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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.

Vesicular stomatitis (VS), characterized by vesicular lesions, produces significant economic losses in livestock industry. Infection by its causative agent, VS virus (VSV), has been previously shown to be mediated by the glycoprotein (G) during attachment, endocytosis and membrane fusion. In the current study, we revealed a novel role of VSV G protein in negative regulation of host cell pro-inflammatory responses. We determined that VSV G protein inhibited lipopolysaccharide (LPS)-induced pro-inflammatory responses as naïve VSV virions in murine peritoneal macrophage-like cell line RAW 264.7. Furthermore, we identified that VSV G protein suppressed nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK)-mediated pro-inflammatory pathways in a dose-dependent manner. Moreover, we demonstrated that α2-3-linked sialic acids on VSV G protein were involved in antagonizing NF-κB- and MAPK-mediated pro-inflammatory responses. All these results expand the knowledge of VSV pathogenesis and strengthen the importance of VSV G protein in host innate immunity, which support implications for the development of VSV-based vaccination and oncolysis.

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.

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.

Antibody-dependent enhancement (ADE) in porcine reproductive and respiratory syndrome virus (PRRSV) infection is a significant obstacle to the development of effective vaccines for controlling PRRS. Our previous results have demonstrated that porcine FcγRIIb (poFcγRIIb) play an important role in mediating ADE of PRRSV infection in vitro. However, the underlying mechanisms involved in poFcγRIIb mediated-ADE are still not clear. In this study, MARC-145 cel1 lines stably expressing mutated poFcγRIIb (MARC-poFcγRIIb-T and MARC-poFcγRIIb-CT) in cytoplasm were established and the capacity of poFcγRIIb mutants in mediating ADE of PRRSV was investigated. Our results showed that removal of cytoplasmic domain or disruption the tyrosine residue within ITIM (immunoreceptor tyrosine-based inhibition motif) of the poFcγRIIb abolished the ability of poFcγRIIb to mediate ADE of PRRSV. Furthermore, we found that SHIP1 and TBK1 were involved in poFcγRIIb-mediated ADE of PRRSV infection. Taken together, our findings indicated that poFcγRIIb mediated the ADE pathway of PRRSV infection through recruiting SHIP-1, which further inhibited of TBK-1-IRF3-IFN-β signaling pathway to enhance PRRSV infection. These findings will contribute to the molecular mechanism of ADE infection and provide some implications for vaccine development.

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499-638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.

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.

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.

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.

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., 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.

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 (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines. IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography. Single crystals were obtained using 20% PEG 4000, 0.15 M potassium sodium tartrate tetrahydrate pH 8.5 and diffracted to 3.30 Å resolution. As the first view of a long-range SRCR repeat, this work lays the structural basis for a deep understanding of SRs and their multiple functions.

Recently, NADC30-like porcine reproductive and respiratory syndrome viruses (PRRSVs), which are genetically similar to the NADC30 strain isolated in the United States of America in 2008, have become prevalent in China. Here, a novel variant PRRSV strain named HNhx was successfully isolated on porcine alveolar macrophages from Henan province and the full-length genome sequence was determined. Phylogenetic analysis indicated that HNhx strain was classified into the NADC30-like PRRSV subgroup, in which all the strains had the unique discontinuous 131-amino acid deletion relative to that of the nonstructural protein 2 (Nsp2) of the VR2332 strain. Genetically, HNhx shared 92.9% nucleotide similarity to NADC30. Furthermore, HNhx strain contained extensive amino acid mutations in GP5. In particular, the S32H, N33D, D34N, and S36G variations resulted in that HNhx lost all the putative N-linked glycosylation sites at amino acid positions 30, 32, 33, 34, and 35. Recombination analysis revealed that HNhx was the result of recombination between the NADC30 strain and the highly pathogenic PRRSV vaccine strain circulating in China in Nsp4 (nt 5261) to Nsp9 (nt 7911). The novel genome data of HNhx will be helpful for understanding the evolution and epidemiology of PRRSV in China.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Prompt detection of PRRS virus (PRRSV) in the field samples is important for effective PRRS control. Droplet digital PCR (ddPCR) is a novel PCR technology, which offers good precision and direct quantification without using calibration curves. In this study, we established a ddPCR system for the sensitive and accurate quantification of PRRSV. Specificity of the assay was determined by the failure of amplification of other relevant viruses. Quantitative linearity, sensitivity and accuracy of ddPCR were compared to those of real time PCR for PRRSV testing. Both methods showed a high degree of linearity (R2=∼1) and quantitative correlation, although ddPCR showed somewhat higher sensitivity than real time PCR. Collectively, our findings indicate that ddPCR might offer improved analytical sensitivity and specificity for PRRSV measurements.

