Pseudorabies virus (PRV), known as porcine herpesvirus type I, belongs to the α-herpesvirus subfamily of the herpesviridae family. Its virions are oval or round, with a genome of approximately 143 000 bp and a high guanine-cytosine (GC) content of 73% (Pomeranz et al., 2005). While swine are the natural host and the virus has caused significant economic loss to the pig industry, PRV can infect other mammals, causing acute infectious diseases characterized by neurological symptoms (He et al., 2019; Cheng et al., 2020). Young pigs are more susceptible to PRV and the infection in piglets is nearly 100% fatal, whereas adult pigs exhibit reproductive disorders such as abortion, stillbirths, and reduced fertility, ultimately decreasing farm productivity (Zuckermann, 2000). Due to its global impact, PRV is classified as a Class B infectious disease by the World Organization for Animal Health (WOAH).

Porcine circovirus type 2 (PCV2) is one of the main pathogens causing porcine circovirus-associated diseases (PCVAD). We recently reported the immunogenicity of the recombinant PRV with an envelope-embedded Cap protein of PCV2 (PRV-Cap) in mice. Here, we further evaluated the immunoprotective efficacy of PRV-Cap virus in pigs. Following vaccination, the PRV-Cap stimulated the production of neutralizing antibodies against PRV and PCV2, along with protected piglets from the challenge of the lethal PRV, the virulent PCV2b and PCV2d. Peripheral blood mononuclear cells analysis revealed that PRV-Cap virus effectively induced proliferation and activation of CD4 and CD8 T cells, as well as an increase in T follicular helper cells, although γδ T and B cells did not show significant differences. Compared to DMEM control piglets, the expanded CD4 and CD8 T cells exhibited an effector memory T cell phenotype, and in vitro stimulation led to PRV- and PCV2-specific IFN-γ and TNF-α secretion, peaking at 21 days post-immunization. In summary, PRV-Cap virus effectively prevents PRV, PCV2b and PCV2d challenges in piglets by simultaneously inducing both PRV- and PCV2-specific humoral and cellular immunity, indicating that PRV-Cap virus is a promising and safe candidate vaccine for combined PRV and PCV2 immunization.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

Pectinases are useful biocatalysts for pectic biomass processing and are extensively used in the food/feed, textile and papermaking industries. Two pectinase genes, a pectate lyase (SbPL1CE8) and a polygalacturonase (SbGH28GH105) were isolated from Segatella bryantii and functionally characterized. Recombinant rSbPL1CE8 was most active against polygalacturonic acid (PGA) and pectin with a 60 % degree of esterification, with kcat/Km values of 721.18 +/- 64.77 and 327.02 +/- 22.44 mL/s/mg, respectively. Truncated rSbPL1 acted as a mesophilic alkaline pectate lyase, which was highly resistant to inactivation by methanol and ethanol. The rSbPL1CE8 exclusively digested PGA and pectin into unsaturated digalacturonate (uG2), which was further converted into galacturonic acid by rSbGH28GH105. The rSbPL1CE8 was highly effective for saccharification of waste materials from Zea mays, Oryza sativa and Arachis hypogaea processing, and for ramie fiber degumming. This novel pectate lyase has great potential for application in industrial pectic biomass processing.

IntroductionVariant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion.MethodsWe reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus.ResultsAn immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells.ConclusionCollectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

Circular RNAs (circRNAs) exert diverse biological functions in different processes. However, the role of circRNAs during virus infection is mostly unknown. Herein, we explored the characteristics of host circRNAs using alphaherpesvirus pseudorabies virus (PRV) as a model. PRV infection upregulated the expression of circRNA circ29164, which does not encode a protein. RNA pulldown assays identified that circ29164 interacts with the microRNA ssc-miRNA-24-3p. Further analysis indicated that ssc-miR-24-3p targets the mRNA encoding kelch-like ECH-associated protein 1 (KEAP1), and circ29164 competitively binds to ssc-miR-24-3p to prevent it binding to Keap1. Apoptosis detection demonstrated that circ29164 or Keap1 overexpression, but not knockdown, induced caspase 3 activity and the release of cytochrome C from mitochondria, and inhibited PRV replication. Taken together, these data identified a previously undiscovered circRNA, circ29164, which inhibits PRV replication by competitively binding to ssc-24-3p to maintain KEAP1 levels.

