Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha

Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-alpha production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-alpha antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-alpha generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs.     

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages. Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli. At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells. However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.     

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

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.     

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.     

Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis by porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection in young piglets is frequently associated with secondary infection due to various pathogens, especially those of the respiratory tract. One of the most important mechanisms in respiratory diseases is related to the alteration of function of porcine alveolar macrophages (PAMs). The objective of this study was to determine how PRRS virus infection affects the capabilities of PAMs in the phagocytosis and destruction of Haemophilus parasuis. Phagocytosis percentages were determined in vitro and ex vivo, after collected PAMs were directly exposed to the virus of if PAMs were collected from piglets previously infected with PRRSV. In vitro experiments demonstrated that H. parasuis uptake by PAMs is only increased in the early stages of PRRSV infection (2 h post-infection). In contrast, in the ex vivo experiments it was shown that PAMs from PRRSV-infected piglets do not seem to change in their phagocytic rate until the later stages of infection. Together with a decrease in the phagocytic rate, a marked decrease in the functional ability of PAMs to kill bacteria was observed 7 d post-infection. It is hypothesized that when animals are exposed to PRRSV, there is a marked decrease in the functional ability of PAMs to kill bacteria through the release of superoxide anion, indicating a possible negative effect of the virus, at least at the macrophage level.     

Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus

Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis.     

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.     

Increase of CD163 but not sialoadhesin on cultured peripheral blood monocytes is coordinated with enhanced susceptibility to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism mainly for porcine alveolar macrophages (PAMs), but not for peripheral blood monocytes (BMo) in vivo. Previous research showed that only a few BMo became susceptible to PRRSV infection after 1 day culture. Porcine sialoadhesin (PoSn) and CD163 are identified to be the two main PRRSV receptors for binding and internalization. Both receptors are not expressed on BMo, or only expressed at low levels, which may explain why PRRSV cannot infect them. The relationship of BMo differentiation/aging, PRRSV receptor level, and susceptibility to PRRS virus infection has not been thoroughly investigated. In this study, BMo were successfully cultured with pig serum plus L929 cell culture supernatant. Our results showed that both the mRNA and protein expression levels of PoSn were significantly increased after 5-day culture. The mRNA level of CD163 was enhanced more than 20-fold after 1-day culture; CD163-positive BMo increased dramatically from about 2% after 2h- culture to about 50% after 96-h culture. Furthermore, cultured BMo became much more permissive to PRRSV infection, and the percentage of PRRSV-infected BMo was at least the same as PAMs, if not higher, when infected with CH-1a, the first PRRSV strain isolated in China, or HV, a highly virulent strain. Three other PRRSV strains including VR2332, and two classical Chinese isolates could also infect cultured BMo as well. Most importantly, PRRS virus was successfully isolated from 14 of 15 antibody-positive serum samples using cultured BMo. These results suggest that the enhanced susceptibility of cultured BMo to PRRS virus is coordinated with increased CD163 expression, but less related to the delayed (day 5) increased expression of PoSn. Thus, cultured BMo could be an alternative choice for PRRS virus isolation and identification.     

氧化应激对PRRSV致PAM细胞TLR3/NF-κB分子转录的影响

为探讨氧化应激对猪繁殖与呼吸综合征病毒(PRRSV)感染猪肺泡巨噬细胞(PAM)TLR3/NF-κB信号分子转录的影响,体外分离培养PAM,分为对照组、PRRSV感染组、抗氧化剂NAC+PRRSV组,促氧化剂H2O2+RRRSV组。分别在培养6、12、24、48、72h收集细胞,观察各组的细胞病变、real-time PCR检测PRRSV、TLR3、TRIF和NF-κB mRNA转录量的变化。结果显示,PRRSV感染组PRRSV、TLR3、TRIF及NF-κB mRNA的转录量与对照组相比随感染时间的延长显著升高(P0.05),48h达到最大值;NAC处理接毒组各信号分子mRNA的转录量比PRRSV感染组同时间点略低;H_2O_2处理接毒组比PRRSV感染组的略高。结果表明,氧化应激可增强PRRSV致PAM细胞TLR3/NF-κB分子mRNA的转录量,NF-κB的活化可能是PRRSV导致细胞损伤的机制之一。     

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-na?ve regulatory T cells to proliferate

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)⁺ and N-protein⁻ moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein⁺ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein⁻ moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3⁺ regulatory T cells present within CD4⁺ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4⁺ T cells showed differences in regard to proliferation and frequency of Foxp3⁺ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.     

