lymphocytes in the blood; and increased the body weight of the weaning piglets. In addition, the serum of newborn piglets also showed enhanced RAW264.7 macrophage phagocytic activity. These results demonstrated that tylvalosin administration in pregnant sows attenuates the enlargement and bluish coloration of inguinal lymph nodes in newborn piglets.

Porcine Reproductive and Respiratory Syndrome Viruses (Porcine Arteriviruses)

Chapter

Mar 2019

Control of porcine reproductive and respiratory syndrome virus (PRRSV) remains problematic, and economic studies have uniformly shown that PRRSV inflicts major losses on swine health and productivity. A fuller picture of PRRSV genetic relationships and evolutionary origins may be facilitated by whole genome analyses and comparisons of multiple protein coding regions, including the polymerase gene, which is widely used in RNA viral evolutionary analyses. PRRSV viral infection can be divided into three distinct stages: acute infection, persistence, and extinction. Acute infection follows exposure and is characterized by rapid spread to primary sites of replication in lung and lymphoid tissues. PRRSV markedly alters innate immunity and inflammatory and immunoregulatory cytokines in a strain‐ specific manner. Infection with PRRSV induces immunity that eventually controls the initial infection, eliminates the virus, and establishes memory that is variably protective against future infection.

Immune System

Chapter

Mar 2019

The continuing explosion of knowledge on the immune system has substantial implications for swine health. This contains key concepts, including immune proteins and cell subsets, genetics, microbiome–immune interactions, and vaccine responses. The immune system develops in the fetus, and immune responses begin where microorganisms and/or their products interact with epithelial cells of the mucosa and skin. Pattern recognition receptors, including Toll‐like receptors, monitor pathogen‐associated molecular patterns and induce different signaling pathways to activate the immune system against infection. Immune system functions require energy, protein, vitamins, and trace minerals. Immune dysfunction or decreased immunocompetence can be due to a variety of factors including stress, malnutrition, and concurrent infections; immaturity or senescence of the immune system may also lead to vaccination failure. If the immune dysfunction occurs at the time of vaccination, the vaccine may fail to induce an adequate immune response.

Dietary arachidonate in milk replacer triggers dual benefits of PGE2 signaling in LPS-challenged piglet alveolar macrophages

Article

Full-text available

Feb 2019

Background Respiratory infections challenge the swine industry, despite common medicinal practices. The dual signaling nature of PGE2 (supporting both inflammation and resolution) makes it a potent regulator of immune cell function. Therefore, the use of dietary long chain n-6 PUFA to enhance PGE2 effects merits investigation. Methods Day-old pigs (n = 60) were allotted to one of three dietary groups for 21 d (n = 20/diet), and received either a control diet (CON, arachidonate = 0.5% of total fatty acids), an arachidonate (ARA)-enriched diet (LC n-6, ARA = 2.2%), or an eicosapentaenoic (EPA)-enriched diet (LC n-3, EPA = 3.0%). Alveolar macrophages and lung parenchymal tissue were collected for fatty acid analysis. Isolated alveolar macrophages were stimulated with LPS in situ for 24 h, and mRNA was isolated to assess markers associated with inflammation and eicosanoid production. Culture media were collected to assess PGE2 secretion. Oxidative burst in macrophages was measured by: 1) oxygen consumption and extracellular acidification (via Seahorse), 2) cytoplasmic oxidation and 3) nitric oxide production following 4, 18, and 24 h of LPS stimulation. Results Concentration of ARA (% of fatty acids, w/w) in macrophages from pigs fed LC n-6 was 86% higher than CON and 18% lower in pigs fed LC n-3 (P < 0.01). Following LPS stimulation, abundance of COX-2 and TNF-α mRNA (P < 0.0001), and PGE2 secretion (P < 0. 01) were higher in LC n-6 PAM vs. CON. However, ALOX5 abundance was 1.6-fold lower than CON. Macrophages from CON and LC n-6 groups were 4-fold higher in ALOX12/15 abundance (P < 0.0001) compared to LC n-3. Oxygen consumption and extracellular acidification rates increased over 4 h following LPS stimulation (P < 0.05) regardless of treatment. Similarly, increases in cytoplasmic oxidation (P < 0.001) and nitric oxide production (P < 0.002) were observed after 18 h of LPS stimulation but were unaffected by diet. Conclusions We infer that enriching diets with arachidonic acid may be an effective means to enhance a stronger innate immunologic response to respiratory challenges in neonatal pigs. However, further work is needed to examine long-term safety, clinical efficacy and economic viability. Electronic supplementary material The online version of this article (10.1186/s40104-019-0321-1) contains supplementary material, which is available to authorized users.

Co-administration of saponin quil A and PRRSV-1 modified-live virus vaccine up-regulates gene expression of type I interferon-regulated gene, type I and II interferon, and inflammatory cytokines and reduces viremia in response to PRRSV-2 challenge

Article

Oct 2018
VET IMMUNOL IMMUNOP

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating virus which suppresses the expression of type I and II interferons (IFNs) as well as several pro-inflammatory cytokines. Our previous study reported that saponin quil A had a potential to up-regulate the expression of type I IFN-regulated genes and type I and II IFNs in porcine peripheral blood mononuclear cells (PBMC) inoculated with PRRSV. The present study evaluated the immunostimulatory effect of quil A on potentiating cross protective immunity of PRRSV-1 modified-live virus (MLV) vaccine against PRRSV-2 challenge. Twenty-four 4-week-old PRRSV-seronegative pigs were divided into four groups of six pigs. Group 1 and group 2 pigs were vaccinated with PRRSV-1 MLV vaccine at 0 dpv (day post vaccination), and additionally group 2 pigs were injected intramuscularly with quil A at −1, 0, 1 dpv. Group 3 pigs were injected with PRRSV-1 MLV vaccine solvent at 0 dpv and served as challenge control, while group 4 pigs served as strict control. Group 1–3 pigs were challenged intranasally with PRRSV-2 at 28 dpv and immune and clinical parameters were observed from 0 until 49 dpv. Group 1 pigs showed significantly reduced PRRSV viremia, number of viremic pigs, and clinical scores, and significantly improved average daily weight gain (ADWG), compared to group 3 pigs. Group 2 pigs showed significantly increased mRNA expressions of interferon regulatory factor 3, 2′-5′-oligoadenylatesynthetase 1, osteopontin, IFNα IFNβ IFNγ interleukin-2 (IL-2), IL-13 and tumor necrosis factor alpha, compared to group 1 pigs. The animals demonstrated significantly reduced PRRSV viremia and number of viremic pigs, but did not demonstrate any further improved PRRSV-specific antibody levels, neutralizing antibody titers, rectal temperature, clinical scores, and ADWG as compared to group 1 pigs. Our findings suggest that quil A up-regulates type I IFN-regulated gene, type I and II IFNs, and inflammatory cytokine expressions which may contribute to further reducing PRRSV viremia and number of viremic pigs which were conferred by PRRSV-1 MLV vaccine. Our findings also suggest that quil A may serve as an effective immunostimulator for potentiating cell-mediated immune defense to PRRSV.

Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro

Article

Full-text available

Jun 2018
ARCH VIROL

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-β) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

The viral innate immune antagonism and an alternative vaccine design for PRRS virus

Article

Mar 2017

Generation of potent porcine monocyte-derived dendritic cells (MoDCs) by modified culture protocol

Article

Oct 2016
VET IMMUNOL IMMUNOP

In vitro derivation of dendritic cells (DCs) is an alternative approach to overcome the low frequency of primary DCs and the difficulty of isolation techniques for studying DC immunobiology. To date, the conventional culture protocol of porcine monocyte-derived DCs (MoDCs) has been widely used. However, this protocol is not practical due to the requirement of a substantial number of blood monocytes, and the process often interferes with DC maturation. To improve in vitro porcine MoDC generation, we modified the previous conventional DC generation protocol, based on the human and mouse primary DC culture system, and compared phenotypic and functional features of MoDCs derived from the modified protocol to the conventional protocol. The modified protocol consumed fewer monocytes but generated higher CD1⁺ cells with DC-like morphology and the ability of maturation. In addition, MoDCs from the modified protocol exhibited increased antigen uptake and IFN-γ production in response to LPS stimulation. Our findings indicate that the modified protocol is expedient and reliable for generating potent MoDCs that substitute for primary DCs. This will be a valuable platform for future research in antigen delivery, vaccines and immunotherapy in pigs, as well as relevant veterinary species.

Effects of mycoplasmal pneumonia of swine (MPS) lung lesion-selected Landrace pigs on MPS resistance and immune competence in three-way crossbred pigs

Article

Sep 2016

To clarify the genetic influence of mycoplasmal pneumonia of swine (MPS) lesion-selected Landrace (La) on MPS resistance and immune characteristics in three-way crossbred pigs (LaWaDa), the LaWaDa pigs were compared with the non-selected crossbred (LbWbDb) and purebred (La) pigs. The MPS lesion score in the three lines was as follows: La line < LaWaDa line < LbWbDb line, with significant differences among the lines. The proportions of myeloid cells and T cells were lower and higher, respectively, in the LaWaDa pigs compared with those in the other two lines. Messenger RNA (mRNA) expression of interleukin (IL)-6, IL-10, transforming growth factor-β, and interferon-γ in peripheral blood was significantly increased after vaccination in the La and LaWaDa lines. IL-4 mRNA expression in the LaWaDa line was intermediate to the La and LbWbDb lines. Furthermore, principal component analysis for immune traits and MPS lesions was executed to clarify the characteristics of each pig line. These findings suggest that the immune responses in the three pig lines are genetically distinct and that MPS resistance and some immunity characteristics from the La line were transmitted to the three-way crossbred pigs.

Effect of amino acids residues 323–433 and 628–747 in Nsp2 of representative porcine reproductive and respiratory syndrome virus strains on inflammatory response in vitro

Article

Jun 2015

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that is responsible for large economic losses in the swine industry worldwide. In PRRSV strains, many genetic variations occur in the central hypervariable region (HV2) of the Nsp2 gene, which encodes non-structural protein 2. For example, PRRSV strains VR2332, Em2007, MN184C, and TJM-F92 contained variations in the Nsp2 sequences and exhibited differing levels of virulence in adult pigs. However, the role of HV2 with respect to PRRSV immunity is unclear. In this study, four recombinant PRRSV strains (rBB/+30aa, rBB/Δ68aa, rBB/Δ111aa, and rBB/Δ120aa) were rescued using a highly pathogenic type 2 PRRSV cDNA clone (pBB). All rescued strains displayed similar growth characteristics to the parental rBB virus in pulmonary alveolar macrophages (PAMs). Expression levels of inflammatory cytokines IL-β, IL-6, and TNF-α were significantly lower, at the mRNA and protein level, for groups infected with rBB/Δ111aa and rBB/Δ120aa than those in the rBB group. Levels of these inflammatory cytokines in the rBB/+30aa and rBB/Δ68aa groups were not significantly different with those in the rBB group. Phosphorylation levels of IκB were decreased to a greater extent in the rBB/Δ111aa and rBB/Δ120aa groups compared with those in the rBB/+30aa, rBB/Δ68aa, and rBB groups. Our results indicate that amino acids 323-433 and 628-747 of Nsp2 failed to exert significant effects on PRRSV replication in PAMs, but modulated the expression of inflammatory cytokines in vitro. Copyright © 2015. Published by Elsevier B.V.

Antiviral Activity of Four Commercial Tilmicosin Preparations against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): An In Vitro Study

Article

Full-text available

Jun 2014
THAI J VET MED

The efficacy of four commercial tilmicosin preparations (A, B, C and D) against type 2 porcine reproductive and respiratory syndrome virus (PRRSV), 01NP1 (Thai isolate), infection in cultured pulmonary alveolar macrophages (PAMs) was conducted in vitro. Primary PAMs were collected from four 4-week-old PRRSV-free pigs. After separately plated onto four 24-well plates, PAMs were treated separately by 4 commercial tilmicosin preparations of 2 concentrations each (0.1 mg/ml and 0.01 mg/ml). The treated PAMs were inoculated with 0.05 MOI of 01NP1 strain and were stained with PRRSV specific antibody using immunoperoxidase monolayer assay (IPMA) to evaluate the quantity of PRRSV infected cells after 12 hours post infection (HPI). Comparing to the untreated-tilmicosin PRRSV-infected PAMs, all tilmicosin-treated preparations exhibited significant virus titer reduction against 01NP1. Based on the results, 0.01 mg/ml of tilmicosin B solution exhibited the greatest PRRSV-titer reduction (65%), but was not statistically different from the others. The results indicated that tilmicosin could be one of the effective chemotherapy in reducing type 2 PRRSV infection in vitro regardless of differences in the preparations. The information obtained is of interest for practitioners for future study of implementation of tilmicosin use in PRRSV-positive farms.

Adaptable inactivated vaccine for combating Porcine Reproductive and Respiratory Syndrome Virus

