Infection Of Porcine Alveolar Macrophages Research Articles

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-β. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Identification of New Compounds against PRRSV Infection by Directly Targeting CD163.

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 μM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Incomplete autophagy promotes the proliferation of Mycoplasma hyopneumoniae through the JNK and Akt pathways in porcine alveolar macrophages

Autophagy is an important conserved homeostatic process related to nutrient and energy deficiency and organelle damage in diverse eukaryotic cells and has been reported to play an important role in cellular responses to pathogens and bacterial replication. The respiratory bacterium Mycoplasma hyopneumoniae has been identified to enter porcine alveolar macrophages, which are considered important immune cells. However, little is known about the role of autophagy in the pathogenesis of M. hyopneumoniae infection of porcine alveolar macrophages. Our experiments demonstrated that M. hyopneumoniae infection enhanced the formation of autophagosomes in porcine alveolar macrophages but prevented the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux and preventing the acidification and destruction of M. hyopneumoniae in low-pH surroundings. In addition, using different autophagy regulators to intervene in the autophagy process, we found that incomplete autophagy promoted the intracellular proliferation of M. hyopneumoniae. We also found that blocking the phosphorylation of JNK and Akt downregulated the autophagy induced by M. hyopneumoniae, but pathways related to two mitogen-activated protein kinases (Erk1/2 and p38) did not affect the process. Collectively, M. hyopneumoniae induced incomplete autophagy in porcine alveolar macrophages through the JNK and Akt signalling pathways; conversely, incomplete autophagy prevented M. hyopneumoniae from entering and degrading lysosomes to realize the proliferation of M. hyopneumoniae in porcine alveolar macrophages. These findings raise the possibility that targeting the autophagic pathway may be effective for the prevention or treatment of M. hyopneumoniae infection.

A synthetic toll-like receptor 7 agonist inhibits porcine reproductive and respiratory syndrome virus replication in piglets

Toll-like receptor 7 (TLR7) agonists have been shown to exert therapeutic effects against several viruses. However, antiviral potential of TLR7 agonist in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection has not been assessed in vivo. In our previous study, a synthetic TLR7 agonist, SZU101, was confirmed to inhibit PRRSV infection of porcine alveolar macrophages (PAMs). Here, antiviral effects of SZU101 were evaluated in PRRSV-challenged piglets based on assessments of rectal temperature, viremia, gross and microscopic lung lesions, PRRSV-specific antibodies, PRRSV-specific lymphocyte proliferation and serum IFN-β level. Our results revealed that SZU101 treatment alleviated PRRSV-induced rectal temperature spikes, pulmonary pathologic changes, and serum viral load. Meanwhile, administration of SZU101 led to increased proliferation of PRRSV-specific lymphocytes and serum IFN-β levels, but did not enhance PRRSV-specific antibody production. These results demonstrate that SZU101 has potential as a therapeutic treatment for PRRS.

Genome-wide transcriptomic analysis of highly virulent African swine fever virus infection reveals complex and unique virus host interaction

African swine fever virus (ASFV), one of the most devastating emerging swine pathogens in China, causes nearly 100 % mortality in naive herds. Here, whole-transcriptome RNA-seq analysis was conducted in porcine alveolar macrophages (PAMs) infected with Pig/Heilongjiang/2018 (Pig/HLJ/18) ASFV at different time points. Our data suggested that ASFV genes expression demonstrated a time-depended pattern and ASFV early genes were involved in antagonizing host innate immunity. Moreover, viral small RNA (vsRNA) was generated as well. Meanwhile, transcriptome analysis of host genes suggested a strong inhibition host immunity-related genes by ASFV infection in PAMs, while enhanced chemokine-mediated signaling pathways and neutrophil chemotaxis were observed in ASFV infected PAMs. Furthermore, ASFV infection also down-regulated host microRNAs (miRNAs) that putatively targeted viral genes, while also triggering dysregulation of host metabolism that promoted virus replication at transcription level. Most importantly, infection of PAMs with ASFV induced a different transcriptome pattern from that of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), which is known to trigger a host cytokine storm. In conclusion, our transcriptome data implied that ASFV infection in PAMs appeared to be associated with strong inhibition of host immune responses, dysregulation of host chemokine axis and metabolic pathways.

