Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Induces Interleukin-17 Production via Activation of the IRAK1-PI3K-p38MAPK-C/EBPß/CREB Pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) is widely prevalent in pigs, resulting in significant economic losses worldwide. A compelling impact of PRRSV infection is severe pneumonia. In the present study, we found that interleukin-17 (IL-17) was upregulated by PRRSV infection. Subsequently, we demonstrated that PI3K and p38MAPK signaling pathways were essential for PRRSV-induced IL-17 production as addition of phosphatidylinositol 3-kinase (PI3K) and p38MAPK inhibitors dramatically reduced IL-17 production. Furthermore, we show here that deleting the C/EBPß and CREB binding motif in porcine IL-17 promoter abrogated its activation and that knockdown of C/EBPß and CREB remarkably impaired PRRSV-induced IL-17 production, suggesting that IL-17 expression was dependent on C/EBPß and CREB. More specifically, we demonstrate that PRRSV nonstructural protein 11 (nsp11) induced IL-17 production, which was also dependent on PI3K-p38MAPK-C/EBPß/CREB pathways. We then show that Ser74 and Phe76 amino acids were essential for nsp11 to induce IL-17 production and viral rescue. In addition, IRAK1 was required for nsp11 to activate PI3K and enhance IL-17 expression by interacting with each other. Importantly, we demonstrate that PI3K inhibitor significantly suppressed IL-17 production and lung inflammation caused by HP-PRRSV in vivo, implicating that higher IL-17 level induced by HP-PRRSV might be associated with severe lung inflammation. These findings provide new insights onto the molecular mechanisms of the PRRSV-induced IL-17 production and help us further understand the pathogenesis of PRRSV infection.IMPORTANCE Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) associated with severe pneumonia has been one of the most important viral pathogens in pigs. IL-17 is a proinflammatory cytokine that might be associated with the strong inflammation caused by PRRSV. Therefore, we sought to determine whether PRRSV infection affects IL-17 expression, and if so, determine this might partially explain the underlying mechanisms for the strong inflammation in HP-PRRSV-infected pigs, especially in lungs. Here, we show that PRRSV significantly induced IL-17 expression, and we subsequently dissected the molecular mechanisms about how PRRSV regulated IL-17 production. Furthermore, we show that Ser74 and Phe76 in nsp11 were indispensable for IL-17 production and viral replication. Importantly, we demonstrated that PI3K inhibitor impaired IL-17 production and alleviated lung inflammation caused by HP-PRRSV infection. Our findings will help us for a better understanding of PRRSV pathogenesis.

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4-5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

The Postfusion Structure of the Heartland Virus Gc Glycoprotein Supports Taxonomic Separation of the Bunyaviral Families Phenuiviridae and Hantaviridae.

Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low-pH conditions. Here, we describe the crystal structure of HRTV Gc in its postfusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct features in the aspects of subdomain orientation, N-linked glycosylation, the interaction pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens.IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee on Taxonomy of Viruses reclassification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

MicroRNA-30c targets the interferon-alpha/beta receptor beta chain to promote type 2 PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases in pigs. MicroRNAs (miRNAs) have emerged as an important regulator of virus-host cell interactions and miR-30c has been found to facilitate PRRSV replication. Here, we found that the interferon-alpha/beta receptor beta chain (IFNAR2) was down-regulated, while miR-30c was up-regulated during HV (a highly pathogenic type 2 PRRSV strain) and CH-1a (a classic type 2 PRRSV strain) infection. Subsequently, using bioinformatics analysis, we predicted that the IFNAR2 was targeted by miR-30c. A luciferase assay verified that the 3' UTR of IFNAR2 was targeted by miR-30c, as a mutation on either the target sequence or the miR-30c seed sequence reversed the luciferase activity. In addition, miR-30c and IFNAR2 mRNA were physically co-localized in RNA-induced silencing complex (RISC). Importantly, we showed that miR-30c also impaired the induction of IFN-stimulated genes (ISGs) by targeting IFNAR2. Our findings further reveal the mechanism of miR-30c promoting PRRSV replication.

MicroRNA-30c Modulates Type I IFN Responses To Facilitate Porcine Reproductive and Respiratory Syndrome Virus Infection by Targeting JAK1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen and has evolved several mechanisms to evade IFN-I responses. We report that a host microRNA, miR-30c, was upregulated by PRRSV via activating NF-κB and facilitated its ability to infect subject animals. Subsequently, we demonstrated that miR-30c was a potent negative regulator of IFN-I signaling by targeting JAK1, resulting in the enhancement of PRRSV infection. In addition, we found that JAK1 expression was significantly decreased by PRRSV and recovered when miR-30c inhibitor was overexpressed. Importantly, miR-30c was also upregulated by PRRSV infection in vivo, and miR-30c expression corresponded well with viral loads in lungs and porcine alveolar macrophages of PRRSV-infected pigs. Our findings identify a new strategy taken by PRRSV to escape IFN-I-mediated antiviral immune responses by engaging miR-30c and, thus, improve our understanding of its pathogenesis.