Thesis

Jan 2013

(PRRSV) is betrokken bij reproductiestoornissen bij zeugen en beren en bij respiratoire problematiek bij biggen van alle leeftijden, wat resulteert in een grote economische verliezen in de varkensindustrie. Een grote genetische variabiliteit is aangetoond onder de verschillende PRRSV isolaten en dit heeft zijn weerslag op hun virulentie, pathogeniciteit, immunogeniciteit,… Deze diversiteit is een groot probleem betreffende PRRSV preventie en controle. Het gebruik van de huidige commerciële vaccins in diverse vaccinatiestrategieën is de meest voorkomende methode om de klinische en economische impact van PRRSV te onderdrukken. Momenteel is er nood aan nieuwe en veilige vaccins die bescherming kunnen bieden tegen de PRRSV stammen die ontsnappen aan de immuniteit geïnduceerd door de huidige beschikbare commerciële vaccins. De doelstelling van deze thesis was het evalueren van de werkzaamheid van een geïnactiveerd bedrijfsspecifiek PRRSV vaccin - op basis van een eerder geoptimaliseerde virus- inactivatiemethode – en deze te vergelijken met die van commercieel beschikbare geattenueerde / geïnactiveerde PRRSV vaccins in naïeve en PRRSV-immune dieren. Tot op heden is het niet gekend welke voordelen er verbonden zijn aan het gebruik van deze bedrijfsspecifieke vaccins. De eerste studie richtte zich voornamelijk op het vergelijken van de werkzaamheid van de homologe BEI-geïnactiveerde vaccins met heterologe BEIgeïnactiveerde vaccins en commerciële geïnactiveerde en geattenueerde vaccins na een homologe of heterologe challenge. De homologe BEIgeïnactiveerde vaccins zorgden na infectie voor een significante reductie in viremie. Ondanks de twijfel betreffende de werkzaamheid van de commercieel geattenueerde vaccins - die ook worden gebruikt op de bedrijven waar de PRRSV-isolaten vandaan komen – hadden de commerciële geattenueerde vaccins toch een invloed op de duur van de viremie na infectie. De heterologe BEI-geïnactiveerde en commercieel geïnactiveerde vaccins daarentegen hadden geen of beperkte invloed op de viremie. In een tweede luik werden de BEI-geïnactiveerde bedrijfsspecifieke vaccins vergeleken met commercieel beschikbare geattenueerde / geïnactiveerde vaccins in PRRSV-immune zeugen qua boosten van de humorale immuniteit tegen huidig circulerende PRRSV isolaten. In een eerste deel van deze studie werd de PRRSVspecifieke antistoffenrespons na booster vaccinatie met commerciële vaccins en BEI-geïnactiveerde bedrijfsspecifieke vaccins geëvalueerd in niet drachtige reforme zeugen van 3 bedrijven met PRRS-problemen ondanks vaccinatie van zeugen en gelten. Een boost in virus-neutraliserende antistoffen tegen de bedrijfsspecifieke PRRSV isolaten werd opgemerkt bij alle zeugen die werden gevaccineerd met het respectievelijke geïnactiveerde bedrijfsspecifieke vaccin. De commerciële geattenueerde en geïnactiveerde vaccins gaven gemengde resultaten qua boosten van neutraliserende antistoffen tegen het bedrijfsspecifieke isolaat. In het tweede deel van de studie werd een veldproef uitgevoerd op 1 van bovenvermelde bedrijven om het booster effect van het BEI-geïnactiveerd bedrijfsspecifiek vaccin en het commercieel geattenueerd EU-type vaccin bij PRRSVimmune zeugen op 60 dagen dracht te evalueren. De impact van deze vaccinatie op de maternale immuniteit en op het PRRSV infectiepatroon bij de biggen gedurende hun eerste levensweken werd geëvalueerd. Na vaccinatie met het bedrijfsspecifieke vaccin werd een sterke stijging in bedrijfsspecifieke virusneutraliserende antistoffen opgemerkt bij alle zeugen. Tot 5 weken na de geboorte werden virusneutraliserende antistoffen gevonden in het bloed van biggen afkomstig van deze zeugen. Niet alle zeugen gevaccineerd met het commercieel geattenueerd EUtype vaccin toonden een stijging in bedrijfsspecifieke virus-neutraliserende antistoffen en de biggen afkomstig van deze zeugen hadden in het algemeen minder virus-neutraliserende antistoffen. Het aantal viremische biggen was significant lager bij biggen van beide gevaccineerde zeugengroepen dan bij biggen afkomstig van de controle zeugen en dit bleef zo tot op 9 weken na hun geboorte. In een laatste studie werd de werkzaamheid van het BEI-geïnactiveerd vaccin virus geproduceerd op de nieuwe gevoelige cellijn PK15Sn-CD163 vergeleken met het BEI-geïnactiveerd vaccin virus geproduceerd op MARC-145 cellen in een homologe en heterologe situatie bij niet-immune biggen. In de homologe PK15Sn-CD163 gegroeide en MARC-145 gegroeide BEIgeïnactiveerde vaccins, was na infectie de duur van de viremie significant gereduceerd met gemiddeld een tweetal weken. Een gelijkaardige resultaat werd bekomen met het heteroloog PK15Sn-CD163 gegroeide vaccin. Geen significante reductie in viremie werd geobserveerd met het heteroloog MARC-145 gegroeid vaccin na heterologe infectie. Finaal worden in de thesis de belangrijkste bevindingen samengevat en bediscussieerd en wordt het potentieel van bedrijfsspecifieke geïnactiveerde vaccins besproken.

The N-N non-covalent domain of the nucleocapsid protein of type 2 porcine reproductive and respiratory syndrome virus enhances induction of IL-10 expression

Article

Jan 2015
J GEN VIROL

Porcine reproductive and respiratory syndrome virus (PRRSV) usually establishes a prolonged infection and causes an immunosuppressive state. It has been proposed that interleukin-10 (IL-10) plays an important role in PRRSV-induced immunosuppression. However, this mechanism has not been completely elucidated. In this study, we found that transfection of 3D4/2 macrophages with the N protein gene of type 2 PRRSV significantly upregulated IL-10 expression at the transcriptional level. Moreover, alanine substitution mutation analysis revealed that the N protein residues 33-37, 65-68, and 112-123 were related to the upregulation of IL-10 promoter activity. Recombinant PRRSV with mutations at residues 33-37 in the N protein (rQ33-5A and rS36A), recovered from corresponding infectious cDNA clones, induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells, as compared to their revertants rQ33-5A(R) and rS36A(R), and the wild-type recombinant PRRSV strain rNT/wt. These data indicate that type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.

Regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus

Article

Dec 2014
VIRUS RES

Investigating the Role of Cytokines in Immunity to Marek's Disease

Article

Kamran Haq

P5 Induction of Porcine Interleukin-10 by Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Article

Full-text available

Mar 2009

Introduction and Objectives Porcine Reproductive and Respira-tory Syndrome Virus (PRRSV) causes economic loss in the pig industry worldwide including Thailand. Many studies indicate that PRRSV can increase IL-10 production in pigs during the early infection period (1), during the same period PRRSV also expresses high quantities of the nucleocapsid protein (N-protein) (2). This information suggests that N-protein might induce IL-10 production in pigs. In this study, the effect of N-protein on IL-10 production in porcine PBMC was investigated. Materials and Methods The ORF7 specific primers were designed for cloning the N-protein gene (ORF7, native conformation) and truncated N-protein (ORF7t, linear conformation) from the Thai isolate US-PRRSV (01NP1). The PCR products were cloned into pGEM?-T easy vector (Promega, USA). Subsequently ORF7 and ORF7t genes were subcloned into pQE31 vector (Qiagen, German) as a fusion protein with 6x-Histidine (6x-His) and expressed in the Escherichia coli strain M15. Porcine peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood of PRRSV-seronegative pigs by density gradient centrifugation. The PBMC were co-cultured with the 5 g/ml purified recom-binant N-protein (ORF7) and truncated N-protein (ORF7t) for 48 hr. Negative control groups included either the PBMC cultured with elution buffer or protein expressed from pQE31. The positive control was the PBMC cultured with PRRSV. The expressions of IL-10 genes were analysed by semi-quantitative RT-mPCR (3). The intracellular expression of IL-10 was analyzed by flow cytometry (4). Results and Discussion N-protein (ORF7) induced IL-10 gene expression and increased the numbers of IL-10+ cells in the PBMC culture. However, truncated N-protein (ORF7t) could not induce IL-10 gene expression nor increase the number of IL-10+ cells (Figs. 1 & 2). The results suggest that, PRRSV N-protein can induce IL-10 production in the PBMC and the conformation of N-protein plays significant role in induction of IL-10. This study identified the novel immunomodulatory mechanism of PRRSV.