PRRSV Vaccine Strain-Induced Secretion of Extracellular ISG15 Stimulates Porcine Alveolar Macrophage Antiviral Response against PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) has disrupted the global swine industry since the 1980s. PRRSV-host interactions are largely still unknown but may involve host ISG15 protein. In this study, we developed a monoclonal antibody (Mab-3D5E6) specific for swine ISG15 (sISG15) by immunizing mice with recombinant sISG15. A sandwich enzyme-linked immunosorbent assay (ELISA) incorporating this sISG15-specific Mab was developed to detect sISG15 and provided a lower limit of sISG15 detection of 200 pg/mL. ELISA results demonstrated that infection of porcine alveolar macrophages (PAMs) with low-virulence or attenuated PRRSV vaccine strains induced intracellular ISG15 expression that was independent of type I IFN production, while PAMs infection with a PRRSV vaccine strain promoted extracellular ISG15 secretion from infected PAMs. Conversely, the addition of recombinant sISG15 to PAMs mimicked natural extracellular ISG15 effects whereby sISG15 functioned as a cytokine by activating PAMs. Once activated, PAMs could inhibit PRRSV replication and resist infection with PRRSV vaccine strain TJM. In summary, a sandwich ELISA incorporating homemade anti-ISG15 Mab detected ISG15 secretion induced by PAMs infection with a PRRSV vaccine strain. Recombinant ISG15 added to cells exhibited cytokine-like activity that stimulated PAMs to assume an anti-viral state that enabled them to inhibit PRRSV replication and resist viral infection.

Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells

BackgroundPorcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease.MethodsThrough artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs).ResultsUsing the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection.ConclusionsOur study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.

Notch signaling contributes to the expression of inflammatory cytokines induced by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection in porcine alveolar macrophages

Notch signaling, an evolutionarily conserved signal pathway has emerged as a key signal pathway to regulate host immune response but the contribution of Notch signaling to immune response in pigs remains unknown. Infection of porcine alveolar macrophages (PAM) with porcine reproductive and respiratory syndrome virus (PRRSV) triggers expression of Jagged1 mRNA, suggesting that Notch signaling might play a role in the immune response to PRRSV infection. To further explore it, we examined the expression profile of Notch molecules in PAM following a highly pathogenic PRRSV (HP-PRRSV) strain infection. We demonstrated that HP-PRRSV infection resulted in the induction of Notch ligands (Jagged1, Dll3, Dll4), the transcription factor RBP-J, and the target gene Hes1, consistent with activation of Notch signaling. Next, using DAPT treatment and the knockdown of RBP-J illustrated that inhibition of activation of Notch signaling attenuated induction of the inflammatory cytokines (TNF-α and IL-1β) instead of viral replication in PAM during HP-PRRSV infection. Furthermore, the knockdown of Jagged1, the most induced ligand not only inhibited activation of Notch signaling, but also reduced the expression of inflammatory cytokines without any influence in viral replication. Moreover, our data revealed that several signaling including NF-κB, MAPK and Notch signaling contributed to the induction of Jagged1 in PAM during HP-PRRSV infection. In summary, these findings reveal that Notch as an important signaling pathway could contribute to the regulation of inflammatory response induced by HP-PRRSV infection.

Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway.

Porcine circovirus type 2 (PCV2) is one of the major threats to pig farms worldwide. Although PCV2 has been identified to promote IL-10 production, the detailed regulatory roles of PCV2 Rep for IL-10 production remain unclear. Herein, we first found that PCV2 Rep, rather than PCV1 Rep, enhanced IL-10 expression at the later phase of PCV2 infection in porcine alveolar macrophages (PAMs). Furthermore, we found that PCV2 Rep directly activated the p38-MAPK pathway to promote transcription factors NF-κB p50 and Sp1 binding to the il10 promoter, but PCV1 Rep did not. During PCV2 infection, however, PCV2 Rep promoted the binding activities of NF-κB p50 and Sp1 with the il10 promoter only at the later phase of PCV2 infection, since Rep proteins only expressed at the later phase of the infection. Moreover, silence of the thymine DNA glycosylase (TDG), a Rep-binding protein, significantly reduced the binding activities of NF-κB p50 and Sp1 with il10 promoter, resulting in the reduction of IL-10 production in PCV2-inoculated PAMs at the later phase of infection. Taken together, our results demonstrate that Rep proteins enhance IL-10 production during PCV2 infection of PAMs via activation of p38-MAPK pathways, in which host TDG is a critical mediator.