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-ß production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-ß expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKß. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-ß production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-ß production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-ß production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

Highly pathogenic porcine reproductive and respiratory syndrome virus induces prostaglandin E2 production through cyclooxygenase 1, which is dependent on the ERK1/2-p-C/EBP-ß pathway.

UNLABELLED: Atypical porcine reproductive and respiratory syndrome (PRRS) caused by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is characterized by high fever and high mortality. However, the mechanism underlying the fever induction is still unknown. Prostaglandin E2 (PGE2), synthesized by cyclooxygenase type 1/2 (COX-1/2) enzymes, is essential for inducing fever. In this study, we found that PGE2, together with COX-1, was significantly elevated by HP-PRRSV. We subsequently demonstrated that extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK (p-ERK) were the key nodes to trigger COX-1 expression after HP-PRRSV infection. Furthermore, we proved the direct binding of p-C/EBP-ß to the COX-1 promoter by luciferase reporter and chromatin immunoprecipitation assays. In addition, silencing of C/EBP-ß remarkably impaired the enhancement of COX-1 production induced by HP-PRRSV infection. Taken together, our results indicate that HP-PPRSV elicits the expression of COX-1 through the ERK1/2-p-C/EBP-ß signaling pathway, resulting in the increase of PGE2, which might be the cause of high fever in infected pigs. Our findings might provide new insights into the molecular mechanisms underlying the pathogenesis of HP-PRRSV infection. IMPORTANCE: The atypical PRRS caused by HP-PRRSV was characterized by high fever, high morbidity, and high mortality in pigs of all ages, yet how HP-PRRSV induces high fever in pigs remains unknown. In the present study, we found out that HP-PRRSV infection could increase PGE2 production by upregulation of COX-1, and we subsequently characterized the underlying mechanisms about how HP-PRRSV enhances COX-1 production. PGE2 plays a critical role in inducing high temperature in hosts during pathogen infections. Thus, our findings here could help us have a better understanding of HP-PRRSV pathogenesis.

Increasing expression of microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Emerging evidence indicates that the host microRNAs (miRNAs) are involved in host-pathogen interactions. However, whether host miRNAs can target PRRSV and be used to inhibit PRRSV infection has not been reported. Recently, microRNA 181 (miR-181) has been identified as a positive regulator of immune response, and here we report that miR-181 can directly impair PRRSV infection. Our results showed that delivered miR-181 mimics can strongly inhibit PRRSV replication in vitro through specifically binding to a highly (over 96%) conserved region in the downstream of open reading frame 4 (ORF4) of the viral genomic RNA. The inhibition of PRRSV replication was specific and dose dependent. In PRRSV-infected Marc-145 cells, the viral mRNAs could compete with miR-181-targeted sequence in luciferase vector to interact with miR-181 and result in less inhibition of luciferase activity, further demonstrating the specific interactions between miR-181 and PRRSV RNAs. As expected, miR-181 and other potential PRRSV-targeting miRNAs (such as miR-206) are expressed much more abundantly in minimally permissive cells or tissues than in highly permissive cells or tissues. Importantly, highly pathogenic PRRSV (HP-PRRSV) strain-infected pigs treated with miR-181 mimics showed substantially decreased viral loads in blood and relief from PRRSV-induced fever compared to negative-control (NC)-treated controls. These results indicate the important role of host miRNAs in modulating PRRSV infection and viral pathogenesis and also support the idea that host miRNAs could be useful for RNA interference (RNAi)-mediated antiviral therapeutic strategies.

MicroRNA 181 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163.

We previously showed that microRNA 181 (miR-181) can inhibit PRRSV replication by directly targeting its genomic RNA. Here, we report that miR-181 can downregulate the PRRSV receptor CD163 in blood monocytes and porcine alveolar macrophages (PAMs) through targeting the 3' untranslated region (UTR) of CD163 mRNA. Downregulation of CD163 leads to the inhibition of PRRSV entry into PAMs and subsequently suppresses PRRSV infection. Our findings indicate that delivery of miR-181 can be used as antiviral therapy against PRRSV infection.