Autophagy plays a dual role in coronavirus infection, facilitating the elimination of either proviral components (virophagy) or antiviral factors such as mitochondria (mitophagy), leading to complex mechanisms of immune evasion. Understanding the mechanisms that govern the switch between the autophagic degradation of deleterious or beneficial substrates in coronavirus infection is crucial for developing precise drug targets to treat virus-induced diseases. However, this switch remains largely unknown. Using a dual split-fluorescence assay, we identify PDPK1 as a negative regulator of innate immunity, directing the transition from virophagy to mitophagy through the phosphorylation of SQSTM1 at T138. Remarkably, a PDPK1-targeting peptide inhibits the replication of various RNA viruses by restoring innate immunity through enhanced virophagy and suppressed mitophagy, thereby protecting female mice from lethal infections. These findings underscore the detrimental role of PDPK1 in innate immunity by orchestrating the shift from virophagy to mitophagy, positioning PDPK1 as a promising pharmacological target for effectively combating a broad spectrum of virus infections. Autophagy plays a dual role in coronavirus infection. Here, the authors identify PDPK1 as a key regulator that switches between virophagy and mitophagy and demonstrate that a PDPK1-targeting peptide restored immunity and protected mice from lethal viral infections.

Pseudorabies virus (PRV), causing Aujeszky’s disease in swine, has important economic impact on the pig industry in China and even poses a threat to public health. Although this disease has been controlled by vaccination with PRV live attenuated vaccines (LAVs), the potency of PRV LAVs in inducing cellular immunity has not been well characterized. In this study, using PRV Bartha K61 strain (BK61), the most-used PRV LAVs, as a model, we re-examined the cellular immune response elicited by the BK61 in mice and pigs by multicolor flow cytometry. We found that phenotypic activation of T cells, NK cells and B cells was hardly detected after vaccination. However, antigen-specific IFN-γ-producing CD4 T cells rather than CD8 T cells were dominantly detected but at low frequency upon restimulation with live BK61 virus. These BK61-specific CD4 T cells are also able to simultaneously produce TNF-α and IL-2, showing characteristics of multifunctional T cells. However, BK61-specific CD4 T cells showed weak secondary response upon challenge with PRV DX strain. Further vaccination with PRV-infected dendritic cells (DCs) transiently increased the percentage of IFN-γ-positive CD4 and CD8 T cells but eventually restored to low frequency and did not improve the protective efficacy of BK61 against challenge, suggesting that PRV BK61 induced a relatively weak cellular immunity that could not be overcome by the DC vaccination. Similar immune responses were also observed following vaccination with another PRV LAV HD/c in mice and pigs, suggesting that this may be an intrinsic drawback of PRV LAVs in inducing cellular immunity. Our results demonstrated that PRV LAVs elicited a CD4 Th1-biased weak cellular immunity which is implicative for the development of PRV-vectored vaccine.

RIG-I-like receptors (RLRs), retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), are pattern recognition receptors through which cells initially sense pathogenic RNA and trigger interferon (IFN) signaling. Herein, we report that interferon induced protein 35 (IFI35) activates the ring finger protein 125 (RNF125)-UbcH5c-dependent degradation of RLRs and represses the recognition by RIG-I and MDA5 of viral RNA to inhibit innate immunity. Furthermore, IFI35 binds selectively to different subtypes of influenza A virus (IAV) nonstructural protein 1 (NS1) with asparagine residue207 (N207). Functionally, the NS1(N207)-IFI35 interaction restores the activity of RLRs, and IAV with NS1(non-N207) showed high pathogenicity in mice. Big data analysis showed that the 21st century pandemic IAV are almost all characterized by NS1 protein with non-N207. Collectively, our data uncovered the mechanism of IFI35 restricting the activation of RLRs and provides a new drug target comprising the NS1 protein of different IAV subtypes.