Porcine reproductive and respiratory syndrome virus (PRRSV) is acknowledged a fulminating infectious pathogen affecting the pig farming industry, and current vaccines and drugs could hardly inhibit this virus. The 2', 5'-oligoadenylate synthetase (OASs) have antiviral activities, but the role(s) played by porcine OAS2 in protection against PRRSV infection are unknown. Here we found that endogenous expression of the porcine OAS2 gene could be promoted by interferon (IFN)-beta or PRRSV infection in porcine alveolar macrophages. Knockdown of porcine OAS2 led to increases in PRRSV replication, and OAS2 expression suppressed replication of PRRSV in a retinoic acid inducible gene I (RIG-I)-dependent manner, anti-PRRSV activity of porcine OAS2 would be lost if RNase L and OAS2 were both silenced. This discovery illustrates a pathway that porcine OAS2 responses to host anti-PRRSV function.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses to global swine industry. Porcine sialoadhesin (poSn) was previously reported to be a putative receptor for the causative agent, PRRS virus (PRRSV). In the current study, we first observed that PRRSV infection up-regulated expression of poSn in a dose- and time-dependent manner. Subsequently, we found that PRRSV-triggered transcription of type I interferons (IFNs) was involved in poSn up-regulation through the IFN-signal transducer and activator of transcription (STAT) signaling cascade. Interestingly, poSn up-regulation was shown to promote PRRSV infection during post-entry process. Taken together, this work deepens our understanding of PRRSV pathogenesis and provides a novel idea on its establishment of persistent infection, which will be interesting to unravel the detailed mechanisms in the future.

Atypical porcine pestivirus (APPV) is recognised as the etiology of congenital tremor (CT) Type A-II and poses a challenge to pig production. Here, we described a CT case in piglets caused by APPV infection in central China in 2017. Interestingly, different from a previous report, more CT litters were observed in the second and third parity sows compared to the first and fourth parity. Evolutionary analysis and recombination evaluation were conducted for the isolate and 61 APPV genomes were available in GenBank. Phylogenetic analysis revealed a high level of genetic variation of APPV and the coexistence of three clades (Clades I–III) in China. The isolate was clustered into Clade I, which seemed to be prevalent worldwide and displayed higher genetic variability (Subgroups 1–4) compared with Clade II and Clade III, both of which were only reported in China. Notably, three putative recombinants were identified and characterized in APPV. The recombination events occurred in inter-clades (Clade II and III) or intra-clades (Clade I). To the best of our knowledge, this study presents the first evidence of homologous recombination within Pestivirus K. These results provide new clinical presentations of APPV infection and may be helpful in better understanding the large amount of genetic variations in this genus.

Porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) are important swine viruses that threaten the swine industry worldwide. Here, we evaluated the co-infection status of PCV2, PCV3 and PEDV in 76 enteric samples from piglets with severe diarrhea disease in Henan, China. All samples were tested by PCR/RT-PCR. Our results showed that the infection rate of PCV2, PCV3 and PEDV was 82.89%, 76.32% and 68.42%, respectively. Interestingly, most of these samples exhibited mixed infections. The co-infection rates of PCV2 and PCV3, PCV2 and PEDV, PCV3 and PEDV were 69.74%, 57.89% and 53.95%, respectively. And the triple infection rate was 48.68%. Furthermore, the genetic characteristics of PCV2 and PCV3 were analyzed based on the cap genes. Two PCV2 genotypes, PCV2b and PCV2d, were circulating in the fields. The cap gene of PCV2b and PCV2d isolates only shared 94.6%-95.0% nucleotide identities. The PCV3 isolates together with the reference strains could be divided into four clades (clade1-4), and the cap genes of these isolates have 98.6%-100% nucleotide identities to each other. Distinctive amino acid substitutions were also characterized on the cap protein of PCV2 and PCV3 isolates. Our studies provide the new knowledge on the co-infectious status of PCV2, PCV3 and PEDV in China. The results also provide insight into the genetic diversity and molecular epidemiology of PCV2 and PCV3.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses in the swine industry since its emergence in the late 1980s. PRRSV exploits various strategies to evade immune responses and establish chronic persistent infections. Suppressor of cytokine signaling (SOCS) 1, a member of the SOCS family, is a crucial intracellular negative regulator of innate immunity. In this study, it was shown that SOCS1 can be co-opted by PRRSV to evade host immune responses, facilitating viral replication. It was observed that PRRSV induced SOCS1 production in porcine alveolar macrophages, monkey-derived Marc-145 cells, and porcine-derived CRL2843-CD163 cells. SOCS1 inhibited the expression of IFN-β and IFN-stimulated genes, thereby markedly enhancing PRRSV replication. It was observed that the PRRSV N protein has the ability to upregulate SOCS1 production and that nuclear localization signal–2 (NLS-2) is essential for SOCS1 induction. Moreover, SOCS1 upregulation was dependent on p38/AP-1 and JNK/AP-1 signaling pathways rather than classical type I IFN signaling pathways. In summary, to our knowledge, the findings of this study uncovered the molecular mechanism that underlay SOCS1 induction during PRRSV infection, providing new insights into viral immune evasion and persistent infection.

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.

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.

Once infected by viruses, cells can detect pathogen-associated molecular patterns (PAMPs) on viral nucleic acid by host pattern recognition receptors (PRRs) to initiate the antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory diseases in pigs of different ages. To date, the sensing mechanism of PRRSV has not been elucidated. Here, we reported that the pseudoknot region residing in the 3′ untranslated regions (UTR) of the PRRSV genome, which has been proposed to regulate RNA synthesis and virus replication, was sensed as nonself by retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3) and strongly induced type I interferons (IFNs) and interferon-stimulated genes (ISGs) in porcine alveolar macrophages (PAMs). The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction, since disruption of the pseudoknot interaction powerfully dampened the IFN induction. Furthermore, transfection of the 3′ UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro . Importantly, the predicted similar structures of other arterivirus members, including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV), also displayed strong IFN induction activities. Together, in this work we identified an innate recognition mechanism by which the PRRSV 3′ UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication. All of these results provide novel insights into innate immune recognition during virus infection. IMPORTANCE PRRS is the most common viral disease in the pork industry. It is caused by PRRSV, a positive single-stranded RNA virus, whose infection often leads to persistent infection. To date, it is not yet clear how PRRSV is recognized by the host and what is the exact mechanism of IFN induction. Here, we investigated the nature of PAMPs on PRRSV and the associated PRRs. We found that the 3′ UTR pseudoknot region of PRRSV, which has been proposed to regulate viral RNA synthesis, could act as PAMPs recognized by RIG-I and TLR3 to induce type I IFN production to suppress PRRSV infection. This report is the first detailed description of pattern recognition for PRRSV, which is important in understanding the antiviral response of arteriviruses, especially PRRSV, and extends our knowledge on virus recognition.