ASFV pA151R negatively regulates type I IFN production via degrading E3 ligase TRAF6.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly mortal and hemorrhagic infectious disease in pigs. Previous studies have indicated that ASFV modulates interferon (IFN) production. In this study, we demonstrated that ASFV pA151R negatively regulated type I IFN production. Ectopic expression of pA151R dramatically inhibited K63-linked polyubiquitination and Ser172 phosphorylation of TANK-binding kinase 1 (TBK1). Mechanically, we demonstrated that E3 ligase TNF receptor-associated factor 6 (TRAF6) participated in the ubiquitination of TBK1 in cGAS-STING signaling pathway. We showed that pA151R interacted with TRAF6 and degraded it through apoptosis pathway, leading to the disruption of TBK1 and TRAF6 interaction. Moreover, we clarified that the amino acids H102, C109, C132, and C135 in pA151R were crucial for pA151R to inhibit type I interferon production. In addition, we verified that overexpression of pA151R facilitated DNA virus Herpes simplex virus 1 (HSV-1) replication by inhibiting IFN-ß production. Importantly, knockdown of pA151R inhibited ASFV replication and enhanced IFN-ß production in porcine alveolar macrophages (PAMs). Our findings will help understand how ASFV escapes host antiviral immune responses and develop effective ASFV vaccines.

Complete genome sequence of a novel highly pathogenic porcine reproductive and respiratory syndrome virus variant.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) emerged in China in 2006, and HP-PRRS virus (HP-PRRSV) has evolved continuously. Here, the complete genomic sequence of a novel HP-PRRSV field strain, JX, is reported. The present finding will contribute to further studies focusing on the evolutionary mechanism of PRRSV.

Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-κB signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-κB inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-κB was indeed activated upon PRRSV infection, as evidenced by IκB phosphorylation and degradation. Moreover, we revealed an NF-κB binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-κB. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-κB activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.

African swine fever virus S273R protein antagonizes type I interferon production by interfering with TBK1 and IRF3 interaction.

African swine fever (ASF) is originally reported in East Africa as an acute hemorrhagic fever. African swine fever virus (ASFV) is a giant and complex DNA virus with icosahedral structure and encodes a variety of virulence factors to resist host innate immune response. S273R protein (pS273R), as a SUMO-1 specific cysteine protease, can affect viral packaging by cutting polymeric proteins. In this study, we found that pS273R was an important antagonistic viral factor that suppressed cGAS-STING-mediated type I interferon (IFN-I) production. A detailed analysis showed that pS273R inhibited IFN-I production by interacting with interferon regulatory factor 3 (IRF3). Subsequently, we showed that pS273R disrupted the association between TBK1 and IRF3, leading to the repressed IRF3 phosphorylation and dimerization. Deletion and point mutation analysis verified that pS273R impaired IFN-I production independent of its cysteine protease activity. These findings will help us further understand ASFV pathogenesis.

miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3'UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.

African swine fever virus QP383R dampens type I interferon production by promoting cGAS palmitoylation.

Cyclic GMP-AMP synthase (cGAS) recognizes viral DNA and synthesizes cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING/MITA) and downstream mediators to elicit an innate immune response. African swine fever virus (ASFV) proteins can antagonize host immune responses to promote its infection. Here, we identified ASFV protein QP383R as an inhibitor of cGAS. Specifically, we found that overexpression of QP383R suppressed type I interferons (IFNs) activation stimulated by dsDNA and cGAS/STING, resulting in decreased transcription of IFNß and downstream proinflammatory cytokines. In addition, we showed that QP383R interacted directly with cGAS and promoted cGAS palmitoylation. Moreover, we demonstrated that QP383R suppressed DNA binding and cGAS dimerization, thus inhibiting cGAS enzymatic functions and reducing cGAMP production. Finally, the truncation mutation analysis indicated that the 284-383aa of QP383R inhibited IFNß production. Considering these results collectively, we conclude that QP383R can antagonize host innate immune response to ASFV by targeting the core component cGAS in cGAS-STING signaling pathways, an important viral strategy to evade this innate immune sensor.

Induction of interleukin-10 is dependent on p38 mitogen-activated protein kinase pathway in macrophages infected with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory illness in pigs and usually establishes a persistent infection. Previous studies suggested that interleukin-10 (IL-10) could play a critical role in PRRSV-induced immunosuppression. However, the ability of PRRSV to induce IL-10 in infected cells is controversial. In this study, we further investigated this issue using PRRSV strain CH-1a, which is the first North American genotype strain isolated in China. RESULTS: PRRSV strain CH-1a could significantly up-regulate IL-10 production both at mRNA and protein levels in porcine alveolar macrophages (PAMs), bone marrow-derived macrophages (BMDMs), and monocyte-derived macrophages (MDMs). However, up-regulation of IL-10 by PRRSV was retarded by specific inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580) and NF-κB (BAY11-7082). Additionally, p38 MAPK and NF-κB pathways but not ERK1/2 MAPK were actually activated in PRRSV-infected BMDMs as demonstrated by western blot analysis, suggesting that p38 MAPK and NF-κB pathways are involved in the induction of IL-10 by PRRSV infection. Transfection of PAMs and PAM cell line 3D4/21 (CRL-2843) with viral structural genes showed that glycoprotein5 (GP5) could significantly up-regulate IL-10 production, which was dependent on p38 MAPK and signal transducer and activator of transcription-3 (STAT3) activation. We also demonstrated that a full-length glycoprotein was essential for GP5 to induce IL-10 production. CONCLUSIONS: PRRSV strain CH-1a could significantly up-regulate IL-10 production through p38 MAPK activation.

