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Multifunctional CD4 + T helper 1 (Th1) cells, producing IFN-γ, TNF-α and IL-2, define a correlate of vaccine-mediated protection against intracellular infection. In our previous study, we found that CVC1302 in oil formulation promoted the differentiation of IFN-γ + /TNF-α + /IL-2 + Th1 cells. In order to extend the application of CVC1302 in oil formulation, this study aimed to elucidate the mechanism of action in improving the Th1 immune response. Considering the signals required for the differentiation of CD4 + T cells to Th1 cells, we detected the distribution of innate immune cells and the model antigen OVA-FITC in lymph node (LN), as well as the quantity of cytokines produced by the innate immune cells. The results of these experiments show that, cDC2 and OVA-FITC localized to interfollicular region (IFR) of the draining lymph nodes, inflammatory monocytes localized to both IFR and T cell zone, which mainly infiltrate from the blood. In this inflammatory niche within LN, CD4 + T cells were attracted into IFR by CXCL10, secreted by inflammatory monocytes, then activated by cDC2, secreting IL-12. Above all, CVC1302 in oil formulation, on the one hand, targeted antigen and inflammatory monocytes into the LN IFR in order to attract CD4 + T cells, on the other hand, targeted cDC2 to produce IL-12 in order to promote optimal Th1 differentiation. The new finding will provide a blueprint for application of immunopotentiators in optimal formulations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could appear to have influenced the work reported in this paper., (Copyright © 2024 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication., Competing Interests: The authors declare no conflict of interest.

Background: Porcine deltacoronavirus (PDCoV), a novel swine enteropathogenic coronavirus, challenges the global swine industry. Currently, there are no approaches preventing swine from PDCoV infection., Methods: A new PDCoV strain named JS2211 was isolated. Next, the dimer receptor binding domain of PDCoV spike protein (RBD-dimer) was expressed using the prokaryotic expression system, and a novel nanoparticle containing RBD-dimer and ferritin (SC-Fe) was constructed using the SpyTag/SpyCatcher system. Finally, the immunoprotection of RBD-Fe nanoparticles was evaluated in mice., Results: The novel PDCoV strain was located in the clade of the late Chinese isolate strains and close to the United States strains. The RBD-Fe nanoparticles were successfully established. Immune responses of the homologous prime-boost regime showed that RBD-Fe nanoparticles efficiently elicited specific humoral and cellular immune responses in mice. Notably, high level PDCoV RBD-specific IgG and neutralizing antibody (NA) could be detected, and the histopathological results showed that PDCoV infection was dramatically reduced in mice immunized with RBD-Fe nanoparticles., Conclusion: This study effectively developed a candidate nanoparticle with receptor binding domain of PDCoV spike protein that offers protection against PDCoV infection in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Song, Deng, Wang, Zhang, Liu, Tang, Liu, Zhou, Tong, Li and Yu.)

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Importance: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication., Competing Interests: The authors declare no conflict of interest.

African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as 542 VTAHGINLIDKF 553 , 568 GNAIKTP 574 , and 584 FALKPREEY 592 . All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, 568 GNAIKTP 574 and 584 FALKPREEY 592 , were internal to the p72 trimer, while the epitope 542 VTAHGINLIDKF 553 was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1β, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v., (© 2023. The Author(s).)

This study found a higher percentage of CD8 + T cells in piglets immunized with a CVC1302-adjuvanted inactivated foot-and-mouth disease virus (FMDV) vaccine. We wondered whether the CVC1302-adjuvanted inactivated FMDV vaccine promoted cellular immunity by promoting the antigen cross-presentation efficiency of ovalbumin (OVA) through dendritic cells (DCs), mainly via cytosolic pathways. This was demonstrated by the enhanced levels of lysosomal escape of OVA in the DCs loaded with OVA and CVC1302. The higher levels of ROS and significantly enhanced elevated lysosomal pH levels in the DCs facilitated the lysosomal escape of OVA. Significantly enhanced CTL activity levels was observed in the mice immunized with OVA-CVC1302. Overall, CVC1302 increased the cross-presentation of exogenous antigens and the cross-priming of CD8 + T cells by alkalizing the lysosomal pH and facilitating the lysosomal escape of antigens. These studies shed new light on the development of immunopotentiators to improve cellular immunity induced by vaccines.

