Porcine deltacoronavirus nucleocapsid protein antagonizes JAK-STAT signaling pathway by targeting STAT1 through KPNA2 degradation.

Porcine deltacoronavirus (PDCoV) is an enteric pathogenic coronavirus that causes acute and severe watery diarrhea in piglets and has the ability of cross-species transmission, posing a great threat to swine production and public health. The interferon (IFN)-mediated signal transduction represents an important component of virus-host interactions and plays an essential role in regulating viral infection. Previous studies have suggested that multifunctional viral proteins encoded by coronaviruses antagonize the production of IFN via various means. However, the function of these viral proteins in regulating IFN-mediated signaling pathways is largely unknown. In this study, we demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I IFN-mediated JAK-STAT signaling pathway. We identified that PDCoV infection stimulated but delayed the production of IFN-stimulated genes (ISGs). In addition, PDCoV inhibited JAK-STAT signal transduction by targeting the nuclear translocation of STAT1 and ISGF3 formation. Further evidence showed that PDCoV N is the essential protein involved in the inhibition of type I IFN signaling by targeting STAT1 nuclear translocation via its C-terminal domain. Mechanistically, PDCoV N targets STAT1 by interacting with it and subsequently inhibiting its nuclear translocation. Furthermore, PDCoV N inhibits STAT1 nuclear translocation by specifically targeting KPNA2 degradation through the lysosomal pathway, thereby inhibiting the activation of downstream sensors in the JAK-STAT signaling pathway. Taken together, our results reveal a novel mechanism by which PDCoV N interferes with the host antiviral response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that receives increased attention and seriously threatens the pig industry and public health. Understanding the underlying mechanism of PDCoV evading the host defense during infection is essential for developing targeted drugs and effective vaccines against PDCoV. This study demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I interferon signaling by targeting STAT1, which is a crucial signal sensor in the JAK-STAT signaling pathway. Further experiments suggested that PDCoV N-mediated inhibition of the STAT1 nuclear translocation involves the degradation of KPNA2, and the lysosome plays a role in KPNA2 degradation. This study provides new insights into the regulation of PDCoV N in the JAK-STAT signaling pathway and reveals a novel mechanism by which PDCoV evades the host antiviral response. The novel findings may guide us to discover new therapeutic targets and develop live attenuated vaccines for PDCoV infection.

Serial passage of PDCoV in cell culture reduces its pathogenicity and its damage of gut microbiota homeostasis in piglets.

Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that mainly causes diarrhea in suckling piglets, and also has the potential for cross-species transmission. However, there are still no commercial vaccines available to prevent and control PDCoV infection. In this study, PDCoV strain HNZK-02 was serially propagated in vitro for up to 150 passages and the amino acid changes have mainly occurred in the S protein during serial passage which caused structure change. PDCoV HNZK-02-passage 5 (P5)-infected piglets exhibited acute and severe watery diarrhea, an obvious intestinal damage, while the piglets infected with PDCoV HNZK-02-P150 showed no obvious clinical signs, weak intestinal lesions, and lower viral loads in rectal swabs and various tissues. Compared with the PDCoV HNZK-02-P5 infection, HNZK-02-P150 infection resulted in a decrease in intestinal mucosal permeability and pro-inflammatory cytokines. Moreover, PDCoV HNZK-02-P5 infection had significantly reduced bacterial diversity and increased relative abundance of opportunistic pathogens, while PDCoV HNZK-02-P150 infection did not significantly affect the bacterial diversity, and the relative abundance of probiotics increased. Furthermore, the alterations of gut microbiota were closely related to the change of pro-inflammatory factor. Metagenomics prediction analysis demonstrated that HNZK-02-P150 modulated the tyrosine metabolism, Nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway, and lipopolysaccharide biosynthesis, which coincided with lower inflammatory response and intestinal permeability in the piglets infected with HNZK-02-P150. In conclusion, the PDCoV HNZK-02 was successfully attenuated by serial passage in vitro, and the changes of S gene, metabolic function, and gut microbiota may contribute to the attenuation. The PDCoV HNZK-02-P150 may have the potential for developing live-attenuated vaccine.IMPORTANCEPorcine deltacoronavirus (PDCoV) is an enteropathogen causing severe diarrhea, dehydration, and death in nursing piglets, devastating great economic losses for the global swine industry, and has cross-species transmission and zoonotic potential. There are currently no approved treatments or vaccines available for PDCoV. In addition, gut microbiota has an important relationship with the development of many diseases. Here, the PDCoV virulent HNZK-02 strain was successfully attenuated by serial passage on cell cultures, and the pathogenesis and effects on the gut microbiota composition and metabolic function of the PDCoV HNZK-02-P5 and P150 strains were investigated in piglets. We also found the genetic changes in the S protein during passage in vitro and the gut microbiota may contribute to the pathogenesis of PDCoV, while their interaction molecular mechanism would need to be explored further.