Recombinant adenovirus-delivered soluble CD163 and sialoadhesin receptors protected pigs from porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases affecting pig industry worldwide. Sialoadehesin (Sn) and CD163 are the two specific receptors for PRRSV infection of porcine alveolar macrophages. Our previous study showed that the soluble Sn receptor Sn4D-Fc and soluble CD163 receptor SRCR59-Fc expressed by the two recombinant adenoviral (rAd) vectors have an additive anti-PRRSV effect in vitro. In the present study, rAd-Sn4D-Fc and rAd-SRCR59-Fc were inoculated into pigs, and the efficient expression of Sn4D-Fc and SRCR59-Fc proteins was detected by ELISA. Then, PRRSV-naïve pigs were inoculated with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc before contagious infection with different PRRSV strains. Among the three rAd inoculation groups, simultaneous inoculation with the two rAd vectors provided the best protection against highly pathogenic JXA1 strain PRRSV, followed by rAd-SRCR59-Fc inoculation and rAd-Sn4D-Fc inoculation. Clinical observation and quantitative RT-PCR analyses showed that all of the double rAd-inoculated pigs (n = 9) survived from the contagious infection with highly pathogenic JXA1, JS07 or SH1705 strain PRRSV with significantly alleviated clinical scores, viremia, fecal viral emission and tissue virus loads. These data suggest that rAd-Sn4D-Fc and rAd-SRCR59-Fc can be developed further as the universal therapeutic vaccine to facilitate PRRSV eradication.

Cholesterol 25-hydroxylase acts as a host restriction factor on pseudorabies virus replication.

Cholesterol 25-hydroxylase (CH25H) catalyses the production of 25-hydroxycholesterol (25HC) from cholesterol by adding a second hydroxyl group at position 25. The aim of this study was to examine the antiviral effect of CH25H on pseudorabies virus (PRV), a swine pathogen that can cause devastating disease and economic losses worldwide. The results showed that porcine ch25h was induced by either interferon or PRV infection. PRV infection of porcine alveolar macrophages (3D4/21 cells) was attenuated by CH25H overexpression and enhanced by silencing of CH25H. Furthermore, treatment of 3D4/21 cells with 25HC inhibited the growth of PRV in vitro, suggesting that CH25H may restrict PRV replication by 25HC production. We further identified that the anti-PRV role of CH25H and 25HC was subject to their inhibitory effect on PRV attachment and entry. Collectively, these findings demonstrate that CH25H is an intrinsic host restriction factor in PRV infection of porcine alveolar macrophages.

The activation of the IFNβ induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable.

It has been recognized that the expression of type I interferon (IFNα/β) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNβ) and downstream signaling events. In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNβ protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNβ protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNβ signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNβ production. The results of this study demonstrate that the induction of IFNβ and signaling in PAMs after PRRSV infection is variable.

Combining laboratory and mathematical models to infer mechanisms underlying kinetic changes in macrophage susceptibility to an RNA virus.

