Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Evasion strategies of porcine reproductive and respiratory syndrome virus.

During the co-evolution of viruses and their hosts, viruses have developed various strategies for overcoming host immunological defenses so that they can proliferate efficiently. Porcine reproductive and respiratory syndrome virus (PRRSV), a significant virus to the swine industry across the world, typically establishes prolonged infection via diverse and complicated mechanisms, which is one of the biggest obstacles for controlling the associated disease, porcine reproductive and respiratory syndrome (PRRS). In this review, we summarize the latest research on how PRRSV circumvents host antiviral responses from both the innate and adaptive immune systems and how this virus utilizes other evasion mechanisms, such as the manipulation of host apoptosis and microRNA. A thorough understanding of the exact mechanisms of PRRSV immune evasion will help with the development of novel antiviral strategies against PRRSV.

Development of an enzyme-linked immunosorbent assay based on viral antigen capture by anti-spike glycoprotein monoclonal antibody for detecting immunoglobulin A antibodies against porcine epidemic diarrhea virus in milk.

BACKGROUND: Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. RESULTS: The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. CONCLUSIONS: The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

Reappraising host cellular factors involved in attachment and entry to develop antiviral strategies against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a highly contagious disease that brings tremendous economic losses to the global swine industry. As an intracellular obligate pathogen, PRRSV infects specific host cells to complete its replication cycle. PRRSV attachment to and entry into host cells are the first steps to initiate the replication cycle and involve multiple host cellular factors. In this review, we recapitulated recent advances on host cellular factors involved in PRRSV attachment and entry, and reappraised their functions in these two stages, which will deepen the understanding of PRRSV infection and provide insights to develop promising antiviral strategies against the virus.

Proteomic Investigation Reveals Eukaryotic Translation Initiation Factor 5A Involvement in Porcine Reproductive and Respiratory Syndrome Virus Infection in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV), one of the most serious animal pathogens in the world, has caused enormous global swine industry losses. An in-depth investigation of the PRRSV-host interaction would be beneficial for preventing and controlling PRRSV infections and transmission. In this study, we performed label-free quantitative proteomic assays to investigate proteome dynamics of porcine alveolar macrophages (PAMs) during infection with highly pathogenic PRRSV (HP-PRRSV) strain HN07-1. Analysis of the results led to identification of 269 significantly differentially expressed host cellular proteins, of which levels of proteins belonging to the eukaryotic translation initiation factor (eIF) family were found to be decreased in abundance in HP-PRRSV-infected PAMs. Furthermore, knockdown of eIF5A expression was demonstrated to markedly suppress HP-PRRSV propagation, as reflected by reduced progeny virus titers in vitro. These results highlight the importance of eIF5A in PRRSV infection, while also demonstrating that PAMs down-regulate eIF5A expression as a host cell antiviral strategy. Results of the current study deepen our understanding of PRRSV pathogenesis and provide novel insights to guide development of effective strategies to combat the virus.

Development of a p72 trimer-based colloidal gold strip for detection of antibodies against African swine fever virus.

African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV. KEY POINTS : • ASFV p72 trimers were successfully generated. • A colloidal gold strip was developed based on ASFV p72 trimers. • The strip is appropriate for detecting ASFV antibodies in the field.

Heat Shock Protein Member 8 (HSPA8) Is Involved in Porcine Reproductive and Respiratory Syndrome Virus Attachment and Internalization.