In several parts of China, there have been a large number of pseudorabies (PR) outbreaks which have devastated many swine farms even though the herds had been previously immunized with gE-deleted vaccines (Bartha-K61). The emergence of these outbreak-associated PRV strains might indicate that Bartha-K61 vaccine could not provide effective protection and poses challenges for current serologic diagnostics of anti-PRV antibodies. Here, we performed phylogenetic analyses based on partial gE, gB, and gC genes to provide information about the molecular epidemiology, diagnostics, and immune protection in these outbreak-associated PRV strains. Our results indicated that the maximal nucleotide sequence divergence for gE, gB, and gC genes are 1.7, 0.4, and 2.7 % within the cluster where outbreak-associated PRV strains were located, and are 2.3, 2.7, and 7.6 % with other clusters in the phylogenetic trees, respectively. Phylogenetic analyses revealed that gE, gB, and gC genes of the twelve outbreak-associated PRV strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor with strains Ea-China-1999, Fa-China-2001, and BJ-China-2008. The genetic relationship between these outbreak-associated PRV strains and strain Bartha is not close which may genetically explain the emergence of PR outbreaks in Bartha-K61-vaccinated swine farms. We suggest that these outbreak-associated PRV strains originate from earlier strains in local regions in China.

Porcine epidemic diarrhea (PED) has caused huge economic losses to the global pork industry. Infection by its causative agent PED virus (PEDV), an Alpha-coronavirus, was previously proven to be mediated by its spike (S) glycoprotein and a cellular receptor porcine aminopeptidase N (pAPN). Interestingly, some recent studies have indicated that pAPN is not a functional receptor for PEDV. To date, there is a lack of a direct evidence for the interaction between pAPN and PEDV S protein in vitro. Here, we prepared pAPN ectodomain and the truncated variants of PEDV S protein in Drosophila S2 cells. These recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. We then assayed the purified target proteins through immunogenicity tests, PEDV binding interference assays, circular dichroism (CD) measurements, pAPN activity assay and structural determination, demonstrating that they were biologically functional. Finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR) analyses. The results showed that their affinities were too low to form complexes, which suggest that pAPN may be controversial as the genuine receptor for PEDV. Therefore, further research needs to be carried out to elucidate the interaction between PEDV and its genuine receptor.

Accurate and real-time traffic flow forecasting is critical to the control and development of intelligent transportation systems (ITS). In recent years, many methods based on deep learning have been used in the prediction of short-term traffic flows, LSTM is also used in short-term traffic flow forecasts because of its powerful ability to process timing data. As the traffic flow data has different short-term characteristics and periodic characteristics, different characteristics may interact with each other. In this paper, we propose a parallel long and short memory neural network model, which is used to obtain the short-time characteristics and periodic characteristics of traffic flow, the experimental results on the real data set show that the model can achieve better prediction results; Finally, in order to evaluate the performance of our model, we compared the prediction effect between the model and the other different models, and explore the reason of proposing parallel network.

Since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central China. To investigate the epidemic characteristics of PEDV, the complete ORF3 genes of 14 PEDV field strains from central China during 2012 to 2013 were cloned, sequenced and compared with reference strains. Phylogenetic analysis based on the complete ORF3 gene showed that the PEDVs in central China and the reference strains could be divided into three groups: G1, G2, and G3. The 14 PEDV isolates were classified as G1 and showed a close relationship to some Chinese strains isolated previously in central China and differed genetically from recent isolates from southern China, Korean strains (SM98 and DB1865, 2012), the Chinese LZC strain (2007), and the vaccine strain (CV777) being used in China. Our findings suggested that the PEDVs circulating between 2012 and 2013 in central China might have evolved from earlier strains in the local region. To determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the S gene. The results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. We speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure. Together, these findings may be useful for understanding the epidemiology of PEDV and may be relevant for designing of new and more efficacious vaccines.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused one of the most economically devastating and pandemic diseases of swine. Previous studies have documented that PRRSV nonstructural protein-1α (nsp1α) was an interferon antagonist, but the mechanism by which nsp1α inhibited the interferon (IFN)-β production was unclear. Here, by site-directed mutagenesis of the predicted zinc-coordinating residues of the zinc-finger (ZF) domain of nsp1α or by deletion of the ZF domain of nsp1α, we explored whether the ZF domain was required for nsp1α to disrupt the IFN-β production. The results showed that both mutagenesis of the predicted zinc-coordinating residues of the ZF domain and deletion of the ZF domain made nsp1α lose its interferon antagonism activity. In conclusion, our present work indicated that the ZF domain of nsp1α was necessary for nsp1α to inhibit the IFN-β induction.