Regulation of antiviral immune response by African swine fever virus (ASFV).

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality approaching 100% in domestic pigs. ASF is an endemic in countries in sub-Saharan Africa. Now, it has been spreading to many countries, especially in Asia and Europe. Due to the fact that there is no commercial vaccine available for ASF to provide sustainable prevention, the disease has spread rapidly worldwide and caused great economic losses in swine industry. The knowledge gap of ASF virus (ASFV) pathogenesis and immune evasion is the main factor to limit the development of safe and effective ASF vaccines. Here, we will summarize the molecular mechanisms of how ASFV interferes with the host innate and adaptive immune responses. An in-depth understanding of ASFV immune evasion strategies will provide us with rational design of ASF vaccines.

miR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been recognized as one of the severest epidemics in pigs worldwide. microRNAs (miRNAs) play important roles in a variety of biological processes, including cell differentiation, proliferation and death, as well as viral infections and antiviral immune responses. In this study, we found that miR-142-3p was expressed lower in cells susceptible to PRRSV infection than in cells less or no permissive to PRRSV infection. Subsequently, we showed that overexpression of miR-142-3p remarkably inhibited PRRSV infection in PAMs, while blockage of endogenous miR-142-3p significantly enhanced PRRSV replication. Then, we demonstrated that miR-142-3p directly targeted Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of Rho GTPases family, by using luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay. Importantly, we verified that miR-142-3p inhibited PRRSV entry into PAMs and accordingly suppressed PRRSV infection by downregulating Rac1 expression. These findings reveal an important role of miR-142-3p in modulating PRRSV infection and provide us with some ideas for developing novel antiviral therapy against PRRSV infection.

Porcine Reproductive and Respiratory Syndrome Virus Evades Antiviral Innate Immunity via MicroRNAs Regulation.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases in pigs, leading to significant economic losses in the swine industry worldwide. MicroRNAs (miRNAs) are small single-stranded non-coding RNAs involved in regulating gene expressions at the post-transcriptional levels. A variety of host miRNAs are dysregulated and exploited by PRRSV to escape host antiviral surveillance and help virus infection. In addition, PRRSV might encode miRNAs. In this review, we will summarize current progress on how PRRSV utilizes miRNAs for immune evasions. Increasing knowledge of the role of miRNAs in immune evasion will improve our understanding of PRRSV pathogenesis and help us develop new treatments for PRRSV-associated diseases.

Ergosterol peroxide exhibits antiviral and immunomodulatory abilities against porcine deltacoronavirus (PDCoV) via suppression of NF-κB and p38/MAPK signaling pathways in vitro.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that poses economic and public health burdens. Currently, there are no effective antiviral agents against PDCoV. Cryptoporus volvatus often serves as an antimicrobial agent in Traditional Chinese Medicines. This study aimed to evaluate the antiviral activities of ergosterol peroxide (EP) from C. volvatus against PDCoV infection. The inhibitory activity of EP against PDCoV was assessed by using virus titration and performing Quantitative Reverse transcription PCR (RT-qPCR), Western blotting and immunofluorescence assays in LLC-PK1 cells. The mechanism of EP against PDCoV was analyzed by flow cytometry, RT-qPCR and Western blotting. We found that EP treatment inhibited PDCoV infection in LLC-PK1 cells in a dose-dependent manner. Subsequently, we demonstrated that EP blocked virus attachment and entry using RT-qPCR. Time-of-addition assays indicated that EP mainly exerted its inhibitory effect at the early and middle stages in the PDCoV replication cycle. EP also inactivated PDCoV infectivity directly as well as suppressed PDCoV-induced apoptosis. Furthermore, EP treatment decreased the phosphorylation of IκBα and p38 MAPK induced by PDCoV infection as well as the mRNA levels of cytokines (IL-1ß, IL-6, IL-12, TNF-α, IFN-α, IFN-ß, Mx1 and PKR). These results imply that EP can inhibit PDCoV infection and regulate host immune responses by downregulating the activation of the NF-κB and p38/MAPK signaling pathways in vitro. EP can be used as a potential candidate for the development of a new anti-PDCoV therapy.