Porcine reproductive and respiratory syndrome virus (PRRSV), a porcine arterivirus, causes severe financial losses to global swine industry. Despite much research, the molecular mechanisms of PRRSV infection remains to be fully elucidated. In the current study, we uncovered the involvement of heat shock protein member 8 (HSPA8) in PRRSV attachment and internalization during infection for the first time. In detail, HSPA8 was identified to interact with PRRSV glycoprotein 4 (GP4), a major determinant for viral cellular tropism, dependent on its carboxy-terminal peptide-binding (PB) domain. Chemical inhibitors and specific small interference RNAs (siRNAs) targeting HSPA8 significantly suppressed PRRSV infection as indicated by decreased viral RNA abundance, infectivity, and titers. Especially, PRRSV attachment was inhibited by interference of its binding to HSPA8 with mouse anti-HSPA8 polyclonal antibodies (pAbs) and recombinant soluble HSPA8 protein. HSPA8 was further shown to participate in PRRSV internalization through clathrin-dependent endocytosis (CME). Collectively, these results demonstrate that HSPA8 is important for PRRSV attachment and internalization, which is a potential target to prevent and control the viral infection. IMPORTANCE PRRSV has caused huge economic losses to the pork industry around the world. Currently, safe and effective strategies are still urgently required to prevent and control PRRSV infection. As the first steps, PRRSV attachment and internalization are initiated by interactions between viral envelope proteins and host cell receptors/factors, which are not fully understood yet. Here, we identified the interaction between PRRSV GP4 and HSPA8, and demonstrated that HSPA8 was involved in PRRSV attachment and internalization. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection, and provides novel insights for the development of antiviral drugs and vaccines against the virus.

Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to global swine industry. As an intracellular obligate pathogen, PRRSV exploits host cellular machinery to establish infection. The endocytic sorting complex required for transport (ESCRT) system has been shown to participate in different life cycle stages of multiple viruses. In the present study, a systematic small interference RNA screening assay identified that certain ESCRT components contributed to PRRSV infection. Among them, tumor susceptibility gene 101 (TSG101) was demonstrated to be important for PRRSV infection by knockdown and overexpression assays. TSG101 was further revealed to be involved in virion formation rather than viral attachment, internalization, RNA replication and nucleocapsid (N) protein translation within the first round of PRRSV life cycle. In detail, TSG101 was determined to specially interact with PRRSV N protein and take effect on its subcellular localization along with the early secretory pathway. Taken together, these results provide evidence that TSG101 is a proviral cellular factor for PRRSV assembly, which will be a promising target to interfere with the viral infection. IMPORTANCE PRRSV infection results in a serious swine disease affecting pig farming in the world. However, efficient prevention and control of PRRSV is hindered by its complicated infection process. Until now, our understanding of PRRSV assembly during infection is especially limited. Here, we identified that TSG101, an ESCRT-I subunit, facilitated virion formation of PRRSV via interaction with the viral N protein along with the early secretory pathway. Our work actually expands the knowledge of PRRSV infection and provides a novel therapeutic target for prevention and control of the virus.

Efficacy of a live attenuated highly pathogenic PRRSV vaccine against a NADC30-like strain challenge: implications for ADE of PRRSV.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection can cause severe reproductive failure in sows and respiratory distress in pigs of all ages, leading to major economic losses. To date, there are still no effective strategies to prevent and control PRRSV. Antibody-dependent enhancement (ADE), a phenomenon in which preexisting non-neutralizing antibodies or sub-neutralizing antibodies facilitate virus entry and replication, may be a significant obstacle in the development of effective vaccines for many viruses, including PRRSV. However, the contribution of ADE to PRRSV infection remains controversial, especially in vivo. Whether attenuated PRRSV vaccines prevent or worsen subsequent disease in pigs infected by novel PRRSV strains requires more research. In the present study, in vivo experiments were conducted to evaluate ADE under different immune statuses, which were produced by waiting different lengths of time after vaccination with a commercially available attenuated highly pathogenic PRRSV (HP-PRRSV) vaccine (JXA1-R) before challenging the pigs with a novel heterologous NADC30-like strain. RESULTS: Piglets that were vaccinated before being challenged with PRRSV exhibited lower mortality rates, lower body temperatures, higher bodyweight gain, and lower viremia. These results demonstrate that vaccination with JXA1-R alleviated the clinical signs of PRRSV infection in all vaccinated groups. CONCLUSIONS: The obtained data indicate that the attenuated vaccine test here provided partial protection against the NADC30-like strain HNhx. No signs of enhanced PRRSV infection were observed under the applied experimental conditions. Our results provide some insight into the molecular mechanisms underlying vaccine-induced protection or enhancement in PRRSV.

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.

Vimentin rearrangement by phosphorylation is beneficial for porcine reproductive and respiratory syndrome virus replication in vitro.

Vimentin, a member of intermediate filaments, has been documented to be involved in viral infections. Despite several studies focusing on its involvement in porcine reproductive and respiratory syndrome virus (PRRSV) infection, the detailed mechanisms by which vimentin takes effect remain to be fully elucidated. In the present study, we identified a previously unrecognized role of vimentin rearrangement in PRRSV replication. We monitored that PRRSV infection induced vimentin reorganization during post-entry stage, which was beneficial for viral replication. In detail, the serine residue of vimentin was phosphorylated at position 38 (Ser38) by calcium calmodulin-dependent protein kinase II gamma (CaMKIIγ), and vimentin filaments reorganized into cage-like structures enwrapping PRRSV replication complex (RC) at the perinuclear location. Taken together, these results expand the knowledge of PRRSV replication, and provide novel targets for prevention and control of PRRSV.

Porcine reproductive and respiratory syndrome virus increases SOCS3 production via activation of p38/AP-1 signaling pathway to promote viral replication.

SOCS3 belongs to the suppressor of cytokine signaling (SOCS) family, which function as negative factors in host immune responses. Prior studies have noted the importance of SOCS family proteins in immunosuppression induced by some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine-borne viruses and has threatened the global swine industry with huge economic losses since it was first described in the 1980s. PRRSV is the etiological agent of PRRS, which causes reproductive failure and respiratory disorders. PRRSV causes immunosuppression thus establishing persistent infection. In this study, it was observed that SOCS3 was upregulated in PRRSV-infected primary porcine alveolar macrophages (PAMs) and Marc-145 cells with dose-dependent effects, which depends on virus replication. Deletion of AP-1 binding motif located in SOCS3 promoter inhibited promoter activities, which indicates that AP-1 is essential for PRRSV-induced SOCS3. This result was confirmed by experiments using AP-1 inhibitor, whose pretreatment suppressed SOCS3 mRNA and protein expression. Further research showed that p38 was crucial for PRRSV-induced SOCS3 production. Importantly, SOCS3 enhanced PRRSV replication during infection. Taken together, this study indicates that PRRSV infection induced SOCS3 expression through p38/AP-1 signaling pathway. These results revealed the molecular basis of SOCS3 upregulation and would advance further understanding of the strategy for viral immune evasion.

Quantitative Proteomic Analysis of Global Protein Acetylation in PRRSV-Infected Pulmonary Alveolar Macrophages.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a serious viral disease affecting global swine industry. Due to the lack of effective vaccines, new antiviral strategies to compensate for the inefficacy of available vaccines are urgently required. Lysine acetylation, as an important post-translational modification during infection, plays a key regulatory role in host antiviral responses. In this study, the global acetylome is profiled using acetylation specific antibody-based enrichment and tandem mass tag label high-affinity purification liquid chromatography-mass spectrometry in PRRSV-infected pulmonary alveolar macrophages (PAMs). As a result, 3731 lysine acetylation sites on 1421 cellular proteins are identified. Bioinformatics analysis of the different acetylated proteins revealed their involvement in various biological processes, including the host immune response and energy metabolism. These findings will contribute to the understanding of PRRSV pathogenesis and identify new cellular targets for anti-PPRSV therapeutics.

Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a widespread viral disease that has led to huge economic losses for the global swine industry. Non-structural protein 9 (Nsp9) of PRRSV possesses essential RNA-dependent RNA polymerase (RdRp) activity for viral RNA replication. Our previous report showed that Nsp9-specific nanobody, Nb6, was able to inhibit PRRSV replication. In this study, recombinant Nsp9 and Nsp9-Nb6 complex were prepared then characterized using bio-layer interferometry (BLI) and dynamic light scattering (DLS) analyses that demonstrated high-affinity binding of Nb6 to Nsp9 to form a homogeneous complex. Small-angle X-ray scattering (SAXS) characterization analyses revealed that spatial interactions differed between Nsp9 and Nsp9-Nb6 complex molecular envelopes. Enzyme-linked immunosorbent assays (ELISAs) revealed key involvement of Nsp9 residues Ile588, Asp590, and Leu643 and Nb6 residues Tyr62, Trp105, and Pro107 in the Nsp9-Nb6 interaction. After reverse genetics-based techniques were employed to generate recombinant Nsp9 mutant viruses, virus replication efficiencies were assessed in MARC-145 cells. The results revealed impaired viral replication of recombinant viruses bearing I588A and L643A mutations as compared with replication of wild type virus, as evidenced by reduced negative-strand genomic RNA [(-) gRNA] synthesis and attenuated viral infection. Moreover, the isoleucine at position 588 of Nsp9 was conserved across PRRSV genotypes. In conclusion, structural analysis of the Nsp9-Nb6 complex revealed novel amino acid interactions involved in viral RNA replication that will be useful for guiding development of structure-based anti-PRRSV agents.

Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses.

BACKGROUND: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. RESULTS: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. CONCLUSION: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

Elastase-mediated membrane fusion of highly pathogenic porcine reproductive and respiratory syndrome virus at host cell surface.

Infection by enveloped viruses includes endocytosis and/or membrane fusion at the plasma membrane, where host cell proteases play an essential role. Among them, elastase-mediated infection has been documented for several enveloped viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically critical factor in global swine industry, is previously reported to infect host cells via low pH-dependent clathrin-mediated endocytosis (CME) and undergo membrane fusion in recycling endosomes. In the current study, we identified that elastase was significantly elevated in the lung tissues of highly pathogenic PRRSV (HP-PRRSV)-infected pigs compared to the mock-infected ones. We subsequently demonstrated that elastase contributed to HP-PRRSV infection in both MARC-145 cells and porcine alveolar macrophages (PAMs). Mechanistically, HP-PRRSV entered host cells at the cell surface via elastase-mediated membrane fusion, independent of low pH and CME, and its glycoprotein 5 (GP5) was cleaved by the protease during this process. All these findings deepen our understanding of HP-PRRSV infection, and are beneficial for prevention and control of the disease.

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Viral Apoptotic Mimicry as an Alternative Pathway To Infect Host Cells.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), has led to enormous economic losses in global swine industry. Infection by PRRSV is previously shown to be via low pH-dependent clathrin-mediated endocytosis, and CD163 functions as an essential receptor during viral infection. Despite much research focusing on it, PRRSV infection remains to be fully elucidated. In this study, we demonstrated that PRRSV externalized phosphatidylserine (PS) on the envelope as viral apoptotic mimicry and infected host cells through T-cell immunoglobulin and mucin domain (TIM)-induced and CD163-involved macropinocytosis as an alternative pathway. In detail, we identified that PS receptor TIM-1/4 recognized and interacted with PRRSV as viral apoptotic mimicry and subsequently induced macropinocytosis by the downstream Rho GTPases Rac1, cell division control protein 42 (Cdc42), and p21-activated kinase 1 (Pak1). Altogether, these results expand our knowledge of PRRSV infection, which will support implications for the prevention and control of PRRS.IMPORTANCE PRRS has caused huge economic losses to pig farming worldwide. Its causative agent, PRRSV, infects host cells through low pH-dependent clathrin-mediated endocytosis and CD163 is indispensable during the process. Whether there exist alternative infection pathways for PRRSV arouses our interest. Here, we found that PRRSV exposed PS on its envelope and disguised as apoptotic debris. The PS receptor TIM-1/4 recognized PRRSV and induced the downstream signaling pathway to mediate viral infection via CD163-dependent macropinocytosis. The current work deepens our understanding of PRRSV infection and provides clues for the development of drugs and vaccines against the virus.