Rapid Construction of Recombinant PDCoV Expressing an Enhanced Green Fluorescent Protein for the Antiviral Screening Assay Based on Transformation-Associated Recombination Cloning in Yeast.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that mainly causes diarrhea and death in suckling piglets and also has the potential for cross-species transmission, threatening public health. However, there is still no effective vaccine or drug to prevent PDCoV infection. In order to accelerate the development of antiviral drugs, we established a high-throughput screening platform using a novel genome editing technology called transformation-associated recombination cloning in yeast. The recombinant PDCoV and PDCoV reporter virus expressing enhanced green fluorescent protein were both rapidly rescued with stable genealogical characteristics during passage. Further study demonstrated that the reporter virus can be used for high-throughput screening of antiviral drugs with a Z-factor of 0.821-0.826. Then, a medicine food homology compound library was applied, and we found that three compounds were potential antiviral reagents. In summary, we have established a fast and efficient reverse genetic system of PDCoV, providing a powerful platform for the research of antiviral drugs.

Chlorogenic acid inhibits porcine deltacoronavirus release by targeting apoptosis.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae, genus Deltacoronavirus, can cause acute diarrhea in piglets, and also possesses cross-species transmission potential, leading to severe economic losses and threatening public health. However, no approved drug against PDCoV infection is available. Here, we investigated the antiviral effect of chlorogenic acid (CGA), the main active component of Lonicerae Japonicae Flos, against PDCoV infection. The results showed that CGA inhibited the replication of PDCoV significantly both in LLC-PK1 and ST cells, with a selectivity index greater than 80. CGA decreased the synthesis of PDCoV viral RNA and protein, and viral titers in a dose-dependent manner. The results of the time-of-addition assay indicated that CGA mainly affected the early stage of virus replication and viral release. Moreover, CGA significantly reduced apoptosis caused by PDCoV infection, and the application of apoptosis agonist and inhibitor revealed that apoptosis could promote progeny virus release. Further study demonstrated that CGA can inhibit virus release by directly targeting apoptosis caused by PDCoV infection. In conclusion, CGA is an effective agent against PDCoV, which provides a foundation for drug development for the treatment of PDCoV and other coronavirus infections.

Porcine ß-defensin-2 alleviates aflatoxin B1 induced intestinal mucosal damage via ROS-Erk1/2 signaling pathway.

Aflatoxin B1 (AFB1) is a highly toxic fungal toxin that causes severe damage to animal intestines. Porcine beta-defensin-2 (pBD-2) is a well-studied antimicrobial peptide in pigs that can protect animal intestines and improve productivity. This study aimed to investigate the molecular mechanisms of pBD-2 in alleviating AFB1-induced oxidative stress and intestinal mucosal damage using porcine intestinal epithelial cells (IPEC-J2 cells) and Kunming (KM) mice. The maximum destructive concentration of AFB1 for IPEC-J2 cells and the optimal therapeutic concentration of pBD-2 were determined by CCK-8 and RT-qPCR. We then investigated the oxidative stress and intestinal damage induced by AFB1 and the alleviating effect of pBD-2 by detecting changes of reactive oxygen species (ROS), inflammatory cytokines, tight junction proteins (TJPs) and mucin. Finally, the molecular mechanism of pBD-2 mitigates AFB1-induced oxidative stress and intestinal mucosal damage were explored by adding ROS and Erk1/2 pathway inhibitors to comparative analysis. In vivo, the therapeutic effect of pBD-2 on AFB1-induced intestinal damage was analyzed from aspects such as average daily gain (ADG), pathological damage, inflammation, and mucosal barrier in KM mice. The study found that low doses of pBD-2 promoted cell proliferation and prevented AFB1-induced cell death, and pBD-2 significantly restored the feed conversion rate and ADG of KM mice reduced by long-term exposed AFB1. Increasing the intracellular ROS and the expression and phosphorylation of Erk1/2, AFB1 promoted inflammation by altering inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8, and disrupted the mucosal barrier by interfering with Claudin-3, Occludin, and MUC2, while pBD-2 significantly reduced ROS and decreased the expression and phosphorylation of Erk1/2 to restored their expression to alleviate AFB1-induced oxidative stress and intestinal mucosal damage in IPEC-J2 cells and the small intestine of mice.