BackgroundMacrophages are essential to innate immunity against many pathogens, but some pathogens also target macrophages as routes to infection. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an RNA virus that infects porcine alveolar macrophages (PAMs) causing devastating impact on global pig production. Identifying the cellular mechanisms that mediate PAM susceptibility to the virus is crucial for developing effective interventions. Previous evidence suggests that the scavenger receptor CD163 is essential for productive infection of PAMs with PRRSV. Here we use an integrative in-vitro–in-silico modelling approach to determine whether and how PAM susceptibility to PRRSV changes over time, to assess the role of CD163 expression on such changes, and to infer other potential causative mechanisms altering cell susceptibility.ResultsOur in-vitro experiment showed that PAM susceptibility to PRRSV changed considerably over incubation time. Moreover, an increasing proportion of PAMs apparently lacking CD163 were found susceptible to PRRSV at the later incubation stages, thus conflicting with current understanding that CD163 is essential for productive infection of PAMs with PRRSV. We developed process based dynamic mathematical models and fitted these to the data to assess alternative hypotheses regarding potential underlying mechanisms for the observed susceptibility and biomarker trends. The models informed by our data support the hypothesis that although CD163 may have enhanced cell susceptibility, it was not essential for productive infection in our study. Instead the models promote the existence of a reversible cellular state, such as macrophage polarization, mediated in a density dependent manner by autocrine factors, to be responsible for the observed kinetics in cell susceptibility.ConclusionsOur dynamic model–inference approach provides strong support that PAM susceptibility to the PRRS virus is transient, reversible and can be mediated by compounds produced by the target cells themselves, and that these can render PAMs lacking the CD163 receptor susceptible to PRRSV. The results have implications for the development of therapeutics aiming to boost target cell resistance and prompt future investigation of dynamic changes in macrophage susceptibility to PRRSV and other viruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0345-5) contains supplementary material, which is available to authorized users.

Caveolin-1-mediated endocytic pathway is involved in classical swine fever virus Shimen infection of porcine alveolar macrophages

Macrophages are at the frontline of defense against pathogenic microorganisms. However, very little is known about the cell invasion mechanism of classical swine fever virus (CSFV) Shimen strain. To elucidate the infective mechanism of this important pathogen, we screened deep-sequencing data derived from macrophages infected with CSFV Shimen and uninfected macrophages, and identified a role of caveolin-1 (CAV1). Digital gene expression profiling showed that CAV1 was differentially expressed in CSFV Shimen-infected macrophages. Quantitative polymerase chain reaction and western blot analyses showed that the transcription and translation of CAV1 were significantly up-regulated in CSFV Shimen-infected macrophages. In addition, immunofluorescent confocal microscopy analysis suggested that CAV1 was temporally colocalized with CSFV E2 throughout the course of the infection. Through the overexpression of recombinant CAV1 or the silencing of CAV1 expression using small interfering RNA in macrophages, we demonstrated that CAV1 expression is beneficial for the replication of CSFV Shimen. However, RNA silencing of CAV1 did not prevent viral replication, which may indicate that CSFV can also enter macrophages by other mechanisms. Our findings suggest that CAV1-mediated endocytosis is advantageous for productive CSFV Shimen infection in macrophages, providing a new insight into the mechanisms of evasion of host immunity for successful viral infection.

Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) can predispose pigs to secondary respiratory infection with bacteria such as Haemophilus parasuis. Animals infected with both pathogens develop more severe clinical disease. The immune response of porcine alveolar macrophages (PAMs) to simultaneous infection with PRRSV and H. parasuis was analysed in vitro, describing cytokine production, expression of cell surface molecules, and production of reactive oxygen species (ROS). Concurrent infection with PRRSV and H. parasuis increased gene expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-8) in PAMs in comparison with PAMs infected with PRRSV or H. parasuis alone. An additive effect of dual infection on IL-1β production was confirmed at the protein level. PAMs infected with PRRSV showed increased production of ROS compared to controls. Conversely, simultaneous infection of PAMs with PRRSV and H. parasuis decreased production of ROS, indicating the presence of an H. parasuis defence mechanism against respiratory burst. Concurrent infection of PAMs with PRRSV and H. parasuis was shown to elicit a pro-inflammatory immune response represented by significant IL-1β production. Severe multifactorial respiratory disease in natural conditions caused by both pathogens could be the consequence of pro-inflammatory mediated immunopathology.

The effects of H3N2 swine influenza virus infection on TLRs and RLRs signaling pathways in porcine alveolar macrophages.

BackgroundSwine influenza is an economically important respiratory disease of swine resulting from infection with influenza A virus. Swine influenza virus (SIV) becomes the focus as pigs have been hypothesized to serve as an intermediate host for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses. The ability of the innate immune system to detect and respond to pathogens is important for survival. Therefore, there is a critical need to evaluate the immediate response to viral infection, especially the role of the toll-like receptors (TLRs) and RNA helicase RIG-I-like receptors (RLRs) innate immunity signaling pathways in H3N2 swine influenza virus infection.MethodIn this study, porcine alveolar macrophages (PAMs) were obtained from porcine lungs and were infected with SIV at a multiplicity of infection (MOI) of 5 in vitro. The changes of the related receptors, signaling proteins and effector molecules of TLRs and RLRs signaling pathways post H3N2 virus infection of PAMs were quantified by Real-time quantitative RT-PCR and western blotting.ResultsThe results showed that H3N2 SIV infection significantly increased mRNA expression of TLR-3, TLR-7, RIG- I and MDA5 after 4 hpi (P < 0.05). Western blotting showed that the protein levels of TLR-3, TLR-7 and RIG-I also had a significantly increase after PAM exposed to virus. A significant change of MyD88, MAVS, IRF-3 and IRF-7 mRNA expression were present at 8 hpi. More than a 4-fold increase was induced for TNF-α and IL-1β mRNA expression. And the concentration of TNF-α and IL-1β peaked at 12 and 24 hpi, respectively. IFN-α, IFN-β mRNA and protein levels increased after SIV infection and significant differences was observed at 8, 12 and 24 hpi.ConclusionThese results indicate that H3N2 swine influenza virus infection significantly influences the expression of the receptors, adapter proteins and downstream effector molecules of RLRs and TLRs signaling pathways. This study enhances our understanding of innate immunity signaling pathways in PAM anti-infection of H3N2 SIV.

Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

BackgroundThe current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.MethodsThe candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.ResultsAll 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.ConclusionThese results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

RNA-Sequence Analysis of Primary Alveolar Macrophages after In Vitro Infection with Porcine Reproductive and Respiratory Syndrome Virus Strains of Differing Virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects porcine alveolar macrophages (PAMs), resulting in porcine reproductive and respiratory syndrome (PRRS) in pigs. Most of the transcriptomic studies on PAMs infected with PRRSV conducted thus far have made use of microarray technology. Here, we investigated the transcriptome of PAMs in vitro at 12 h post-infection with two European PRRSV strains characterized by low (Lelystad, LV) and high (Lena) virulence through RNA-Seq. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with the two strains. Gene ontology analysis confirmed that infection of PAMs with both the Lena and LV strains affected signaling pathways directly linked to the innate immune response, including interferon regulatory factors (IRF), RIG1-like receptors, TLRs and PKR pathways. The results confirmed that interferon signaling is crucial for transcriptional regulation during PAM infection. IFN-β1 and IFN-αω, but not IFN-α, were up-regulated following infection with either the LV or Lena strain. The down-regulation of canonical pathways, such as the interplay between the innate and adaptive immune responses, cell death and TLR3/TLR7 signaling, was observed for both strains, but Lena triggered a stronger down-regulation than LV. This analysis contributes to a better understanding of the interactions between PRRSV and PAMs and outlines the differences in the responses of PAMs to strains with different levels of virulence, which may lead to the development of new PRRSV control strategies.

Infection of porcine alveolar macrophages with recombinant chimeric porcine reproductive and respiratory syndrome virus: Effects on cellular gene transcription and virus growth

Porcine reproductive and respiratory syndrome virus (PRRSV) genetic determinants affecting the response of the host primary target cell, the macrophage, to infection are yet to be defined. Here we have used recombinant viruses encompassing ORF 1A to identify PRRSV determinants associated with growth and modulation of pro- and anti-inflammatory cytokine expression in primary pulmonary alveolar macrophages (PAMs) cultures. Three genomic chimeras encompassing ORF 1A of PRRSV live attenuated vaccine Prime Pac (LAV SP) in the genetic background of pathogenic strain NVSL 97-7895 (FL12v) were characterized in vitro. Unlike parental viruses, two of the recombinant viruses encompassing the area of the genome encoding for NSP2 to NSP8 showed reduced growth in PAM cultures. The effect of virus infections on gene activation was studied for 25 immunomodulatory cellular genes in PAMs at 24 and 48 h post-infection (hpi). Steady state mRNA levels in PAMs infected with recombinant and LAV SP viruses were compared to levels observed in cells infected with parental virus FL12v. Recombinant viruses induced patterns of transcriptional activation differing from patterns induced by parental FL12v, suggesting a regulatory role of PRRSV ORF1A on PAM gene expression.

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.