Comparative Analysis of Immune Responses in Pigs to High and Low Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses Isolated in China

Article

Full-text available

Dec 2013
TRANSBOUND EMERG DIS

The CH-1a and HuN4 strains of porcine reproductive and respiratory syndrome virus (PRRSV) show different pathogenicities in pigs. To understand host immune responses against these viruses, we investigated the dynamic changes in cytokine levels produced in peripheral blood of piglets infected with the highly pathogenic PRRSV HuN4 strain or the CH-1a strain. Clinical signs, virus loads and serum cytokine levels [interferon(IFN)-α, Interleukin (IL)-1, TNF-α, IL-6, IL-12, IFN-γ, IL-10 and TGF-β] were tested. The results showed that while piglets developed effective cellular immune responses against CH-1a infection, those infected with HuN4 displayed ineffective cellular immunity, organ lesions and persistent elevated levels of immunoregulatory cytokines (IL-10 and TGF-β), which delayed the development of PRRSV-specific immune responses. These results demonstrated that HuN4 infection induced higher cytokine levels than that of CH-1a infection induced. The changes in inflammatory cytokines intensified the inflammatory reaction and damaged the tissues and organs.

The challenges of classical swine fever control: Modified live and E2 subunit vaccines

Article

Nov 2013

Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely-used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA.

Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection

Article

Full-text available

Aug 2013

PRRSV is a member of the genus Arterivirus, family Arteriviridae, order Nidovirales. PRRSV is the causative agent of the most important infectious disease that affects swine herds world-wide, producing great economic losses. Since its emergence over 25 years ago, much has been learned about the immunobiology of PRRSV. The immune response to PRRSV infection is weak and relatively late. This results in long-term persistence of viral load (4 – 5 weeks), and infectious virions are detected in lymphoid tissue up to 157 days post-infection. Although the organism's immune response to PRRSV infection and the mechanisms of resistance to infection have not been fully explored, commercial vaccines are available on the market. These vaccines do not provide full and universal protection against PRRSV infection. The present review discusses current knowledge on the virus immunobiology, including the innate and adaptive immune responses to the virus and the regulation of the immune response by PRRSV.

Seroprevalance of bovine viral respiratory diseases

Article

Full-text available

Jan 2007

In this study, sera from 188 unvaccinated cattle from Middle Black Sea Region of Turkey were investigated. Serum samples were tested for antibodies against five viruses which cause respiratory diseases in cattle, including bovine herpesvirus type 1 (BHV-1), bovine viral diarrhoea virus (BVDV), parainfluenzavirus type 3 (PIV-3), bovine adenovirus type 1 (BAdV-1) and bovine adenovirus type 3 (BAdV-3) by using a conventional method, i.e. the serum neutralization (SN) test. The antibody seroprevalence found in cattle against 5 viruses (BHV-1, BVDV, PIV-3, BAdV-1 and BAdV-3) were found to be: 61.17%, 53.19%, 88.82%, 72.34% and 81.38%, respectively.

Transmission of porcine reproductive and respiratory syndrome virus by fomites (boots and coveralls)

Article

Full-text available

May 2002
J SWINE HEALTH PROD

Objectives: To determine if porcine reproductive and respiratory syndrome virus (PRRSV) can be transmitted to susceptible pigs by fomites or people exposed to infected pigs. Methods: Ninety-six 4-week-old pigs from a PRRSV-naive source were organized into six groups individually housed in isolation rooms (four replicate trials, 24 pigs per trial). Group 1 pigs were inoculated intranasally with PRRSV strain VR-2332 (2 mL, 105 median tissue culture infective doses per mL.) On days 5, 6, and 7 post inoculation, investigators exposed to Group 1 pigs attempted to transmit PRRSV to sentinel pigs (Groups 2 to 5) by contact. After exposure to the infected pigs, an investigator entered the Group 2 room (Direct Contact group) wearing contaminated boots and coveralls and without washing hands. In contrast, investigators who entered the rooms housing Groups 3 to 5 were required to complete specific sanitation protocols, which included changing boots and coveralls and washing hands (Danish System, Group 3); changing boots and coveralls, showering, and 12 hours down time (Standard Protocol, Group 4); and changing boots and coveralls and showering, with no down time (Alternative Protocol, Group 5). Results: The PRRSV was detected on contaminated coveralls, boots, and hands of investigators who had contacted Group 1 pigs. Transmission of PRRSV occurred between Groups 1 and 2, but not between Group 1 and Group 3, 4, or 5. Implications: The PRRSV can be transmitted to susceptible pigs by contaminated fomites (boots and coveralls) and hands; however, the use of sanitation protocols appears to limit its spread.

Pathogenesis of Porcine Reproductive and Respiratory Syndrome Virus Infection in Gnotobiotic Pigs

Article

Full-text available

Aug 1995

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) was determined in gnotobiotic pigs by studying the sequential development of microscopic lesions and sites of virus distribution and replication. Thirty-two pigs (three pigs/infected group and one pig/control group) were inoculated by nasal instillation of either PRRSV isolate ATCC VR-2332 (total dose 10(2.6) TCID50) or uninfected cell culture supernatant. Infected and control pigs were euthanized at 12 hours, and 1, 2, 3, 5, 7, 14, and 21 days postexposure (PE). Gnotobiotic pigs experimentally infected with PRRSV were viremic by 12 hours PE and subsequently developed pneumonia, lymphadenopathy, vasculitis, myocarditis and encephalitis. Lung lesions developed by day 3 PE, persisted through day 21 PE and were characterized by alveolar septa thickened by macrophages, alveolar proteinaceous and karyorrhectic debris, alveolar syncytial cells, and multifocal type II pneumocyte hypertrophy. Lymph node lesions varied in distribution and severity and were characterized by germinal center hypertrophy and hyperplasia, lymphocyte necrosis, multiple cystic spaces, and polykaryocytes within the cystic spaces. Heart lesions were a late feature of infection and all infected pigs had heart lesions on day 21 PE characterized by subendocardial, myocardial, and perivascular foci of lymphocytes. Vasculitis also varied in distribution and severity and affected all sizes of vessels. Results of this experiment indicate that PRRSV is a multisystem disease characterized initially by viremia with subsequent virus distribution and replication in multiple organs causing interstitial pneumonia, vasculitis, lymphadenopathy, myocarditis, and encephalitis.

Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): Implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe

Article

Full-text available

Feb 1995

The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96-100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57-59% and 78-81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes.