Vitamin E supplementation does not mitigate the acute morbidity effects of porcine reproductive and respiratory syndrome virus in nursery pigs

The objective of this study was to determine whether feeding a vitamin E-rich diet would benefit nursery pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Sixty-four pigs were subjected to one of four treatment combinations (2 x 2 factorial) of dietary vitamin E (adequate or excess) and PRRSV (medium or inoculation with VR-2385 isolate P-129). Pigs were fed experimental diets during a 3-wk period before inoculation as well as during a 12-d period after inoculation. Growth performance was determined throughout the study, and lipid peroxidation in liver, glutathione peroxidase (GPX) activity in serum, circulating white blood cells, and serum interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) were determined in samples collected from pigs killed 4 or 12 d after inoculation. Infection by PRRSV (P < 0.001) induced a marked decrease in both ADFI and ADG, but neither the main effect of diet nor the diet x PRRSV interaction was significant. Neither diet nor PRRSV affected feed efficiency. At 12 d after inoculation, lipid peroxidation in liver and GPX activity in serum were lower in pigs fed excess vitamin E than in those fed adequate vitamin E (P < 0.01), suggesting that the diet high in vitamin E bolstered the antioxidant status of the pigs. However, PRRSV did not affect lipid peroxidation in liver or serum GPX activity, and the diet x PRRSV interaction was not significant. White blood cell counts were decreased and IFN-gamma, and IL-1beta were increased (P < 0.05) 4 and 12 d after inoculation in PRRSV-infected pigs, but neither diet nor the diet x PRRSV interaction was significant. Collectively, these results indicate that increasing antioxidant defenses by feeding high levels of vitamin E did not ameliorate the effects of PRRSV on decreased growth, leukopenia, and increased serum IL-1beta and IFN-gamma. Thus, feeding nursery pigs a diet high in vitamin E may not be useful for mitigating the acute morbidity effects of PRRSV infection.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.     

The diagnostic sensitivity of immunohistochemistry for the detection of porcine reproductive and respiratory syndrome virus in the lung of vaccinated and unvaccinated swine.

Immunohistochemistry (IHC) is used routinely to detect porcine reproductive and respiratory syndrome virus (PRRSV) in the lung of nursery and grow/finish pigs with respiratory disease and has been reported to be highly specific (100%) but only moderately sensitive (67%). When multiple sections of lung are examined from field cases of porcine pneumonia, it is common to detect PRRSV antigen in only 1 or 2 of the sections. This study was undertaken to determine the impact of the number of lung sections evaluated on the diagnostic sensitivity of IHC for the detection of PRRSV in vaccinated and unvaccinated swine. Five anterioventral sections of lung from animals experimentally challenged with PRRSV were evaluated on a single IHC slide. Utilizing a beta binomial model, observed results were used to calculate the probability of detecting PRRSV with IHC as a function of the number of lung sections assessed. Results demonstrate that the diagnostic sensitivity of PRRSV IHC is dependent on the number of lung sections examined. In unvaccinated pigs, a beta binomial model predicts that if a single lung section were evaluated, PRRSV would likely be confirmed in only 48% of infected animals, and at least 5 sections of anterioventral lung would need to be assessed to detect >90% of PRRSV-infected pigs. Vaccination resulted in significantly lower gross and microscopic lung lesion scores and significantly fewer antigen-positive cells. In vaccinated swine, the calculated probability of detecting a PRRSV-infected pig with IHC when a single lung section is evaluated was only 14%. If PRRSV is a primary concern, diagnosticians should collect at least 5 anterioventral sections of lung from each pig to be evaluated on a single IHC slide. This approach will diminish the number of false-negative results obtained with this method of antigen detection.     

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.     

Phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus

It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48 h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses.