Lactate-lactylation-HSPA6 axis promotes PRRSV replication by impairing IFN-ß production.

Lactate, traditionally considered a metabolic by-product, has recently been identified as a substrate for the induction of lactylation, a newly identified epigenetic modification that plays an important role in the regulation of host gene expression. Our previous study showed that lactate levels were significantly elevated in cells infected with the porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide for over 30 years. However, the role of elevated lactate in PRRSV infections remains unknown. In this study, we found that lactate was required for optimal PRRSV proliferation, and PRRSV infection increased cellular lactylation in a dose-dependent manner. Using the Cleavage Under Targets and Tagmentation (CUT&Tag) combined with RNA sequencing (RNA-seq) to screen the downstream genes regulated by lactylation in PRRSV-infected cells, we found that PRRSV-induced lactylation activated the expression of heat shock 70 kDa protein 6 (HSPA6). Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon (IFN)-ß induction. Mechanistically, HSPA6 impeded the interaction between TNF-receptor-associated factor 3 (TRAF3) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε), thereby hindering the production of IFN-ß. Taken together, these results indicate that the activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-ß induction, providing new therapeutic targets for the prevention and control of PRRSV infection. The results presented here also link lactylation to the virus life cycle, improving our understanding of epigenetic regulation in viral infection.IMPORTANCEAs a newly identified epigenetic modification, lactate-induced lactylation has received attentions because it plays important roles in gene expression and contributes to tumorigenesis and the innate immune response. Previous studies showed that many viruses upregulate cellular lactate levels; however, whether virus-elevated lactate induces lactylation and the subsequent biological significance of the modification to viral infection have not been reported. In this study, we demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection induced cellular lactylation, which, in turn, upregulated the expression of HSPA6, an IFN-negative regulator. We also dissected the mechanism by which HSPA6 negatively regulates IFN-ß production. To our knowledge, this is the first report to study virus-induced lactylation and establish the relationship between lactylation and virus infection.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Identificationof a novel linear epitope on the porcine reproductive and respiratory syndrome virus nucleocapsid protein, as recognized by a specific monoclonal antibody.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most threatening pathogens of swine. The nucleocapsid (N) protein is the major structural protein of the virus and has been used as a PRRSV diagnostic antigen due to its high level of inherent immunogenicity. Methods: The recombinant PRRSV N protein was generated by the prokaryotic expressing system and used to immunized mice. Monoclonal antibodies against PRRSV were produced and validated by western blot analysis and indirect immunofluorescence analysis. In this study, the linear epitope of a specific monoclonal antibody mAb (N06) was subsequently identified by enzyme-linked immunosorbent assays (ELISA) using the synthesized overlapping peptides as antigens. Results: According to the results of western blot analysis and indirect immunofluorescence analysis, mAb (N06) was capable of recognizing the native form as well as the denatured form of PRRSV N protein. The results of ELISA showed that mAb N06 recognized the epitope NRKKNPEKPHFPLATE, which was consistent with BCPREDS predictions of antigenicity. Conclusion: All the data suggested that the mAb (N06) can be used as diagnostic reagents for PRRSV detection, while the recognized linear epitope can be useful in epitope-based vaccines development, which is helpful for the control of local PRRSV infections in swine.

PSMB4 Degrades the Porcine Reproductive and Respiratory Syndrome Virus Nsp1α Protein via the Autolysosome Pathway and Induces the Production of Type I Interferon.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in pigs of all ages and reproductive failure in sows, resulting in great economic losses to the swine industry. In this work, we identified the interaction between PSMB4 and PRRSV Nsp1α by yeast two-hybrid screening. The PSMB4-Nsp1α interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and laser confocal experiments. The PCPα domain (amino acids 66 to 166) of Nsp1α and the C-terminal domain (amino acids 250 to 264) of PSMB4 were shown to be critical for the PSMB4-Nsp1α interaction. PSMB4 overexpression reduced PRRSV replication, whereas PSMB4 knockdown elicited opposing effects. Mechanistically, PSMB4 targeted K169 in Nsp1α for K63-linked ubiquitination and targeted Nsp1α for autolysosomal degradation by interacting with LC3 to enhance the activation of the lysosomal pathway. Meanwhile, we found that PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. In conclusion, our data revealed a new mechanism of PSMB4-mediated restriction of PRRSV replication, whereby PSMB4 was found to induce Nsp1α degradation and type I interferon expression, in order to impede the replication of PRRSV. IMPORTANCE In the swine industry, PRRSV is a continuous threat, and the current vaccines are not effective enough to block it. This study determined that PSMB4 plays an antiviral role against PRRSV. PSMB4 was found to interact with PRRSV Nsp1α, mediate K63-linked ubiquitination of Nsp1α at K169, and thus trigger its degradation via the lysosomal pathway. Additionally, PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. This study extends our understanding of the proteasome subunit PSMB4 against PRRSV replication and will contribute to the development of new antiviral strategies.

PSMB1 Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting NBR1 To Degrade Nonstructural Protein 12 by Autophagy.

The nonstructural proteins (Nsps) of porcine reproductive and respiratory syndrome virus (PRRSV) play essential roles in virus replication-a multistep process that requires the participation of host factors. It is of great significance for the development of antiviral drugs to characterize the host proteins that interact with PRRSV Nsps and their functions in PRRSV replication. Here, we determined that proteasome subunit ß type 1 (PSMB1) interacted with viral Nsp12 to inhibit PRRSV replication in target and permissive cells. PSMB1 could be downregulated by PRRSV infection through interaction with the transcription factor EBF1. Proteasome and autophagy inhibitor assays showed that PSMB1 was regulated by the autophagic pathway to degrade Nsp12. Cotransfection of PSMB1 and Nsp12 increased the level of intracellular autophagy; both molecules were colocated in lysosomes. We also found that the selective autophagy cargo receptor protein NBR1 and E3 ubiquitin ligase STUB1 interacted with PSMB1 and Nsp12, respectively, in the autophagic degradation of Nsp12. Furthermore, the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 at lysine site 130. Our results indicate for the first time that PSMB1 is an anti-PRRSV host protein that inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway. IMPORTANCE PRRS is a major threat to the global pig industry and urgently requires an effective and sustainable control strategy. PRRSV Nsps have important roles in viral RNA synthesis, proteinase activity, induction of replication-associated membrane rearrangements, replicative endoribonuclease activity, determination of virulence, and regulation of host immune response. Research associated with PRRSV Nsps can provide vital guidance to modify the PRRSV genome through reverse genetics in the development of vaccines and diagnostics. The function of Nsp12, which generally plays essential roles in virus replication, remains unclear. We demonstrated that PSMB1 interacted with and degraded Nsp12 through an autophagic pathway to inhibit PRRSV replication. Our data confirmed a novel antiviral function of PSMB1 and allowed us to elaborate on the roles of Nsp12 in PRRSV pathogenesis. These findings suggest a valid and highly conserved candidate target for the development of novel therapies and more effective vaccines and demonstrate the complex cross talk between selective autophagy and PRRSV infection.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Identification of Cryptic Promoter Activity in cDNA Sequences Corresponding to PRRSV 5' Untranslated Region and Transcription Regulatory Sequences.

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5' untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5' UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5' UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

MicroRNA-376b-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting Viral Restriction Factor TRIM22.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically significant pathogen and has evolved several strategies to evade host antiviral response and provide favorable conditions for survival. In the present study, we demonstrated that a host microRNA, miR-376b-3p, was upregulated by PRRSV infection through the viral components, nsp4 and nsp11, and that miR-376b-3p can directly target tripartite motif-containing 22 (TRIM22) to impair its anti-PRRSV activity, thus facilitating the replication of PRRSV. Meanwhile, we found that TRIM22 induced degradation of the nucleocapsid protein (N) of PRRSV by interacting with N protein to inhibit PRRSV replication, and further study indicated that TRIM22 could enhance the activation of the lysosomal pathway by interacting with LC3 to induce lysosomal degradation of N protein. In conclusion, PRRSV increased miR-376b-3p expression and hijacked the host miR-376b-3p to promote PRRSV replication by impairing the antiviral effect of TRIM22. Therefore, our finding outlines a novel strategy of immune evasion exerted by PRRSV, which is helpful for better understanding the pathogenesis of PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes enormous economic losses each year in the swine industry worldwide. MicroRNAs (miRNAs) play important roles during viral infections via modulating the expression of viral or host genes at the posttranscriptional level. TRIM22 has recently been identified as a key restriction factor that inhibited the replication of a number of human viruses, such as HIV, encephalomyocarditis virus (ECMV), hepatitis C virus (HCV), HBV, influenza A virus (IAV), and respiratory syncytial virus (RSV). In this study, we showed that host miR-376b-3p could be upregulated by PRRSV and functioned to impair the anti-PRRSV role of TRIM22 to facilitate PRRSV replication. Meanwhile, we found that TRIM22 inhibited the replication of PRRSV by interacting with viral N protein and accelerating its degradation through the lysosomal pathway. Collectively, the findings reveal a novel mechanism that PRRSV used to exploit the host miR-376b-3p to evade antiviral responses and provide new insight into the study of virus-host interactions.

Induction of HOXA3 by Porcine Reproductive and Respiratory Syndrome Virus Inhibits Type I Interferon Response through Negative Regulation of HO-1 Transcription.

Type I interferons (IFN-Is) play a key role in host defense against virus infection, but porcine reproductive and respiratory syndrome virus (PRRSV) infection does not effectively activate IFN-I response, and the underlying molecular mechanisms are poorly characterized. In this study, a novel transcription factor of the heme oxygenase-1 (HO-1) gene, homeobox A3 (HOXA3), was screened and identified. Here, we found that HOXA3 was significantly increased during PRRSV infection. We demonstrated that HOXA3 promotes PRRSV replication by negatively regulating the HO-1 gene transcription, which is achieved by regulating IFN-I production. A detailed analysis showed that PRRSV exploits HOXA3 to suppress beta interferon (IFN-ß) and IFN-stimulated gene (ISG) expression in host cells. We also provide direct evidence that the activation of IFN-I by HO-1 depends on its interaction with IRF3. Then we further proved that a deficiency of HOXA3 promoted the HO-1-IRF3 interaction and subsequently enhanced IRF3 phosphorylation and nuclear translocation in PRRSV-infected cells. These data suggest that PRRSV uses HOXA3 to negatively regulate the transcription of the HO-1 gene to suppress the IFN-I response for immune evasion. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, causes significant worldwide economic losses in the pork industry. HOXA3 is generally considered to be an important molecule in the process of body development and cell differentiation. Here, we found that a novel transcription factor of the HO-1 gene, HOXA3, can negatively regulate the transcription of the HO-1 gene and play an important role in the suppression of IFN-I response by PRRSV. PRRSV induces the upregulation of HOXA3, which can negatively regulate HO-1 gene transcription, thereby weakening the interaction between HO-1 and IRF3 for inhibiting the type I IFN response. This study extends the function of HOXA3 and provides new insights into the PRRSV immune evasion mechanism.

Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

The Antimalaria Drug Artesunate Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by Activating AMPK and Nrf2/HO-1 Signaling Pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Currently, vaccine strategies provide limited protection against PRRSV transmission, and no effective drug is commercially available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV pandemics. This study showed that artesunate (AS), one of the antimalarial drugs, potently suppressed PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs) at micromolar concentrations. Furthermore, we demonstrated that this suppression was closely associated with AS-activated AMPK (energy homeostasis) and Nrf2/HO-1 (inflammation) signaling pathways. AS treatment promoted p-AMPK, Nrf2, and HO-1 expression and, thus, inhibited PRRSV replication in Marc-145 and PAM cells in a time- and dose-dependent manner. These effects of AS were reversed when the AMPK or HO-1 gene was silenced by short interfering RNA. In addition, we demonstrated that AMPK works upstream of Nrf2/HO-1, as its activation by AS is AMPK dependent. Adenosine phosphate analysis showed that AS activates AMPK via improving the AMP/ADP-to-ATP ratio rather than direct interaction with AMPK. Altogether, our findings indicate that AS is a promising novel therapeutic for controlling PRRSV and that its anti-PRRSV mechanism, which involves the functional link between energy homeostasis and inflammation suppression pathways, may provide opportunities for developing novel antiviral agents. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infections have continuously threatened the pork industry worldwide. Vaccination strategies provide very limited protection against PRRSV infection, and no effective drug is commercially available. We show that artesunate (AS), one of the antimalarial drugs, is a potent inhibitor against PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs). Furthermore, we demonstrate that AS inhibits PRRSV replication via activation of AMPK-dependent Nrf2/HO-1 signaling pathways, revealing a novel link between energy homeostasis (AMPK) and inflammation suppression (Nrf2/HO-1) during viral infection. Therefore, we believe that AS may be a promising novel therapeutics for controlling PRRSV, and its anti-PRRSV mechanism may provide a strategy to develop novel antiviral agents.

Evolutionary Dynamics of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Whole-Genome Analysis.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen in the swine industry, is a genetically highly diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus are not yet fully understood. In this study, we performed an integrated analysis of all available whole-genome sequences of type 2 PRRSV (n = 901) to reveal its evolutionary dynamics. The results showed that there were three distinct phylogenetic lineages of PRRSV in their distribution patterns. We identified that sublineage 2.7 (L2.7), associated with a NADC30 cluster, had the highest substitution rate and higher viral genetic diversity, and inter-lineage recombination is observed more frequently in L2.7 PRRSV compared to other sublineages. Most inter-lineage recombination events detected are observed between L2.7 PRRSVs (as major parents) and L3.4 (a JXA1-R-related cluster)/L3.7 (a WUH3-related cluster) PRRSVs (as minor parents). Moreover, the recombination hotspots are located in the structural protein gene ORF2 and ORF4, or in the non-structural protein gene nsp7. In addition, a GM2-related cluster, L3.2, shows inconsistent recombination modes compared to those of L2.7, suggesting that it may have undergone extensive and unique recombination in their evolutionary history. We also identified several amino acids under positive selection in GP2, GP4 and GP5, the major glycoproteins of PRRSV, showing the driving force behind adaptive evolution. Taken together, our results provide new insights into the evolutionary dynamics of PPRSV that contribute to our understanding of the critical factors involved in its evolution and guide future efforts to develop effective preventive measures against PRRSV.

Porcine Reproductive and Respiratory Syndrome (PRRS) Epidemiology in an Integrated Pig Company of Northern Italy: A Multilevel Threat Requiring Multilevel Interventions.

Porcine reproductive and respiratory syndrome (PRRS) is probably the most relevant viral disease affecting pig farming. Despite the remarkable efforts paid in terms of vaccination administration and biosecurity, eradication and long-term control have often been frustrated. Unfortunately, few studies are currently available that objectively link, using a formal statistical approach, viral molecular epidemiology to the risk factors determining the observed scenario. The purpose of the present study is to contribute to filling this knowledge gap taking advantage of the advancements in the field of phylodynamics. Approximately one-thousand ORF7 sequences were obtained from strains collected between 2004 and 2021 from the largest Italian pig company, which implements strict compartmentalization among independent three-sites (i.e., sow herds, nurseries and finishing units) pig flows. The history and dynamics of the viral population and its evolution over time were reconstructed and linked to managerial choices. The viral fluxes within and among independent pig flows were evaluated, and the contribution of other integrated pig companies and rurally risen pigs in mediating such spreading was investigated. Moreover, viral circulation in Northern Italy was reconstructed using a continuous phylogeographic approach, and the impact of several environmental features on PRRSV strain persistence and spreading velocity was assessed. The results demonstrate that PRRSV epidemiology is shaped by a multitude of factors, including pig herd management (e.g., immunization strategy), implementation of strict-independent pig flows, and environmental features (e.g., climate, altitude, pig density, road density, etc.) among the others. Small farms and rurally raised animals also emerged as a potential threat for larger, integrated companies. These pieces of evidence suggest that none of the implemented measures can be considered effective alone, and a multidimensional approach, ranging from individual herd management to collaboration and information sharing among different companies, is mandatory for effective infection control.