Comparison of the Pathogenicity of Two US Porcine Reproductive and Respiratory Syndrome Virus Isolates with that of the Lelystad Virus

Article

Full-text available

Dec 1995

The Lelystad virus or one of two US isolates (VR2385, VR2431) of porcine reproductive and respiratory syndrome virus were given intranasally to 25 4-week-old cesarian-derived colostrum-deprived pigs. Pigs from these groups were necropsied at 1, 2, 3, 5, 7, 10, 15, 21, or 28 days postinoculation. The Lelystad virus and VR2431 induced mild transient pyrexia, dyspnea, and tachypnea. VR2385 induced labored and rapid abdominal respiration, pyrexia, lethargy, anorexia, and patchy dermal cyanosis. All three isolates induced multifocal tan-mottled consolidation involving 6.8% (n = 9; SEM = 3.4) of the lung for Lelystad, 9.7% (n = 9, SEM = 2.7) of the lung for VR2431, and 54.2% (n = 9, SEM = 4.4) of the lung for VR2385 at 10 days postinoculation. Characteristic microscopic lung lesions consisted of type 2 pneumocyte hypertrophy and hyperplasia, necrotic debris and increased mixed inflammatory cells in alveolar spaces, and alveolar septal infiltration with mononuclear cells. Lymphadenopathy with follicular hypertrophy, hyperplasia, and necrosis was consistently seen. Similar follicular lesions were also seen in Peyer's patches and tonsils. Lymphohistiocytic myocarditis and encephalitis were reproduced with all three isolates. Clinical respiratory disease and gross and microscopic lung lesion scores were considerably and significantly more severe in the VR2385-inoculated pigs. All three viruses were readily isolated from sera, lungs, and tonsils throughout the 28 days of the study. The lymphoid and respiratory systems have the most remarkable lesions and appear to be the major site of replication of these viruses. This work demonstrated a marked difference in pathogenicity of porcine reproductive and respiratory syndrome isolates.

Effects of Low (Modified-live Virus Vaccine) and High (VR-2385)-Virulence Strains of Porcine Reproductive and Respiratory Syndrome Virus on Pulmonary Clearance of Copper Particles in Pigs

Article

Full-text available

Sep 1998

Seventy-five 3-week-old, crossbred pigs from a herd free of porcine reproductive and respiratory syndrome virus (PRSSV) were randomly assigned to three groups: uninfected controls, pigs inoculated intranasally with RespPRRS/Repro modified-live virus vaccine (RespPRRS), and pigs inoculated intranasally with a high-virulence strain of PRRSV (VR-2385). Pigs were intravenously infused with 3% copper phthalocyanine tetrasulfonic acid (0.2 ml/kg) in normal saline 30 minutes before necropsy, which was performed 3, 7, 10, 14, or 28 days postinoculation (DPI) with PRRSV. There were no differences in serum copper concentration in samples collected at 0, 15, or 30 minutes after infusion. Copper concentrations in the lungs of VR-2385-inoculated pigs were significantly lower than levels in the lungs of control and RespPRRS-inoculated pigs at 7, 10, and 14 DPI (P < 0.05). The greatest difference between the groups was observed at 10 DPI. Liver and spleen copper concentrations were slightly, but not significantly, higher in both PRRSV-infected groups. The percentage of lung affected by grossly visible pneumonia ranged from 0 to 5.6% in the RespPRRS-inoculated group and from 15.2 to 46.4% in the VR-2385-inoculated group, with lesions peaking at 7 and 10 DPI, respectively. PRRSV antigen was demonstrated in both pulmonary alveolar macrophages (PAMs) and pulmonary intravascular macrophages (PIMs) by immunohistochemical methods. Copper particles were demonstrated in the PIMs by light microscopy. PRRSV was isolated from bronchoalveolar lavage fluid of VR-2385-infected pigs from 3 to 28 DPI and from RespPRRS-inoculated pigs from 7 to 28 DPI. No PRRSV, PRRSV antibodies, or PRRSV-induced pneumonia was detected in the control group. These results suggest that 1) PRRSV has a detrimental effect on the uptake of copper particles by PIMs, 2) the severity of PRRSV-induced damage to PIMs differs among strains, and 3) demonstration of PRRSV-induced decreased pulmonary clearance supports the hypothesis that PRRSV infection may make pigs more susceptible to bacterial septicemia.

Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: A role for CD8+ cells

Article

Full-text available

Mar 2000

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

The interleukin-10 family of cytokines

Article

Full-text available

Mar 2002
TRENDS IMMUNOL

A family of interleukin-10 (IL-10)-related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence paralogs, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads, through the activation of signal transducer and activator of transcription (STAT) factors, to diverse biological effects. For example, whereas IL-10 is a well-studied pleiotropic immunosuppressive and immunostimulatory cytokine, IL-22/IL-TIF mediates acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes, which has been proposed as a pathogenic mechanism of psoriasis.

Mechanical transmission of porcine reproductive and respiratory syndrome virus by mosquitoes, Aedes vexans (Meigen)

Article

Full-text available

Aug 2002
CAN J VET RES

The objective of this study was to determine whether porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to naive pigs by mosquitoes following feeding on infected pigs. During each of 4 replicates, mosquito-to-pig contact took place on days 5, 6, and 7 after PRRSV infection of the donor pig. A total of 300 mosquitoes [Aedes vexans (Meigen)] were allowed to feed on each viremic donor pig, housed in an isolation room. After 30 to 60 s, feeding was interrupted, and the mosquitoes were manually transferred in small plastic vials and allowed to feed to repletion on a naïve recipient pig housed in another isolation room. Prior to contact with the recipient pig, the mosquitoes were transferred to clean vials. Swabs were collected from the exterior surface of all vials, pooled, and tested for PRRSV. Separate personnel handled the donor pig, the recipient pig, and the vial-transfer procedure. Transmission of PRRSV from the donor to the recipient pig occurred in 2 out of 4 replicates. The PRRSV isolated from the infected recipient pigs was nucleic-acid-sequenced and found to be 100% homologous with the virus used to infect the donor pigs. Homogenates of mosquito tissues collected in all replicates were positive by either polymerase chain reaction or swine bioassay. All control pigs remained PRRSV negative, and PRRSV was not detected on the surface of the vials. This study indicates that mosquitoes (A. vexans) can serve as mechanical vectors of PRRSV.

Cytokine Profiles in Peripheral Blood Mononuclear Cells and Lymph Node Cells from Piglets Infected In Utero with Porcine Reproductive and Respiratory Syndrome Virus

Article

Full-text available

Dec 2002

The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (gamma-IFN), tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, gamma-IFN-, and TNF-alpha-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing gamma-IFN and TNF-alpha. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.

Immunological Responses of Swine to Porcine Reproductive and Respiratory Syndrome Virus Infection

Article

Full-text available

Feb 2002

The immunology of porcine reproductive and respiratory syndrome virus (PRRS) begins with an initial encounter of PRRSV with the pig. Regardless of the route of entry of PRRSV--via inhalation, intramuscular vaccination, insemination, or other routes--productive infection occurs predominately in alveolar macrophages of the lung. Thus, innate responses of the lung and the alveolar macrophage comprise the initial defense against PRRSV. The virus appears not to elicit innate interferon and cytokine responses characteristic of other strongly immunogenic viral pathogens, and its effects are consistent with induction of a weak adaptive immune response. Humoral and cell-mediated immunity is induced in due course, and results in clearance of virus from the circulation but not from lymphoid tissues, where the infection becomes persistent. Subsequent reexposure to PRRSV elicits an anamnestic response that is partially to completely protective. Within this unconventional picture of anti-PRRSV immunity lie a variety of unresolved issues, including the nature of protective immunity within individual pigs and among pigs in commercial populations, the efficacy of protective immunity against genetically different PRRSV isolates, the effects of developmental age, sex, genetics, and other host factors on the immune response to PRRSV, and the possible suppression of host immunity to other pathogens.