Ubiquitination is an important reversible post-translational modification. Many viruses hijack the host ubiquitin system to enhance self-replication. In the present study, we found that Avibirnavirus VP3 protein was ubiquitinated during infection and supported virus replication by ubiquitination. Mass spectrometry and mutation analysis showed that VP3 was ubiquitinated at residues K73, K135, K158, K193, and K219. Virus rescue showed that ubiquitination at sites K73, K193, and K219 on VP3 could enhance the replication abilities of infectious bursal disease virus (IBDV), and that K135 was essential for virus survival. Binding of the zinc finger domain of TRAF6 (TNF receptor associated factor 6) to VP3 mediated K11- and K33-linked ubiquitination of VP3, which promoted its nuclear accumulation to facilitate virus replication. Additionally, VP3 could inhibit TRAF6-mediated NFKB/NF-κB (nuclear factor kappa B) activation and IFNB/IFN-β (interferon beta) production to evade host innate immunity by inducing TRAF6 autophagic degradation in an SQSTM1/p62 (sequestosome 1)-dependent manner. Our findings demonstrated a macroautophagic/autophagic mechanism by which Avibirnavirus protein VP3 blocked NFKB-mediated IFNB production by targeting TRAF6 during virus infection, and provided a potential drug target for virus infection control.Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Cas9: CRISPR-associated protein 9; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; IBDV: infectious bursal disease virus; IF: indirect immunofluorescence; IFNB/IFN-β: interferon beta; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MS: mass spectrometry; NFKB/NF-κB: nuclear factor kappa B; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody; PRRs: pattern recognition receptors; RNF125: ring finger protein 125; RNF135/Riplet: ring finger protein 135; SQSTM1/p62: sequestosome 1; TAX1BP1: tax1 binding protein1; TCID50: 50% tissue culture infective dose; TRAF3: TNF receptor associated factor 3; TRAF6: TNF receptor associated factor 6; TRIM25: tripartite motif containing 25; Ub: ubiquitin; Wort: wortmannin; WT: wild type.

The highly virulent and antigenic variant of Pseudorabies virus (PRV) that emerged from classical Bartha-K61-vaccinated pig herds has caused substantial economic losses to the swine industry in China since 2011. A safe and more effective vaccine is most desirable. In this study, a gE/TK gene-deficient PRV, namely, HD/c, was constructed based on a PRV type II DX strain isolated from a commercial vaccine-immunized farm and the HD/c-based inactivated vaccine was formulated and evaluated for its safety, immunogenicity, and protective efficacy in mice and piglets. The resulting PRV HD/c strain has a similar growth curve to the parental DX strain. After vaccination, the inactivated HD/c vaccine did not cause any visible gross pathological or histopathological changes in the tissues of mice and piglets and provided rapid and potent protection against the challenge of the classical and variant PRVs at day 21 post-vaccination in mice. A single immunization of 10(8.5)TCID(50) inactivated PRV HD/c strain-elicited robust immunity with high titer of neutralizing antibody and provided complete protection from the lethal challenge of PRV DX strain in piglets. These results indicated that the inactivated PRV HD/c vaccine with the deletion of gE/TK genes was a safe and effective PRV vaccine candidate for the control of PRV.

为建立一种同时鉴别致病性猪圆环病毒(PCV)不同型的检测方法,本研究对PCV2、PCV3、PCV4各自Rep基因进行特异性保守序列分析,建立了致病性PCV的TaqMan单重和三重荧光定量PCR检测方法.该方法能特异性区分PCV2、PCV3和PCV4,与PCV1及PRV等临床上常见的猪传染性病原均无交叉反应.建立的单重及三重PCR方法的最低检测限度均为101copies/μL,组内及组间的变异系数均小于1％,表明重复性良好.对354份猪临床组织病料进行三重荧光定量PCR检测,结果显示,PCV2、PCV3和PCV4的阳性率分别为66.7％、26.8％和0;PCV2和PCV3共感染率为15.8％.结果表明,本研究建立的三重荧光定量PCR方法能准确鉴别致病性PCV,灵敏性优于普通PCR法,检测过程更高效、便捷,为致病性PCV的临床诊断及流行病学调查提供了检测工具.

Ischemic cardiomyopathy (ICM) caused by coronary artery disease always leads to myocardial infarction and heart failure. Identification of novel transcriptional regulators in ICM is an effective method to establish new diagnostic and therapeutic strategies. In this study, we used two RNA-seq datasets and one microarray dataset from different studies, including 25 ICM and 21 non-failing control (NF) samples of human left ventricle tissues for further analysis. In total, 208 differentially expressed genes (DEGs) were found by combining two RNA-seq datasets with batch effects removed. GO and KEGG analyses of DEGs indicated that the response to wounding, positive regulation of smooth muscle contraction, chromatin, PI3K-Akt signaling pathway, and transporters pathways are involved in ICM. Simple Enrichment Analysis found that NFIC-binding motifs are enriched in promoter regions of downregulated genes. The Gene Importance Calculator further proved that NFIC is vital. NFIC and its downstream genes were verified in the validating microarray dataset. Meanwhile, in rat cardiomyocyte cell line H9C2 cells, two genes (Tspan1 and Hopx) were confirmed, which decreased significantly along with knocking down Nfic expression. In conclusion, NFIC participates in the ICM process by regulating TSPAN1 and HOPX. NFIC and its downstream genes may be marker genes and potential diagnostic and therapeutic targets for ICM.