Japanese encephalitis virus NS4B inhibits interferon beta production by targeting TLR3 and TRIF.

Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes, causing epidemics of encephalitis in humans and reproductive disorders in pigs. This virus is predominantly distributed in Asian countries and causes tens of thousands of infections in humans annually. Interferon (IFN) is an essential component of host defense against viral infection. Multiple studies have indicated that multifunctional nonstructural proteins of flaviviruses suppress the host IFN response via various strategies to facilitate viral replication. The flaviviruses encoded nonstructural protein 4B (NS4B) is a multifunctional hydrophobic nonstructural protein widely involved in viral replication, pathogenesis and host immune evasion. In this study, we demonstrated that NS4B of JEV suppressed the induction of IFN-ß production, mainly through targeting the TLR3 and TRIF (a TIR domain-containing linker that induces IFN-ß) proteins in the TLR3 pathway. In a dual-luciferase reporter assay, JEV NS4B significantly inhibited the activation of IFN-ß promoter induced by TLR3 and simultaneously treated with poly (I:C). Moreover, NS4B also inhibited the activation of IFN-ß promoter triggered by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in TLR3 signaling pathway. Furthermore, NS4B inhibited the phosphorylation of IRF3 under the stimulation of TLR3 and TRIF molecules. Mechanistically, JEV NS4B interacts with TLR3 and TRIF and confirmed by co-localization and co-immunoprecipitation assay, thereby inhibiting the activation of downstream sensors in the TLR3-mediated pathway. Overall, our results provide a novel mechanism by which JEV NS4B interferes with the host's antiviral response through targeting TLR3 receptor signaling pathway.

Preparation of monoclonal antibody and identification of two novel B cell epitopes to VP1 protein of porcine sapelovirus.

Porcine sapelovirus (PSV) is an important emerging swine pathogen that causes diarrhoea, respiratory distress, severe reproductive system and neurological disorders in pigs, posing huge threat to swine industry. However, there are no effective serological diagnostic products and the epitope characterization of PSV VP1 protein is still largely unknown. In current study, we successfully expressed recombinant His-VP1 protein by prokaryotic expression system and the recombinant VP1 protein had good immunogenicity. BALB/C mice were then selected and immunized with purified recombinant VP1 protein, and two monoclonal antibodies (Mabs) 9F10 and 15E4 against VP1 were successfully prepared by hybrioma technology. The isotype of these two Mabs were identified and showed that Mab 9F10 with the heavy chain subtype was IgG1 and the light chain subtype was kappa. Mab 15E4 was identified as IgG2 for the heavy chain subtype and Kappa for the light chain subtype. The antigen epitopes of prepared two VP1 Mabs were clearly identified. The minimal unit of B cell specific epitope recognized by Mab 15E4 was 203YDGDG207 and conserved in different strain genotypes of PSV, indicating this epitope may be a good target for serological detection of PSV. However, the epitope recognized by Mab 9F10 was 8QAIVNRT14 and varied greatly among different PSV strains. Structural modeling analysis showed that the identified two novel B cell epitopes were located on the surface of VP1. Our study provides useful tool for the establishment the serological detection methods of PSV and may support the study of VP1 protein function.