As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope ( 8 LLSHNLSTREGIK 20 ) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Porcine reproductive and respiratory syndrome virus (PRRSV) has seriously affected the viability of swine industries worldwide, and effective measures to control PRRSV are urgently required. Understanding the mechanisms of action of antiviral proteins is crucial for developing antiviral strategies. Interferon-induced bone marrow stromal cell antigen 2 (BST2) can inhibit the replication of various viruses via different pathways. However, little is known about the effects of BST2 on PRRSV. Therefore, this study aimed to evaluate whether the interferon-induced BST2 can inhibit PRRSV replication. We used western blotting and RT-qPCR techniques to analyze the effect of BST2 overexpression and knockdown on PRRSV replication. Overexpression of BST2 inhibited the replication of PRRSV, whereas knockdown of BST2 by small interfering RNA promoted PRRSV replication. Additionally, the expression of BST2 was upregulated during the early phase of PRRSV infection in porcine alveolar macrophages. Analysis of PRRSV proteins showed that BST2 restricted the expression of several non-structural viral proteins. BST2 downregulated the expression of Nsp12 through a proteasome-dependent pathway and downregulated the expression and transcription of E protein. These findings demonstrate the potential of BST2 as a critical regulator of PRRSV replication., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea (PED) is an acute, highly infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which seriously endangers the healthy development of the pig industry. PEDV N protein is the most abundant viral structural protein, which can be combined with viral genomic RNA to form ribonucleoprotein complexes, thereby participating in the transcription and replication of the virus. However, how PEDV hijacks the host transcription translation system to promote viral proliferation remains unclear. In this study, we found that there is an interaction between PEDV N, polyadenylate-binding protein cytoplasmic 1 (PABPC1) and eukaryotic initiation factor 4F (eIF4F) proteins through coimmunoprecipitation, GST pulldown and fluorescence microscopy experiments. PABPC1 could bind to the poly(A) tail of the mRNA, and eIF4F could bind to the 5' end cap structure of the mRNA, so the interaction of PABPC1 and eIF4F could facilitate mRNA forming a circular shape to promote translation to the proteins. To further explore the effect of N protein capture protein translation element PABPC1 and eIF4F on PEDV replication, we overexpressed PABPC1, eIF4F (containing eIF4A, eIF4E and eIF4G) separately on Vero cells and LLC-PK1 cells, and we found that the PABPC1 and eIF4F protein could promote PEDV replication. Taken together, our data suggested that PEDV N protein promoted cyclization of viral mRNA carried by N protein through binding with PABPC1 and eIF4F proteins, thus promoting viral transcription and facilitating viral replication., Competing Interests: Declaration of Competing Interest The authors declare that they have no potential conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. The protein degradation role of autophagy has been widely used to control viral infection at multiple levels. In the ongoing evolutionary arms race, viruses have developed various ways to hijack and subvert autophagy in favor of its replication. It is still unclear exactly how autophagy affects or inhibits viruses. In this study, we have found a novel host restriction factor, HNRNPA1, that could inhibit PEDV replication by degrading viral nucleocapsid (N) protein. The restriction factor activates the HNRNPA1-MARCHF8/MARCH8-CALCOCO2/NDP52-autophagosome pathway with the help of transcription factor EGR1 targeting the HNRNPA1 promoter. HNRNPA1 could also promote the expression of IFN to facilitate the host antiviral defense response for antagonizing PEDV infection through RIGI protein interaction. During viral replication, we found that PEDV can, in contrast, degrade the host antiviral proteins HNRNPA1 and others (FUBP3, HNRNPK, PTBP1, and TARDBP) through its N protein through the autophagy pathway. These results reveal the dual function of selective autophagy in PEDV N and host proteins, which could promote the ubiquitination of viral particles and host antiviral proteins and degradation both of the proteins to regulate the relationship between virus infection and host innate immunity. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; Baf A1: bafilomycin A 1 ; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PEDV: porcine epidemic diarrhea virus; siRNA: small interfering RNA; TCID 50 : 50% tissue culture infectious doses.

African swine fever is a highly lethal contagious disease of pigs for which there is no vaccine. Its causative agent African swine fever virus (ASFV) is a highly complex enveloped DNA virus encoding more than 150 open reading frames. The antigenicity of ASFV is still unclear at present. In this study, 35 proteins of ASFV were expressed by Escherichia coli , and ELISA was developed for the detection of antibodies against these proteins. p30, p54, and p22 were presented as the major antigens of ASFV, positively reacting with all five clinical ASFV-positive pig sera, and 10 pig sera experimentally infected by ASFV. Five proteins (pB475L, pC129R, pE199L, pE184L, and pK145R) reacted well with ASFV-positive sera. The p30 induced a rapid and strong antibody immune response during ASFV infection. These results will promote the development of subunit vaccines and serum diagnostic methods against ASFV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Hu, Li, Wang, Meng, Chen, Wei, Tong, Kong, Yu, Yu, Shan, Tong, Wang and Zheng.)

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) are two important pathogens, which cause serious impact on swine industry worldwide. In our previous research, rPRRSV-E2, the recombinant PRRSV expressing CSFV E2 protein, could provide sufficient protection against the lethal challenge of highly pathogenic PRRSV and CSFV, and could maintained genetically stable in vitro . Here, to evaluate the virulence reversion potential risk, rPRRSV-E2 had been continuously passaged in vivo , the stability of E2 expression and virulence of the passage viruses were analyzed. The results showed that no clinical symptoms or pathological changes could be found in the inoculated groups, and there were no significant differences of viraemia among the test groups. Sequencing and IFA analysis showed that the coding gene of exogenous CSFV E2 protein existed in the passaged viruses without any sequence mutations, deletions or insertions, and could expressed steadily. It could be concluded that the foreign CSFV E2 gene in the genome of rPRRSV-E2 could be maintained genetically stable in vivo , and rPRRSV-E2 strain had relatively low level of potential risk for virulence reversion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiang, Gao, Li, Zhou, Tong, Yu, Zhang, Zhao, Zhu, Liu, Li and Tong.)