Upregulation of IL-10 gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus

Article

Full-text available

Mar 2003

Several lines of evidence suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system. To determine the effect of PRRSV on cytokine production, a multiplex PCR was established. This allowed a semi-quantitative analysis of IFN-gamma, IL-2, IL-4, IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression levels from porcine peripheral blood mononuclear cells (PBMCs). These results showed that both live and inactivated PRRSV predominantly upregulated IL-10 gene expression in porcine PBMCs. In addition, when PBMCs from pigs immunized previously with classical swine fever virus (CSFV) vaccine were cultivated with the recall antigen, CSFV, in the presence of PRRSV, significant upregulation of IL-10 gene expression and reduction of IFN-gamma gene expression were observed. These findings indicated that the presence of PRRSV in the culture could affect recall antigen response. This study implies that the induction of IL-10 production may be one of the strategies used by PRRSV to modulate host immune responses.

Interleukin-10 and interleukin-10 receptor

Article

Feb 2001
ANNU REV IMMUNOL

Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.

O''Garra A: Interleukin10 and the interleukin-10 receptor

Article

Hijacking and exploitation of IL-10 by intracellular pathogens

Article

Mar 2001
TRENDS MICROBIOL

Macrophages play a central role in infections, as a target for pathogens and in activation of the immune system. Interleukin-10 (IL-10), a cytokine produced by macrophages, is a potent immunosuppressive factor. Some intracellular pathogens specifically target macrophages for infection and use IL-10 to dampen the host immune response and stall their elimination from the host. Certain viruses induce production of cellular IL-10 by macrophages, whereas other viruses encode their own viral IL-10 homologs. Additionally, specific bacteria, including several Mycobacteria spp. and Listeria monocytogenes, can survive and replicate in macrophages while inducing cellular IL-10, highlighting a potential role for IL-10 of macrophage origin in the immunosuppressive etiology of these pathogens. Thus, the exploitation of IL-10 appears to be a common mechanism of immunosuppression by a diverse group of intracellular pathogens that can infect macrophages.

Characterization of the Humoral Immune Response to Porcine Reproductive and Respiratory Syndrome (PRRS) Virus Infection

Article

Aug 1995

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-Mediated Immunity to Porcine Reproductive and Respiratory Syndrome Virus in Swine

Article

Feb 1997

Cell-mediated immunity has been demonstrated to be a necessary component of immunity against viral infection. Methods to detect T-cell mediated immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection were established both in vitro as lymphocyte proliferation and in vivo as delayed-type hypersensitivity response (DTH). Optimal conditions for detection of lymphocyte proliferation were determined by testing different antigen concentrations and various stimulation periods. The proliferation response to PRRSV was antigen-specific and dose-dependent. The kinetics of the T-cell proliferation response to PRRSV were analyzed after primary and secondary exposure to virus. Lymphocyte proliferation was first detected at four weeks post-infection (PI), peaked at 7 weeks PI, and declined after 11 weeks PI. The secondary response increased in magnitude. Experiments with blocking antibodies to porcine leukocyte antigens demonstrated that CD4+ T-cells were the major effector cells in the proliferation response. The in vivo response to PRRSV was shown by detection of a dose-dependent DTH reaction in infected pigs after intradermal challenge with UV-inactivated virus. These results demonstrate that pigs generate specific T-cell responses on PRRSV infection and provide a foundation for studying their role in protection.

Porcine reproductive and respiratory syndrome virus: A persistent infection

Article

May 1997
VET MICROBIOL

Persistent infection with porcine reproductive and respiratory syndrome virus (PRRSV) was shown in experimentally infected pigs by isolation of virus from oropharyngeal samples for up to 157 days after challenge. Four 4 week old, conventional, PRRSV antibody-negative pigs were intranasally inoculated with PRRSV (ATCC VR-2402). Serum samples were collected every 2 to 3 days until day 42 post inoculation (PI), then approximately every 14 days until day 213 PI. Fecal samples were collected at the time of serum collection through day 35 PI. Oropharyngeal samples were collected at the time of serum collection from 56 to 213 days PI by scraping the oropharyngeal area with a sterile spoon, especially targeting the palatine tonsil. Turbinate, tonsil, lung, parotid salivary gland, spleen, lymph nodes and serum were collected postmortem on day 220 PI. Virus isolation (VI) on porcine alveolar macrophage cultures was attempted on all serum, fecal and oropharyngeal samples, as well as tissues collected postmortem. Postmortem tonsil tissues and selected fecal samples were also assayed for the presence of PRRSV RNA by the polymerase chain reaction (PCR). Serum antibody titers were determined by IFA, ELISA and SVN. Virus was isolated from all serum samples collected on days 2 to 11 PI and intermittently for up to 23 days in two pigs. No PRRSV was isolated from fecal samples, but 3 of 24 samples were PCR positive, suggesting the presence of inactivated virus. Oropharyngeal samples from each pig were VI positive 1 or more times between 56 and 157 days PI. Oropharyngeal samples from 3 of 4 pigs were VI positive on days 56, 70 and 84 PI. Virus was isolated from one pig on day 157 PI, 134 days after the last isolation of virus from serum from this animal. Virus was isolated from oropharyngeal samples for several weeks after the maximum serum antibody response, as measured by IFA, ELISA and SVN tests. All tissues collected postmortem were VI negative and postmortem tonsil samples were also negative by PCR. An important element in the transmission of PRRSV is the duration of virus shedding. The results of this study provided direct evidence of persistent PRRSV infection and explain field observations of long-term herd infection and transmission via purchase of clinically normal, but PRRSV infected, animals. Effective prevention and control strategies will need to be developed in the context of these results.

Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV)

Article

Jun 1997
VET MICROBIOL

Sixteen 6 week old conventional pigs were inoculated by aerosol with a European strain of porcine reproductive and respiratory syndrome virus (PRRSV). Virus replication was followed by virus titration and immunofluorescence in the lungs and in associated and distant lymphoid tissues at 3, 14, 21, 35, 42 and 82 days post-inoculation (DPI). PRRSV replication was detected in alveolar macrophages, lungs, tonsils, spleen, retropharyngeal lymph nodes, bronchial lymph nodes and thoracic aortic lymph nodes at 3 DPI. The same tissues, except retropharyngeal and thoracic aortic lymph nodes, were PRRSV positive at 14 DPI. Lungs and alveolar macrophages were PRRSV positive until 35 DPI. PRRSV was not detected in heart, peripheral blood mononuclear cells and bone marrow cells. Viremia was detected from 3 to 28 DPI. Not more than 2% of alveolar macrophages were PRRSV positive even during the acute stage of infection. 80 to 94% of the PRRSV infected cells in the lungs and in lung lavaged cells were identified as macrophages using a porcine macrophage specific monoclonal antibodies. In the lymph nodes and spleen, 100% of the infected cells were macrophages. Anti-PRRSV antibodies were detected by a blocking ELISA as early as 7 DPI. the antibody titre gradually increased to reach a geometric mean titre (GMT) of 160 at 35 DPI. It remained at that level until the end of the study. These findings clearly demonstrate that PRRSV has a tropism for macrophages. PRRSV mainly replicates in macrophages of the lymphoid tissues and lungs in the acute phase of infection and persists in the lung macrophages.

Nidovirales: A new order comprising Coronaviridae and Arteriviridae

Article

Feb 1997

D Cavanagh

Differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: Correlations with pathogenicity

Article

Sep 1999
RES VET SCI

The acute stages of infection with swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive-respiratory syndrome virus (PRRSV) were shown to differ in terms of clinical and lung inflammatory effects and proinflammatory cytokine profiles in bronchoalveolar lavage (BAL) fluids. Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with one of the three viruses. SIV infection was followed within 1 day post inoculation (d PI) by characteristic respiratory and general signs, and excessive lung epithelial desquamation and neutrophil infiltration (38 to 56 per cent of BAL cells at 1 d PI vs 0 to 1 per cent in controls). High concentrations of bioactive interferon-alpha (IFN -alpha), tumour necrosis factor-alpha (TNF -alpha) and interleukin-1 (IL -1) coincided with peak symptoms and neutrophil infiltration. PRCV infection was asymptomatic and produced a mild bronchointerstitial pneumonitis and neutrophil infiltration (13 to 22 per cent of BAL cells at 4 d PI). IFN -alpha titres parallelled those found during SIV infection, TNF -alpha was negligible and IL -1 undetectable. PRRSV infection induced anorexia and lethargy between 3 and 5 d PI. There was marked infiltration with mononuclear cells in alveolar septa and BAL fluids between 7 and 10 d PI, while neutrophils remained at less than 11 per cent of BAL cells at any time. IL -1 was produced from three throughout 10 d PI, while IFN -alpha production was minimal and TNF -alpha undetectable. These data strongly suggest that proinflammatory cytokines can be important mediators of viral respiratory disease.

Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection

Article

Nov 1999
VIRUS RES

The cellular immune response to a European isolate of porcine reproductive and respiratory syndrome (PRRS) virus in animals recovered from the experimental infection has been studied in vitro. Peripheral blood mononuclear cells (PBMC) from these pigs proliferated specifically when they were stimulated with PRRS virus. This response was not detectable until 4 weeks after inoculation and remained for more than 3 months. Addition of blocking monoclonal antibodies to the cultures showed that this proliferation was mainly dependent on CD4(+) cells with the participation of SLA-class II molecules. T-cell cultures established by stimulating responding cells with PRRS virus and maintained in culture for up to 3 weeks showed an increase of CD8(+) CD4(+) and CD4(-) CD8(+) subsets within activated cells, gated according to their light scatter parameters, whereas CD4(+) CD8(-) cells declined along the time in culture. Within the activated cells, those expressing the TcR gammadelta receptor also increased, being most of them also positive for the CD8 marker. By RT-PCR, T-cells responding to the virus showed a Th1 type cytokine production pattern. During the culture period the cytotoxic activity against K-562 cells increased from 15 to 35% of specific lysis. This cellular immune response may play a relevant role in the clearance of PRRS virus and the recovery of the infection.

Proinflammatory cytokines and viral respiratory disease in pigs

Article

Mar 2000

Swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) are enzootic viruses causing pulmonary infections in pigs. The first part of this review concentrates on known clinical and pathogenetic features of these infections. SIV is a primary respiratory pathogen; PRCV and PRRSV, on the contrary, tend to cause subclinical infections if uncomplicated but they appear to be important contributors to multifactorial respiratory diseases. The exact mechanisms whereby these viruses cause symptoms and pathology, however, remain unresolved. Classical studies of pathogenesis have revealed different lung cell tropisms and replication kinetics for each of these viruses and they suggest the involvement of different lung inflammatory responses or mediators. The proinflammatory cytokines interferon-alpha (IFN-alpha), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) have been shown to play key roles in several respiratory disease conditions. The biological effects of these cytokines and their involvement in human viral respiratory disease are discussed in the second part of this review. The third part summarises studies that were recently undertaken in the authors' laboratory to investigate the relationship between respiratory disease in pigs and bioactive lung lavage levels of IFN-alpha, TNF-alpha and IL-1 during single and combined infections with the above viruses. In single SIV infections, typical signs of swine "flu" were tightly correlated with an excessive and coordinate production of the 3 cytokines examined. PRCV or PRRSV infections, in contrast, were subclinical and did not induce production of all 3 cytokines. Combined infections with these 2 subclinical respiratory viruses failed to potentiate disease or cytokine production. After combined inoculation with PRCV followed by bacterial lipopolysaccharide, both clinical respiratory disease and TNF-alpha/IL-1 production were markedly more severe than those associated with the respective single inoculations. Taken together, these data are the first to demonstrate that proinflammatory cytokines can be important mediators of viral respiratory diseases in pigs.

Heterogeneity of porcine reproductive and respiratory syndrome virus: Implications for current vaccine efficacy and future vaccine development

Article

Jul 2000
VET MICROBIOL

Xiang-Jin Meng

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem to the pork industry worldwide. Increasing data indicate that PRRSV strains differ in virulence in infected pigs and are biologically, antigenically, and genetically heterogeneous. It is evident that the current vaccines, based on a single PRRSV strain, are not effective in protecting against infections with the genetically diverse field strains of PRRSV. The recent outbreaks of atypical or acute PRRS in vaccinated pigs have raised a serious concern about the efficacy of the current vaccines and provided the impetus for developing more effective vaccines. Special attention in this review is given to published work on antigenic, pathogenic and genetic variations of PRRSV and its potential implications for vaccine efficacy and development. Although there are ample data documenting the heterogeneous nature of PRRSV strains, information regarding how the heterogeneity is generated and what clinical impact it may have is very scarce. The observed heterogeneity will likely pose a major obstacle for effective prevention and control of PRRS. There remains an urgent need for fundamental research on this virus to understand the basic biology and the mechanism of heterogeneity and pathogenesis of PRRSV.

Transmission of porcine reproductive and respiratory syndrome virus by needles

Article

Feb 2002

Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases

Article

Oct 2002
TRENDS IMMUNOL

Current dogma suggests that immunity to infection is controlled by distinct type 1 (Th1) and type 2 (Th2) subpopulations of T cells discriminated on the basis of cytokine secretion and function. However, a further subtype of T cells, with immunosuppressive function and cytokine profiles distinct from either Th1 or Th2 T cells, termed regulatory T (Tr) cells has been described. Although considered to have a role in the maintenance of self-tolerance, recent studies suggest that Tr cells can be induced against bacterial, viral and parasite antigens in vivo and might prevent infection-induced immunopathology or prolong pathogen persistence by suppressing protective Th1 responses. These observations have significant implications for our understanding of the role of T cells in immunity to infectious diseases and for the development of new therapies for immune-mediated disorders.