伪狂犬病病毒(Pseudorabies virus,PRV)感染猪(Sus scrofa)可引起烈性接触性传染病,即伪狂犬病(Pseudorabies,PR),临床上主要通过疫苗免疫进行防控.PRV变异株(PRVⅡ型)的流行使传统疫苗失去有效保护.近年来出现PRV跨宿主感染人(Homo sapiens)的案例,更使PRV成为全球公共卫生威胁,但其感染致病机制仍不完全清楚.为从分子水平上全面了解PRV感染宿主细胞引起的变化,本研究以PRVⅡ型毒株PRV-DX感染猪肾细胞PK-15,通过高通量测序获得PRV感染后全部的细胞差异转录组数据,并对差异表达基因进行功能富集.结果表明,PRV-DX感染PK-15可引起3595条基因表达上调、3604条基因表达下调.通过qPCR验证随机选择的12条差异表达基因,其变化趋势与转录组数据一致.GO注释和KEGG信号通路富集将差异表达基因聚焦在代谢、基因表达、免疫等信号通路.本研究为深入解析PRV感染的致病机制提供了重要基础数据及研究方向.

The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.

Entry of circovirus into the host cell nucleus is essential for viral replication during the early stage of infection. However, the mechanisms by which nucleolar shuttle proteins are used during viral replication is still not well understood. Here, we report a previously unidentified nucleolar localization signal in circovirus capsid protein (Cap), and that circovirus hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate its replication. Colocalization analysis showed that NPM1 translocates from the nucleolus to the nucleoplasm and cytoplasm during viral infection. Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that Cap interacts directly with NPM1. Binding domain mapping showed that the arginine-rich N-terminal motif 1MTYPRRRYRRRRHRPRSHLG20 of Cap, and residue serine-48 of the N-terminal oligomerization domain of NPM1, are essential for the interaction. Virus rescue experiments showed that all arginine to alanine substitution in the N-terminal arginine-rich motif of Cap resulted in diminished viral replication. Knockdown of NPM1 and substitution of serine-48 in NPM1 to glutamic acid also decreased viral replication. In addition, binding assays showed that the arginine-rich motif of Cap is a nucleolar localization signal. Taken together, our findings demonstrate that circovirus protein Cap is a nucleolus-located, and regulates viral replication by directly binding to NPM1.

Virus-host interaction is a tug of war between pathogenesis and immunity, followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation. Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies. To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein, we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry (LC-MS) approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney (PK-15) cells. Further, a protein-protein interactions (PPIs) network was plotted, and the PCV2 Cap-interacting host proteins were potentially involved in protein binding, DNA transcription, metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment. Verification in vitro assay demonstrated that eight cellular proteins, namely heterogeneous nuclear ribonucleoprotein C, nucleophosmin-1, DEAD-box RNA helicase 21, importin β3, eukaryotic translation initiation factor 4A2, snail family transcriptional repressor 2, MX dynamin like GTPase 2, and intermediate chain 1 interacted with PCV2 Cap. Thus, this work effectively provides useful protein-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and pathogenesis.

Porcine delta coronavirus (PDCoV) is a novel emerging enterocytetropic virus causing diarrhea, vomiting, dehydration, and mortality in suckling piglets. Long non-coding RNAs (lncRNAs) are known to be important regulators during virus infection. Here, we describe a comprehensive transcriptome profile of lncRNA in PDCoV-infected swine testicular (ST) cells. In total, 1,308 annotated and 1,190 novel lncRNA candidate sequences were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these lncRNAs might be involved in numerous biological processes. Clustering analysis of differentially expressed lncRNAs showed that 454 annotated and 376 novel lncRNAs were regulated after PDCoV infection. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to metabolism and TNF signaling. Our study provided comprehensive information about lncRNAs that would be a useful resource for studying the pathogenesis of and designing antiviral therapy for PDCoV infection.

PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. In vivo data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis.Abbreviations: AKT1: AKT serine/threonine kinase 1; ATG14: autophagy related 14; Co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; hpi: hours post-infection; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; pAb: polyclonal antibody; PDPK1: 3-phosphoinositide dependent protein kinase 1; PI3K: phosphoinositide 3-kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic, subunit type 3; RPS6KB1: ribosomal protein S6 kinase B1; SGK: serum/glucocorticoid regulated kinase; SQSTM1: sequestosome 1; SUMO: small ubiquitin like modifier; UBE2I/UBC9: ubiquitin conjugating enzyme E2 I; UVRAG: UV radiation resistance associated.