Influenza A virus (IAV) exploits host metabolic pathways to support its replication. To improve the understanding of lipid metabolic changes that could occur upon IAV infection, a comprehensive analysis of lipid metabolites in A549 cells infected with the avian H9N2 virus at the different time points was performed. It was found that H9N2 infection could largely promote the level of lipid metabolites. Further, these metabolites were mainly included in glycerophospholipids (GPs), sphingolipids (SPs), glycerolipids (GLs), fatty acids (FAs), sterollipids (STs), triglycerides (TGs), and prenol lipids (PRs). Specifically, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these metabolites were mainly associated with the glycerphospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and autophagy. Furthermore, it is interesting to note that these metabolites, including FFA(19:1), PE(P-17:0_20:3), PE(P-18:1_20:2), LPC(14:0/0:0), PE(O-18:0_20:3), and MGDG(16:0_18:1), are upregulated and shared in the top 10 at 12 h, 24 h, 36 h, and 48 h after H9N2 infection, indicative of the possibility of acting as biomarkers for the diagnosis in the lung infected with influenza virus. These pathways and altered metabolites could provide new understandings about biological characteristics and pathogenicity of influenza virus and have the potential to serve as biomarkers for influenza., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope ( 2 VEPREQFFQDLLSAV 16 ) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Qiao, Liu, Zhou, Tong, Dong, Liu, Jiang, Guo, Zheng, Zhao, Tong, Li and Gao.)

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope ( 3 TETSPLLSH 11 ) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF., Competing Interests: The authors declare that they have no conflict of interest associated with this study.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

Giardia duodenalis is an important intestinal protozoan parasite, infections of which are frequently seen in cattle and cause intermittent diarrhea and weight loss in young animals around the world. The release of neutrophil extracellular traps (NETs) is an effector mechanism of neutrophils to fight against invading pathogens including parasites. In this present study, we aimed to investigate the effect of Giardia duodenalis trophozoites on bovine NETs formation, and to further examine its basic characteristics and molecular mechanisms. Scanning electron microscopy analyses displayed that Giardia duodenalis trophozoites exposure triggered NET-like filamentary structures released by bovine polymorphonuclear leukocytes (PMNs), and many trophozoites were entrapped within these structures. Immunofluorescence analyses illustrated that these structures were mainly composed of DNA, histones, Myeloperoxidase (MPO) and neutrophil elastase (NE), which confirmed the classical characteristics of NETs. NETs quantification showed that Giardia duodenalis trophozoites significantly increased NETs formation, and it is in a dose-dependent manner from 4:1-1:2 ratio of PMN: trophozoites. Furthermore, pharmacological inhibitory experiment indicated that P2X1 receptor and PAD4 were essential for Giardia duodenalis trophozoites-triggered NETs formation. Additionally, LC3B-based immunostaining analyses revealed that autophagy and NETs formation occurred simultaneously in Giardia duodenalis trophozoites-exposed bovine PMN, imply that autophagy may play a key role in Giardia duodenalis trophozoites-triggered bovine NETs. In summary, these findings suggest that NETs formation might have a crucial role in innate host defense against Giardia duodenalis trophozoites. Hence, we call for future molecular investigations not only on Giardia duodenalis trophozoites-triggered NETs but also on its potential role in giardiasis-related pathology in vivo., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) is an important viral disease, causing significant economic losses to the swine industry worldwide. Atypical cases caused by highly pathogenic PRRS virus (HP-PRRSV) emerged in 2006 in China. The vaccine strain HuN4-F112 has been developed from the wild-type HP-PRRSV HuN4 through repeated passages on MARC-145 cells. However, the mechanisms of attenuation have yet to be defined. Previous studies have shown that the vaccine strain HuN4-F112 could not effectively replicate in porcine alveolar macrophages (PAMs). In the present study, a series of chimeric and mutant PRRSVs were constructed to investigate regions associated with the virus attenuation. Firstly, the corresponding genome regions (ORF1a, ORF1b and ORFs 2-7) were exchanged between two infectious clones of HuN4 and HuN4-F112, and then the influence of small regions in ORF1a and ORF2-7 was evaluated, then influence of specific amino acids on NSP2 was tested. NSP2 was determined to be the key gene that regulated infection efficiency on PAMs, and amino acids at 893 and 979 of NSP2 were the key amino acids. The results of in vivo study indicated that NSP2 was not only important for infection efficiency in vitro, but also influenced the virulence, which was indicated by the results of survival rate, temperature, viremia, lung score and tissue score.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteric coronavirus currently spreading in several nations and inflicting substantial financial damages on the swine industry. The currently available coronavirus vaccines do not provide adequate protection against the newly emerging viral strains. It is essential to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. This study shows that heterogeneous nuclear ribonucleoprotein K (hnRNP K), the host protein determined by the transcription factor KLF15, inhibits the replication of PEDV by degrading the nucleocapsid (N) protein of PEDV in accordance with selective autophagy. hnRNP K was found to be capable of recruiting the E3 ubiquitin ligase, MARCH8, aiming to ubiquitinate N protein. Then, it was found that the ubiquitinated N protein could be delivered into autolysosomes for degradation by the cargo receptor NDP52, thereby inhibiting PEDV proliferation. Moreover, based on the enhanced MyD88 expression, we found that hnRNP K activated the interferon 1 (IFN-1) signaling pathway. Overall, the data obtained revealed a new mechanism of hnRNP K-mediated virus restriction wherein hnRNP K suppressed PEDV replication by degradation of viral N protein using the autophagic degradation pathway and by induction of IFN-1 production based on upregulation of MyD88 expression. IMPORTANCE The spread of the highly virulent PEDV in many countries is still leading to several epidemic and endemic outbreaks. To elucidate effective antiviral mechanisms, it is important to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. In the work, we detected hnRNP K as a new host restriction factor which can hinder PEDV replication through degrading the nucleocapsid protein based on E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. In addition, via the upregulation of MyD88 expression, hnRNP K could also activate the interferon (IFN) signaling pathway. This study describes a previously unknown antiviral function of hnRNP K and offers a new vision toward host antiviral factors that regulate innate immune response as well as a protein degradation pathway against PEDV infection.

KLF16, a member of KLFs (Krüppel-like factors), contributes to the progression of a variety of cancer types. There is, however, still uncertain regarding the role of KLF16 in viral replication and the signaling mechanism of type I IFN. It was discovered that KLF16 inhibited the replication of porcine epidemic diarrhea virus (PEDV) through the type I IFN signaling pathway. Besides, it can also be found that the expression of KLF16 was down-regulated after PEDV infection of LLC-PK1 cells. Furthermore, overexpression of KLF16 inhibited the replication of PEDV in Vero cells as well as LLC-PK1 cells, whereas the replication of PEDV was promoted by the knockdown of KLF16. KLF16 up-regulated the expression of interferons (IFNs) via the TRAF6-pTBK1-pIRF3 pathway with the aim of promoting the host antiviral innate immune response. In addition, the obtained findings proved that KLF16 plays a novel role in antiviral action, thereby offering novel possibilities for preventing and controlling PEDV., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Influenza a virus exploits host machinery to benefit its replication in host cells. Knowledge of host factors reveals the complicated interaction and provides potential targets for antiviral treatment. Here, instead of the traditional proteomic analysis, we employed a 4D label free proteomic method to identify cellular factors in A549 cells treated with avian H9N2 virus. We observed that 425 proteins were upregulated and 502 proteins were downregulated. Western blotting and quantitative real-time PCR results showed that the zinc-finger CCHC-type containing protein 3 (ZCCHC3) levels were markedly induced by H9N2 infection. Transient expression assay showed that ZCCHC3 expression decreased NP protein levels and viral titers, whereas knockdown of ZCCHC3 enhanced viral growth. Specifically, ZCCHC3 promoted the expression of IFN-β, leading to the increased transcription of IFN downstream antiviral factors. Surprisingly, viral NS1 protein was able to antagonize the antiviral effect of ZCCHC3 by downregulating IFN-β. Eventually, we observed that chicken finger CCCH-type containing protein 3, named ZC3H3, could also suppress the replication of H9N2 virus and the coronavirus-infectious bronchitis virus (IBV) in DF-1 cells. Together, our results showed the cellular proteomic response to H9N2 infection and identified ZCCHC3 as a novel antiviral factor against H9N2 infection, contributing to the understanding of host-virus interaction., (Copyright © 2022. Published by Elsevier B.V.)

Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

ASF has caused huge economic losses to China's swine industry. As clinical symptoms of ASF were difficult to distinguish from classical swine fever and porcine reproductive and respiratory syndrome (PRRS), rapid and effective differential diagnosis of ASFV seems very important to control the spread of the disease. In this study, the ASFV E248R gene was selected to be the target for establishing a real-time PCR method. TaqMan real-time PCR for the detection of ASFV E248R gene did not cross-react with other porcine viruses that could cause similar symptoms. The results of the repeatability test showed that the coefficients of variation between and within groups were lower than 1.977%. This method can be used for the rapid detection and early diagnosis of ASF. Meanwhile, the recombinant PRRS virus (PRRSV)-expressing E248R gene of ASFV was constructed and rescued by using the reverse genetic platform of live-attenuated PRRSV vaccine. The ASFV E248R gene can be detected by using this real-time PCR detection method, confirming that the ASFV E248R gene could be stably amplified in PRRSV genome at least 20 cell passages. The detection methods can be used for the efficient detection of the ASFV infection and recombinant PRRSV live vector virus-expressing ASFV antigen protein.

Senecavirus A (SVA), an important emerging porcine virus, has outbreaks in different regions and countries each year, becoming a virus with global prevalence. SVA infection has been reported to induce macroautophagy/autophagy; however, the molecular mechanisms of autophagy induction and the effect of SVA on autophagy remain unknown. This study showed that SVA infection induced the autophagy process in the early stage of SVA infection, and the rapamycin-induced autophagy inhibited SVA replication by degrading virus 3 C protein. To counteract this, SVA utilized 2AB protein inhibiting the autophagy process from promoting viral replication in the late stage of SVA infection. Further study showed that SVA 2AB protein interacted with MARCHF8/MARCH8 and LC3 to degrade the latter and inhibit the autophagy process. In addition, we found that MARCHF8 was a positive regulator of type I IFN (IFN-I) signaling. During the autophagy process, the SVA 2AB protein targeted MARCHF8 and MAVS forming a large complex for degradation to deactivate IFN-I signaling. Together, our study reveals the molecular mechanisms of selective autophagy in the host against viruses and reveals potential viral strategies to evade the autophagic process and IFN-I signaling for successful pathogenesis. Abbreviations: Baf A 1 : bafilomycin A 1 ; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; hpi: hours post-infection; IFN: interferon; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane associated ring-CH-type finger 8; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; Rapa: rapamycin; RT: room temperature; siRNA: small interfering RNA; SVA: Senecavirus A; TCID 50 : 50% tissue culture infectious doses.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and dehydration in pigs and leads to great economic losses in the commercial swine industry. However, the underlying molecular mechanisms of host response to viral infection remain unclear. In the present study, we investigated a novel mechanism by which RALY, a member of the heterogeneous nuclear ribonucleoprotein family, significantly promotes the degradation of the PEDV nucleocapsid (N) protein to inhibit viral replication. Furthermore, we identified an interaction between RALY and the E3 ubiquitin ligase MARCH8 (membrane-associated RING-CH 8), as well as the cargo receptor NDP52 (nuclear dot protein 52 kDa), suggesting that RALY could suppress PEDV replication by degrading the viral N protein through a RALY-MARCH8-NDP52-autophagosome pathway. Collectively, these results suggest a preventive role of RALY against PEDV infection via the autophagy pathway and open up the possibility of inducing RALY in vivo as an effective prophylactic and preventive treatment for PEDV infection., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Interferons (IFNs) including type I/III IFNs are the major components of the host innate immune response against porcine epidemic diarrhea virus (PEDV) infection, and several viral proteins have been identified to antagonize type I/III IFNs productions through diverse strategies. However, the modulation of PEDV infection upon the activation of the host's innate immune response has not been fully characterized. In this study, we observed that various IFN-stimulated genes (ISGs) were upregulated significantly in a time- and dose-dependent manner in LLC-PK1 cells infected with the PEDV G2 strain FJzz1. The transcriptions of IRF9 and STAT1 were increased markedly in the late stage of FJzz1 infection and the promotion of the phosphorylation and nuclear translocation of STAT1, implicating the activation of the JAK-STAT signaling pathway during FJzz1 infection. In addition, abundant type I/III IFNs were produced after FJzz1 infection. However, type I/III IFNs and ISGs decreased greatly in FJzz1-infected LLC-PK1 cells following the silencing of the RIG-I-like receptors (RLRs), including RIG-I and MDA5, and the Toll-like receptors (TLRs) adaptors, MyD88 and TRIF. Altogether, FJzz1 infection induces the production of type-I/III IFNs in LLC-PK1 cells, in which RLRs and TLRs signaling pathways are involved, followed by the activation of the JAK-STAT signaling cascade, triggering the production of numerous ISGs to exert antiviral effects of innate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Zhu, Yu, Liu, Lao, Yu, Gao, Jiang, Liu, Tong, Liu, Tong and Zhou.)

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), which is highly homologous to the NADC30 strain isolated in the United States. The NADC30-like PRRSV was first reported in 2014 in China, where it spread and gradually caused an epidemic. Currently, growing research has shown that NADC30-like strains have greater propensity to recombine with other PRRSV strains, particularly the PPRSV vaccine virus used clinically, making the prevention and control of PRRSV highly complex. To carry out an in-depth molecular biology and virulence analysis, a full-length infectious clone of the NADC30-like strain was successfully constructed and rescued by reverse genetic manipulation. The rescued virus, rZJqz, was indistinguishable from its parental virus, ZJqz21, based on virological characteristics. Further animal experiments demonstrated that rZJqz retained similar pathogenicity and induced the typical clinical symptoms and viral shedding observed in the ZJqz21 challenge model. Together, these results provide a useful tool for further study of the biological characteristics and pathogenicity of NADC30-like strains. Moreover, these findings also provide a solid foundation for studying the recombination of different PRRSVs and developing new and effective universal vaccines in the future., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) is the globally distributed alphacoronavirus that can cause lethal watery diarrhea in piglets, causing substantial economic damage. However, the current commercial vaccines cannot effectively the existing diseases. Thus, it is of great necessity to identify the host antiviral factors and the mechanism by which the host immune system responds against PEDV infection required to be explored. The current work demonstrated that the host protein, the far upstream element-binding protein 3 (FUBP3), could be controlled by the transcription factor TCFL5, which could suppress PEDV replication through targeting and degrading the nucleocapsid (N) protein of the virus based on selective autophagy. For the ubiquitination of the N protein, FUBP3 was found to recruit the E3 ubiquitin ligase MARCH8/MARCHF8, which was then identified, transported to, and degraded in autolysosomes via NDP52/CALCOCO2 (cargo receptors), resulting in impaired viral proliferation. Additionally, FUBP3 was found to positively regulate type-I interferon (IFN-I) signaling and activate the IFN-I signaling pathway by interacting and increasing the expression of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3). Collectively, this study showed a novel mechanism of FUBP3-mediated virus restriction, where FUBP3 was found to degrade the viral N protein and induce IFN-I production, aiming to hinder the replication of PEDV. IMPORTANCE PEDV refers to the alphacoronavirus that is found globally and has re-emerged recently, causing severe financial losses. In PEDV infection, the host activates various host restriction factors to maintain innate antiviral responses to suppress virus replication. Here, FUBP3 was detected as a new host restriction factor. FUBP3 was found to suppress PEDV replication via the degradation of the PEDV-encoded nucleocapsid (N) protein via E3 ubiquitin ligase MARCH8 as well as the cargo receptor NDP52/CALCOCO2. Additionally, FUBP3 upregulated the IFN-I signaling pathway by interacting with and increasing tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) expression. This study further demonstrated that another layer of complexity could be added to the selective autophagy and innate immune response against PEDV infection are complicated.