Immunohistochemical staining of IFN-γ positive cells in porcine reproductive and respiratory syndrome virus-infected lungs

Article

Jan 2003
VET IMMUNOL IMMUNOP

Paraffin-embedded lungs were obtained from a previous porcine reproductive and respiratory syndrome virus (PRRSV)-challenged experiment involving three groups: an uninfected control group, a low virulence (LV, Resp PRRSV/Repro)-infected group, and a high virulence (HV, VR-2385)-infected group. Tissues were collected at 3, 7, 10, 14 or 28 days post-inoculation (DPI) (n=5). Lungs were examined to detect IFN-gamma positive cells by immunohistochemical staining using polyclonal antibodies to IFN-gamma. The microscopic lung lesions induced by the HV group were more severe than those in the LV group. A significant increase in number of lymphocytes in the HV group was observed at 10 DPI (24.90+/-9.79%), 14 DPI (22.00+/-11.47%) and 28 DPI (28.95+/-15.11%) (P<0.05). A relative decrease in macrophage numbers was observed and correlated well with the increase in lymphocyte numbers when the disease progressed. IFN-gamma positive cells were demonstrated in both lymphocytes and macrophages, particularly pulmonary alveolar macrophages. A significant increase in IFN-gamma positive cells was found at 7 DPI (15.90+/-13.65%), 10 DPI (46.95+/-13.79%), 14 DPI (10.90+/-5.13%) and 28 DPI (13.40+/-4.89%) in the HV group (P<0.05). The results suggested that the increase in IFN-gamma positive cells in the HV group correlated well with the severity of the lung lesions, which may be because of the presence of PRRSV in the lung.

Cytokine mRNA Profiles in Bronchoalveolar Cells of Piglets Experimentally Infected in Utero with Porcine Reproductive and Respiratory Syndrome Virus: Association of Sustained Expression of IFN-γ and IL-10 after Viral Clearance

Article

Feb 2002

An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALC's were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV.

Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

Article

Aug 2003
VET IMMUNOL IMMUNOP

To investigate cytokine alterations in pigs infected in-utero with porcine reproductive and respiratory syndrome virus (PRRSV), constitutive mRNA expression by peripheral blood mononuclear cells (PBMCs) was measured. PBMC from in-utero PRRSV-infected pigs displayed significantly increased IL-6, IL-10, and IFN-gamma mRNA expression at 0 and 14 days of age compared with age-matched control pigs. There were no significant differences in IL-2, IL-4, and IL-12 mRNA expression between in-utero PRRSV-infected and control pigs. However, the IL-10/IL-12 ratio was significantly increased in in-utero PRRSV-infected pigs at 0 and 14 days of age, suggesting the imbalance of IL-10 and IL-12 mRNA production. The abnormal mRNA expression of cytokines in in-utero PRRSV-infected pigs occurred concurrently with a significant decrease in the CD4(+)/CD8(+) T-cell ratio in peripheral blood. PRRSV was not isolated from the sera of pigs at 9 weeks of age that had been viremic at 0 and 14 days old. Delayed type hypersensitivity (DTH) responses to Tuberculin and analysis of cytokine mRNA expression by PBMC showed that cell-mediated immune response and cytokine message profiles in pigs infected in-utero with PRRSV had returned to levels similar to those of control pigs by 9 weeks of age. We conclude that in-utero infection with PRRSV results in significant alteration of cytokine mRNA expression that may cause transient immunomodulation. However, at 10 weeks of age the pigs' immune responses seemed to recover. This may help to understand the immunopathogenesis of in-utero PRRSV infection and the increased susceptibility to secondary bacterial pathogens in neonatal piglets.

Interleukin-10, Interleukin-12, and Interferon- γ Levels in the Respiratory Tract Following Mycoplasma hyopneumoniae and PRRSV Infection in Pigs

Article

Feb 2003

The cytokine profile associated with either a T helper 1 (Th1) or Th2 response in a porcine respiratory disease model was assessed by measuring IL-12, IL-10 and IFN-gamma using RT-PCR and ELISA, respectively. IL-10, IL-12, and IFN-gamma levels in pulmonary alveolar macrophages and bronchial lavage fluid were increased in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, or both pathogens. At 10 days post-infection (DPI), both IL-10 and IL-12 mRNA levels were increased in both groups infected with PRRSV. The IL-12 levels were increased in pigs infected with both pathogens and IFN-gamma protein levels were increased in pigs infected with PRRSV alone and only numerically increased in the dual infection. At 28 DPI, IL-12 mRNA levels and IL-10 protein levels were increased in all infected groups. The mRNA level of IL-12 remained elevated in the group infected with both pathogens at 42 DPI. Production of IFN-gamma did not appear to be closely correlated with elimination of virus from the respiratory tract. However, when the virus existed in the lung, the local IFN-gamma production appeared to increase. Although IL-12 mRNA levels were significantly elevated in the pigs infected with both pathogens, the increased protein levels of IL-12 may compromise the immune system's ability to clear PRRSV from the lung. This could explain the prolonged presence of PRRSV, IFN-gamma production and the increased pneumonia observed in the lungs of dual-infected pigs. The increased levels of cytokines associated with both Th1 and Th2 responses in the respiratory tract of pigs infected with PRRSV and M. hyopneumoniae provides valuable information on the pathogenesis of these diseases.

PRRS virus infection of macrophages: regulation by maturation and activation state

Jan 1996
563-569

T W Molitor
J Xiao
C Choi

Molitor, T. W., Xiao, J. & Choi, C. (1996). PRRS virus infection of macrophages: regulation by maturation and activation state. In Proceedings of the 27th Annual Meeting of the American Association of Swine Practitioners, pp. 563-569. Nashville, Tennessee.

PRRS: nature of the RNA virus and how it causes disease (a keynote paper)

Jan 2000
538

Lager

Lager, K. M. & Mengeling, W. L. (2000). PRRS: nature of the RNA virus and how it causes disease (a keynote paper). In Proceedings of the 16th International Pig Veterinary Society Congress, pp. 538-543. Melbourne, Australia.

Cytokine production by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

Jan 2000
657

W H Feng
M Tompkins
J Xu
T Brown
S Laster
M B Mccaw

Feng, W. H., Tompkins, M., Xu, J., Brown, T., Laster, S. & McCaw, M. B. (2000). Cytokine production by pigs infected in-utero with porcine reproductive and respiratory syndrome virus. In Proceedings of the 16th International Pig Veterinary Society Congress, p. 657. Melbourne, Australia.

Jan 1995

P G Halbur
P S Paul
M L Frey
J Landgraf
K Eernisse
X J Meng
M A Lum
J J Andrews
Rathje

Halbur, P. G., Paul, P. S., Frey, M. L., Landgraf, J., Eernisse, K., Meng, X. J., Lum, M. A., Andrews, J. J. & Rathje, J. A. (1995).

Cytokine production by pigs infected in-utero with porcine reproductive and respiratory syndrome virus.In Proceedings of the 16th International Pig Veterinary Society Congress

Jan 2000
p

Feng

Typing of PRRSV isolates in Thailand by a nested multiplex PCR

Jan 2002
p

Thanawongnuwech

PRRS virus infection of macrophages: regulation by maturation and activation state

Jan 1996
563

Molitor

Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus

Abstract

No full-text available

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