Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Identifying the genetic components involved in host resistance/susceptibility would represent an important step forward in the design of disease control programs. In this study, alveolar macrophages derived from five commercial pig lines were used to study the innate immune response to PRRSV infection in vitro. Analysis by flow cytometry has demonstrated that bronchial alveolar lavage fluid (BALF) preparations were almost exclusively composed of alveolar macrophages and that the pigs tested were free from infection. Macrophages from the Landrace line showed significantly reduced virus replication and poor growth of PRRSV during 30 h of infection. By 72 h, PRRSV viral load was down to 2.5 log(10) TCID(50) compared with an average of 5 log(10) TCID(50) for the other breeds tested. These observations suggest that factors intrinsic to the Landrace breed may be responsible for this reduced or delayed response to PRRSV. Preliminary investigation suggests that the PRRSV coreceptor, sialoadhesin, may not be responsible for the Landrace macrophage phenotype as its abundance and localisation were comparable in all the breeds. Strikingly, we found that the reduced or delayed growth of PRRSV was temporally associated with high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-8 mRNA accumulation and substantial reduction of secretion of IL-8, suggesting a key contributory role for cytokine synthesis and secretion during the innate immune response to PRRSV infection.     

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.     

Porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages

Different viruses have evolved strategies that inhibit apoptosis of the host cell early in infection and/or induce apoptosis in the host cell late in infection. In this study, it was investigated if and when porcine reproductive and respiratory syndrome virus (PRRSV) modulates apoptosis in PRRSV-infected macrophages. The PRRSV replication cycle in macrophages was completed within 12 h post-inoculation (hpi). PRRSV-infected macrophages, treated with staurosporine at 4, 5, 6 and 8 hpi, were significantly protected against staurosporine-induced apoptosis, but PRRSV-infected macrophages, treated with staurosporine at 12 hpi, were not. In contrast, starting from 12 hpi, all PRRSV-infected macrophages died by caspase-dependent apoptosis, which culminated in secondary necrosis. Treatment of PRRSV-infected macrophages with Z-Val-DL-Asp-fluoromethylketone indicated that apoptosis late in infection was not essential for efficient virus release. Anti- and pro-apoptotic activities were also observed in PRRSV-infected Marc-145 cells. In conclusion, this study shows that PRRSV stimulates anti-apoptotic pathways in macrophages early in infection and that PRRSV-infected macrophages die by apoptosis late in infection.     

Heterogeneity of swine lung macrophages inoculated by porcine reproductive and respiratory syndrome virus

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susceptibility to PRRSV.     

Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus

Summary.  The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV. Received April 17, 2002; accepted July 25, 2002     

Modulation of CD163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) has a specific cell tropism for differentiated macrophages, such as porcine alveolar macrophages (PAMs). We analyzed the expression of CD163 on PAMs and macrophages derived from CD14 positive blood monocytes (MDMs), in correlation with PRRSV replication. By flow cytometry analysis, we showed that the levels of CD163 expression correlated well with the overall level of PRRSV replication. We further examined the effects of modulators of macrophage function, including 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS), and interleukin (IL)-10 on the expression of CD163 and PRRSV replication. Pre-treatment of PAMs or MDMs with TPA or LPS resulted in decreased expression of CD163 and reduction in PRRSV replication. On the contrary, the incubation of CD14 positive monocytes with IL-10 during differentiation into MDMs resulted in up-regulated expression of CD163 with a corresponding increase in PRRSV infection. These data indicate that the expression of CD163 on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenecity of PRRSV.     

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.     

Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication

Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway that acts in the maintenance of cellular homeostasis and plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), an agent that has caused devastating losses in the international swine industry since the late 1980s. Using protein quantification and microscopy, we observed that PRRSV infection results in LC3-I/II conversion, an increased accumulation of punctate GFP-LC3-expressing cells, and a higher number of autophagosome-like double-membrane vesicles in the cytoplasm of host cells. Inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNAs targeting ATG7 and Beclin-1 led to a significant reduction in PRRSV titers and protein expression. Conversely, induction of autophagy by rapamycin resulted in increased viral replication. These results demonstrate that PRRSV infection induces autophagy which, in turn, enhances viral replication efficiency.     

Functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. One region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. PRRSV nsp10 was found to possess a thermolabile and pH-sensitive NTPase activity that was modulated by polynucleotides and to unwind dsRNA in a 5' to 3' polarity. These results provide the first evidence of the functional properties of PRRSV helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases.     

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

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.     

Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection

Porcine reproductive and respiratory syndrome virus (PRRSV) displays notorious genetic, antigenic, and clinical variability. Little is known, however, about the nature and extent of viral variation present within naturally infected animals. By amplifying and cloning the open reading frame 5 gene from tonsils of naturally infected swine, and by sequencing individual clones, we characterized viral diversity in nine animals from two farms. All animals harbored multiple PRRSV variants at both the nucleic and the amino acid levels. Structural variation and rates of synonymous and nonsynonymous nucleotide substitution were no different within known epitopes than elsewhere. Analysis of molecular variance indicated that differences between farms, among animals within farms, and within individual animals accounted for 92.94, 3.84, and 3.22% of the total viral genetic variability observed, respectively. PRRSV exists during natural infection as a quasispecies distribution of related genotypes. Positive natural selection for immune evasiveness does not appear to maintain this diversity.     

Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What’s more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.     

Peptide ELISA for measuring antibodies to N-protein of porcine reproductive and respiratory syndrome virus

Indirect and competition ELISAs with synthetic peptides were used to characterize the epitopes of the N-protein of porcine reproductive and respiratory syndrome virus (PRRSV) that are recognized by a battery of monoclonal antibodies (mAbs) and by antibodies from infected pigs. Four linear epitopes recognized by mAbs have been identified in the most hydrophilic segment of the N-protein (AA25-57). Similarly, at least four linear epitopes in this segment are immunogenic in PRRSV-infected pigs, but only one corresponds to an epitope recognized by one of the mAbs (AA36-45). Antibody formation to these epitopes varied greatly between individual pigs. Most infected pigs generated antibodies that bound to both peptides and HerdChek plates, which are commonly used in the sero-diagnosis of PRRSV infections, but the time course of formation of peptide binding antibodies and antibodies that react with HerdChek plates differed greatly between pigs. This suggests that, although the peptide and HerdChek ELISAs may detect antibodies to some of the same epitopes, they also seem to detect antibodies to epitopes that are uniquely expressed by one and not the other. Some mAbs fail to bind to HerdChek ELISA plates and this is also the case for certain pig antibodies. Peptide ELISA results identified four herds in which most or all pigs possessed N-protein peptide binding antibodies, even though they were HerdChek ELISA sero-negative and exhibited no other signs of PRRSV infection. Thus PRRSV infections may be more widespread than presently realized involving strains that cause asymptomatic infections. The peptide ELISA is useful as an adjunct to the HerdChek ELISA or it could replace it since only two serum samples among 450 tested were HerdChek ELISA positive but peptide ELISA negative. The peptide ELISA is also considerably cheaper than the HerdChek ELISA, more flexible and can provide information on the epitope specificity of the reacting antibodies.     

In vitro susceptibility of macrophages to porcine reproductive and respiratory syndrome virus varies between genetically diverse lines of pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be responsible for financial losses in the swine industry worldwide. It remains undetermined whether genetic variability of the host in susceptibility to PRRSV exists and if this variability can be exploited to help control this important disease. The objective of this study was to determine if an in vitro flow cytometry (FACS) assay that detects the percentage of monocyte-derived macrophages (MDM) infected with PRRSV could be utilized to demonstrate genetic variability in the susceptibility between distinct lines of pigs. Over 400 growing pigs from six genetic lines maintained in a single commercial breeding herd were screened using an in vitro FACS assay. From this initial screening, two genetically diverse lines of pigs that were also divergent in their FACS results were selected for further study. An additional 264 pigs from these two lines were subsequently tested for in vitro susceptibility to PRRSV. As in the preliminary screening, the Large White line had significantly higher average percent positive MDM over the Duroc-Pietrain synthetic line. This report suggests a genetic component for susceptibility to PRRSV exists and that the in vitro assay may be useful in predicting the relative susceptibility to PRRSV in large groups of animals.