Zinc finger CCHC-type containing protein 3 (ZCCHC3) acts as an antiviral factor that interacts with RIG-I and cGAS to modulate innate signaling against viral infections. Here, we investigated the role of porcine ZCCHC3 during pseudorabies virus (PRV) proliferation. We found that porcine ZCCHC3 plays an inhibitory role in the proliferation of PRV by regulating cellular innate immune responses. Further, overexpression of ZCCHC3 inhibited gB protein levels and viral titers, whereas knockdown of ZCCHC3 promoted viral growth. ZCCHC3 overexpression increased IFN-β expression to upregulate downstream gene expression, thus leading to the suppression of viral replication. However, PRV infection reduced the endogenous expression of ZCCHC3 in permissive cells. Importantly, PRV-encoded UL13 and UL24 proteins were identified to inhibit the expression of ZCCHC3, thus antagonizing its antiviral effect. Collectively, our data underscore the important role of ZCCHC3 against PRV infection and promote understandings of viral proteins in PRV pathogenesis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In global infection and serious morbidity and mortality, porcine epidemic diarrhea virus (PEDV) has been regarded as a dreadful porcine pathogen, but the existing commercial vaccines are not enough to fully protect against the epidemic strains. Therefore, it is of great necessity to feature the PEDV-host interaction and develop efficient countermeasures against viral infection. As an RNA/DNA protein, the trans -active response DNA binding protein (TARDBP) plays a variety of functions in generating and processing RNA, including transcription, splicing, transport, and mRNA stability, which have been reported to regulate viral replication. The current work aimed to detect whether and how TARDBP influences PEDV replication. Our data demonstrated that PEDV replication was significantly suppressed by TARDBP, regulated by KLF16, which targeted its promoter. We observed that through the proteasomal and autophagic degradation pathway, TARDBP inhibited PEDV replication via the binding as well as degradation of PEDV-encoded nucleocapsid (N) protein. Moreover, we found that TARDBP promoted autophagic degradation of N protein via interacting with MARCHF8, an E3 ubiquitin ligase, as well as NDP52, a cargo receptor. We also showed that TARDBP promoted host antiviral innate immune response by inducing interferon (IFN) expression through the MyD88-TRAF3-IRF3 pathway during PEDV infection. In conclusion, these data revealed a new antiviral role of TARDBP, effectively suppressing PEDV replication through degrading virus N protein via the proteasomal and autophagic degradation pathway and activating type I IFN signaling via upregulating the expression of MyD88. IMPORTANCE PEDV refers to the highly contagious enteric coronavirus that has quickly spread globally and generated substantial financial damage to the global swine industry. During virus infection, the host regulates the innate immunity and autophagy process to inhibit virus infection. However, the virus has evolved plenty of strategies with the purpose of limiting IFN-I production and autophagy processes. Here, we identified that TARDBP expression was downregulated via the transcription factor KLF16 during PEDV infection. TARDBP could inhibit PEDV replication through the combination as well as degradation of PEDV-encoded nucleocapsid (N) protein via proteasomal and autophagic degradation pathways and promoted host antiviral innate immune response by inducing IFN expression through the MyD88-TRAF3-IRF3 pathway. In sum, our data identify a novel antiviral function of TARDBP and provide a better grasp of the innate immune response and protein degradation pathway against PEDV infection.

Background: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife., Results: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident)., Conclusions: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract., (© 2022. The Author(s).)

A reliable and reproducible model in vitro for swine enteric coronaviruses infection would be intestinal models that support virus replication and can be long-term cultured and manipulated experimentally. Here, we designed a robust long-term culture system for porcine intestinal organoids from the intestinal crypt or single LGR5 + stem cell by combining previously defined insights into the growth requirements of the intestinal epithelium of humans. We showed that long-term cultured swine intestinal organoids were expanded in vitro for more than 6 months and maintained the potential to differentiate into different types of cells. These organoids were successfully infected with porcine enteric coronavirus, including porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), and were capable of supporting virus replication and progeny release. RNA-seq analysis showed robust induction of transcripts associated with antiviral signaling in response to enteric coronavirus infection, including hundreds of interferon-stimulated genes and cytokines. Moreover, gene set enrichment analysis indicated that PEDV infection could suppress the immune response in organoids. This 3D intestinal organoid model offers a long-term, renewable resource for investigating porcine intestinal infections with various pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XL declared a shared affiliation with the authors YL, FG, LY, YJ, WT, LLi, GL, GT, and CL to the handling editor at the time of the review., (Copyright © 2022 Zhang, Lv, Cai, Li, Gao, Yu, Jiang, Tong, Li, Li, Tong and Liu.)