Major Vault Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection in CRL2843CD163 Cell Lines and Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type Ⅰ interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type Ⅰ IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.

The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function.

Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

The glycosyltransferase ST3GAL2 modulates virus proliferation and the inflammation response in porcine reproductive and respiratory syndrome virus infection.

ß-galactoside α-2,3-sialyltransferase 2 (ST3GAL2) is a member of the sialyltransferase family that mediates terminal modification of glycoproteins and glycolipids. ST3GAL2 has been found to play a role in obesity, aging, and malignant diseases. In this study, we cloned porcine ST3GAL2 (pST3GAL2) from porcine alveolar macrophages (PAMs), and its role in porcine reproductive and respiratory syndrome virus (PRRSV) infection was investigated by transcriptome analysis. pST3GAL2 was found to be located in the Golgi apparatus, and it was expressed at high levels in PRRSV-infected PAMs. Overexpression of pST3GAL2 resulted in a slight increase in PRRSV proliferation, and the interaction between pST3GAL2 and GP2a of PRRSV was detected by coimmunoprecipitation and confocal microscopy. The expression of pro-inflammatory cytokines (IFN-ß, IL-2, IL-6, IL-18, IL-1ß and TNF-α) was significantly inhibited in pST3GAL2-overexpressing, PRRSV-infected cells and upregulated in PRRSV-infected pST3GAL2-knockout cells, while the pattern of expression of anti-inflammatory cytokines (IL-4 and IL-10) was diametrically opposite. Our results demonstrate that the regulation of pST3GAL2 plays an important role in PRRSV proliferation and functional alterations in virus-infected cells. These results contribute to our understanding of the role of ß-galactoside α-2,3-sialyltransferase 2 in antiviral immunity.

The jigsaw of PRRSV virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the, probably, most economically important disease for the pig industry worldwide. This disease, characterised by producing reproductive failure in sows and respiratory problems in growing pigs, appeared in the late 1980s in the United States and Canada. Since its appearance, strains capable of producing higher mortality rates as well as greater severity in clinical signs and lesions than classical strains have been identified. However, since the first reports of these "virulent" PRRSV outbreaks, no homogeneity and consensus in their description have been established. Moreover, to the authors' knowledge, there is no published information related to the criteria that a PRRSV strain should fulfil to be considered as a "virulent" strain. In this review, we revise the terminology used and gather the information related to the main characteristics and differences in clinical signs, lesions, viral replication and tropism as well as immunological parameters between virulent and classical PRRSV strains and propose a first approximation to the criteria to define a virulent PRRSV strain.

Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro.

Porcine reproductive and respiratory syndrome (PRRS) is a serious disease burdening global swine industry. Infection by its etiological agent, PRRS virus (PRRSV), shows a highly restricted tropism of host cells and has been demonstrated to be mediated by an essential scavenger receptor (SR) CD163. CD163 fifth SR cysteine-rich domain (SRCR5) is further proven to play a crucial role during viral infection. Despite intense research, the involvement of CD163 SRCR5 in PRRSV infection remains to be elucidated. In the current study, we prepared recombinant monkey CD163 (moCD163) SRCR5 and human CD163-like homolog (hCD163L1) SRCR8, and determined their crystal structures. After comparison with the previously reported crystal structure of porcine CD163 (pCD163) SRCR5, these structures showed almost identical structural folds but significantly different surface electrostatic potentials. Based on these differences, we carried out mutational research to identify that the charged residue at position 534 in association with the one at position 561 were important for PRRSV-2 infection in vitro. Altogether the current work sheds some light on CD163-mediated PRRSV-2 infection and deepens our understanding of the viral pathogenesis, which will provide clues for prevention and control of PRRS.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.