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 ​cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Pseudorabies is caused by pseudorabies virus (PRV), a member of the Herpesvirus family, and has caused tremendous damage to the pig industry. Protein unique lone 16 (pUL16) is a conserved envelope protein in all herpesviruses, that is known to play an important role in several aspects, including virus diffusion in cells and virulence in mice. It has been shown that the pUL16 can interact with the virus proteins UL11, UL49, UL21, gD, and gE. However, the research to date on pUL16 has only focused on etiology, without discussing the possible cellular pathways involved in PRV infection. Leucine-rich PPR motif-containing protein (LRPPRC) is a multifunctional cellular protein that participates in various cellular processes, such as RNA processing, splicing, stabilization, editing, translation, and energy metabolism. This was the first caspase-independent apoptosis protein to be identified. In this study, immune precipitation and mass spectrometry was performed to define the function of the pUL16 in PRV infection to study the possible cellular pathways in which pUL16 may participate. It was found that LRPRRC could interact with PRV pUL16, which may indicate that UL16 is involved in a redox reaction or cellular apoptosis. This is the first study of the interaction between pUL16 and host proteins, which has positive significance to gain a further understanding of the pUL16., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The recombinant bivalent live vectored vaccine rPRRSV-E2 has been proved to be a favorable genetic engineering vaccine against classical swine fever (CSF) and highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS). NADC30-like strains have recently emerged in China and caused severe disease, and it is necessary to evaluate the vaccine candidate for the currently circulating viruses. This study established a good challenge model to evaluate the candidate rPRRSV-E2 vaccine in preventing infection with a representative NADC30-like strain (ZJqz21). It was shown that the challenge control piglets displayed clinical signs typical of PRRSV, including a persistent fever, dyspnea, moderate interstitial pneumonia, lymph node congestion, and viremia. In contrast, the rPRRSV-E2 vaccination significantly alleviated the clinical signs, yielded a high level of antibodies, provided adequate protection against challenge with ZJqz21, and inhibited viral shedding and the viral load in target tissues. Our results demonstrated that the recombinant bivalent live vectored vaccine strain rPRRSV-E2 can provide efficient protection against the challenge of heterologous circulating NADC30-like strain and could be a promising vaccine candidate for the swine industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Chen, Cao, Cao, Guo, Tong, Zhou, Li, Jiang, Liu, Yu, Qiao, Liu, Tong and Gao.)

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro.

Currently, various porcine reproductive and respiratory syndrome virus (PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing the difficulty of PRRS control. In this study, a PRRSV strain named HBap4-2018 was isolated from swine herds suffering severe respiratory disease with high morbidity in Hebei Province of China in 2018. The genome of HBap4-2018 is 15,003 nucleotides in length, and compared with NADC30-like PRRSV, nsp2 of HBap4-2018 has an additional continuous deletion of five amino acids. Phylogenetic analysis based on complete genome and ORF5 showed that HBap4-2018 belonged to lineage 8 of PRRSV-2, which was characterized by highly variable genome. However, HBap4-2018 was classified into lineage 1 based on phylogenetic analysis of nsp2, sharing higher amino acid homology (85.3%-85.5%) with NADC30-like PRRSV. Further analysis suggested that HBap4-2018 was a novel natural recombinant PRRSV with three recombinant fragments in the genome, of which highly pathogenic PRRSV (HP-PRRSV) served as the major parental strains, while NADC30-like PRRSV served as the minor parental strains. Five recombination break points were identified in nsp2, nsp3, nsp5, nsp9 and ORF6, respectively, presenting a novel recombinant pattern in the genome. Piglets inoculated with HBap4-2018 presented typical clinical signs with a mortality rate of 60%. High levels of viremia and obvious macroscopic and histopathological lesions in the lungs were observed, revealing the high pathogenicity of HBap4-2018 in piglets., (© 2021. Wuhan Institute of Virology, CAS.)

African swine fever (ASF) is one of the most lethal viral diseases affecting both domestic pigs and wild boars. The acute infection of the ASF disease in domestic pigs leads to a 100% mortality rate with symptoms including high fever, vascular changes, cyanosis of the skin. Until now, there are no commercial vaccines and antiviral drugs available for ASF control. Therefore, the spread of ASF poses great economic losses to the pig industry and the ecosystems in the affected countries. A rapid and capable method was urgently needed to monitor ASFV-specific antibodies for controlling the spread of ASFV. In this study, we obtained one strain of monoclonal antibody (mAb) against the p54 protein of ASFV, and the target epitope of the mAb was determined to be 175 YTHKDLENSL 184 . The experimental results demonstrated that the monoclonal antibody could successfully recognize the exogenously expressed p54 protein and the chimeric virus constructed in our laboratory. The mAb could be used as a detection tool for the development of ASF vaccine strains. In addition, the ELISA established by using the obtained synthetic epitope peptide as the antigen had high sensitivity, good specificity and showed the great potential for ASF epidemic monitoring and control., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera ( Alphacoronavirus , Betacoronavirus , Gammacoronavirus , and Deltacoronavirus ) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera ( Alphacoronavirus , Betacoronavirus , Gammacoronavirus , and Deltacoronavirus ) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.

Introduction: The pseudorabies virus (PRV) gene encoding thymidine kinase (tk) is an important virulence-associated factor. Attenuation of PRV in susceptible animals is a frequent result of tk deletion. The aim of the study was to assess the pathogenicity of tk-deleted PRV in rats., Material and Methods: Sprague Dawley rats were infected with the tk-deleted PRV strain SuHV-1 ΔTK:247 via intranasal or intramuscular inoculation. PRV loads in ten tissues from dead and euthanised rats were determined using real-time PCR., Results: Infection with SuHV-1 ΔTK:247 could cause death in rats. The 50% lethal dose (LD 50 ) of SuHV-1 ΔTK:247 via intranasal inoculation was 10 3.16 TCID 50 in rats. Intramuscular inoculation required a higher dose of SuHV-1 ΔTK:247 (10 5.0 TCID 50 ). A high SuHV-1 ΔTK:247 titre was observed in the trigeminal ganglia or spinal cord of dead rats., Conclusion: The results of this study show that rats are highly susceptible to PRV infection, and tk deletion did not completely diminish the pathogenicity of PRV in rats., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2021 L. Zhang et al. published by Sciendo.)

Host interferon-stimulated gene 20 (ISG20) exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling. Here, we examined the role of ISG20 during pseudorabies virus (PRV) proliferation. We found that ISG20 modulates PRV replication by enhancing IFN signaling. Further, ISG20 expression was upregulated following PRV infection and poly(I:C) treatment. Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells, whereas knockdown of ISG20 promoted PRV proliferation. In addition, ISG20 expression upregulated IFN-β expression and enhanced IFN downstream signaling during PRV infection. Notably, PRV UL24 suppressed the transcription of ISG20, thus antagonizing its antiviral effect. Further domain mapping analysis showed that the N terminus (amino acids 1-90) of UL24 was responsible for the inhibition of ISG20 transcription. Collectively, these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay., (© 2021. Wuhan Institute of Virology, CAS.)

Highly virulent porcine epidemic diarrhea virus (PEDV) strains re-emerged and circulated in China at the end of 2010, causing significant economic losses in the pork industry worldwide. To understand the genetic dynamics of PEDV during its passage in vitro, the PEDV G2 strain FJzz1 was serially propagated in Vero cells for up to 200 passages. The susceptibility and adaptability of the FJzz1 strain increased gradually as it was serially passaged in vitro. Sequence analysis revealed that amino acid (aa) changes were mainly concentrated in the S glycoprotein, which accounted for 72.22%-85.71% of all aa changes. A continuous aa deletion ( 55 I 56 G 57 E →  55 K 56 Δ 57 Δ) occurred in the N-terminal domain of S1 (S1-NTD). To examine how the aa changes affected its virulence, FJzz1-F20 and FJzz1-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets. All the piglets in the FJzz1-F20-infected group showed typical diarrhea at 24 h postinfection, and the piglets died successively by 48 h postinfection. However, the clinical signs of the piglets in the FJzz1-F200-infected group were significantly weaker, and no deaths occurred. The FJzz1-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues, and no obvious histopathological lesions. Type I and III interferon were induced in the FJzz1-F200 infection group, together with pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-8. These results indicate that the identified genetic changes may contribute to the attenuation of FJzz1 strain, and the attenuated FJzz1-F200 may have the potential for developing PEDV live-attenuated vaccines., (© 2021. Wuhan Institute of Virology, CAS.)

Porcine reproductive and respiratory syndrome (PRRS) is one of the major drivers of economic loss in the swine industry worldwide. In commercial pig production, vaccination is the first option in an attempt to control infectious diseases. Pigs are therefore often immunized with different vaccines, and almost all of them are delivered via the intramuscular (IM) route. However, the IM injection may result in physical damage, stress reactions, and is labor demanding. An alternative route is urgently needed to reduce the disadvantages of conventional vaccination. In this study, a needle-free intradermal (ID) delivery system was evaluated for delivering a live PRRS vaccine as compared with the traditional needle-syringe method. Fifty-two 4-week-old piglets were divided into six groups: piglets in groups A-C were immunized using ID delivery system with 10 4 , 10 5 and 10 6 TCID 50 of PRRS candidate vaccine strain rHN-NP49, respectively; piglets in group D were immunized IM with 10 5 TCID 50 of rHN-NP49; and group E and F were used as challenge and control groups, respectively. At 28 days post vaccination, piglets in group A to E were challenged with a lethal dose of highly-pathogenic PRRSV. Similar results were found in viremia and antibody response among the ID and IM groups during the immunization stage. After challenge, similar results were found in average body weight gain, viral shedding, serum viral load, and clinical score among the immunization groups, with a higher protection ratio in the ID group compared with IM group with the same immunization dose. These results demonstrated that the ID delivery system could provide similar or even better protection compared with IM route, and could be an effective route for PRRS vaccination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)