Porcine Deltacoronavirus Utilizes Sialic Acid as an Attachment Receptor and Trypsin Can Influence the Binding Activity.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Studies showed that PDCoV uses porcine aminopeptidase N (pAPN) as an entry receptor, but the infection of pAPN-knockout cells or pigs with PDCoV revealed that pAPN might be not a critical functional receptor, implying there exists an unidentified receptor involved in PDCoV infection. Herein, we report that sialic acid (SA) can act as an attachment receptor for PDCoV invasion and facilitate its infection. We first demonstrated that the carbohydrates destroyed on the cell membrane using NaIO4 can alleviate the susceptibility of cells to PDCoV. Further study showed that the removal of SA, a typical cell-surface carbohydrate, could influence the PDCoV infectivity to the cells significantly, suggesting that SA was involved in the infection. The results of plaque assay and Western blotting revealed that SA promoted PDCoV infection by increasing the number of viruses binding to SA on the cell surface during the adsorption phase, which was also confirmed by atomic force microscopy at the microscopic level. In in vivo experiments, we found that the distribution levels of PDCoV and SA were closely relevant in the swine intestine, which contains huge amount of trypsin. We further confirmed that SA-binding capacity to PDCoV is related to the pre-treatment of PDCoV with trypsin. In conclusion, SA is a novel attachment receptor for PDCoV infection to enhance its attachment to cells, which is dependent on the pre-treatment of trypsin on PDCoV. This study paves the way for dissecting the mechanisms of PDCoV-host interactions and provides new strategies to control PDCoV infection.

Integrated mRNA and microRNA Transcriptome Sequencing Characterizes Sequence Variants and mRNA-microRNA Regulatory Networks in Grass Carp Fibroblasts Infected with Virulent and Attenuated GCRV.

Grass carp hemorrhagic disease is a fatal disease caused by the grass carp reovirus (GCRV). The aberrant regulation of transcripts has been implicated in many types of diseases. In the present study, we characterized mRNA and miRNA transcriptomes of different virulent GCRVs using RNA sequencing (RNA-Seq). One hundred eighteen miRNAs were identified as being differentially expressed between different virulent viruses in grass carp fibroblasts. Eight miRNAs were selected to verify the RNA-Seq results using RT-PCR and mRNA methods. In total, 996 differentially expressed mRNA genes were identified in grass carp fibroblasts, while 901 miRNA-mRNA target pairs were observed to be inversely regulated in grass carp fibroblasts. Integrated mRNA/miRNA expression profiling analysis results showed that the most influenced processes were the immune response and cell death. Three miRNAs were shown to exhibit the same expression patterns when two different methods were used and had important functions during viral infection. These results provide insights into the miRNA-mediated regulation of mRNA and valuable resources on transcript variation and regulation during GCRV infection, which are potentially useful for mechanistic and drug studies.

Duck PIAS2 Promotes H5N1 Avian Influenza Virus Replication Through Its SUMO E3 Ligase Activity.

The protein inhibitor of the activated STAT2 (PIAS2) has been implicated in many cellular processes and can also regulate viral replication in mammals. However, the role of PIAS2 in the highly pathogenic avian influenza virus (HPAIV) H5N1 replication in ducks is still unclear. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, we identified that duck PIAS2 (duPIAS2) was one protein that interacted with the nucleoprotein (NP) from the H5N1 HPAIV strain of DK212. Through confocal microscopy images and Co-IP assay, we confirmed NP could interact with duPIAS2. Overexpression of duPIAS2 in primary duck embryo fibroblast (DEF) cells was shown to promote DK212 replication, and knockdown of duPIAS2 could repress DK212 replication. We further found duPIAS2 could promote NP SUMOylation through duck SUMO1 (duSUMO1), and the potential SUMOylation sites of NP were at lysines 7, 48, and 87. Furthermore, duPIAS2 promoted the replication of DK212, here relying on the activity of its SUMO E3 ligase. Duck SENP1 (duSENP1), a deSUMOylation enzyme, could repress NP SUMOylation and also inhibit DK212 replication. Together, we identified duPIAS2 could interact with NP and that duPIAS2 promoted H5N1 HPAIV replication, which might be related to NP SUMOylation.

Duck TRIM32 Functions in IFN-ß Signaling Against the Infection of H5N6 Highly Pathogenic Avian Influenza Virus.

In mammals, tripartite motif 32 (TRIM32) is essential for regulating host innate immune responses to viral infections. However, the antiviral effect of TRIM32 in birds has not been reported. Here, we cloned the full-length duck TRIM32 (duTRIM32) cDNA from total spleen RNA of Peking duck. DuTRIM32 consists of 682 amino acids and has 95.5% similarity in amino acid sequences with chicken TRIM32 and 84.9% similarity with human TRIM32, respectively. DuTRIM32 mRNA was found to be ubiquitously expressed in all tested tissues from healthy ducks. Overexpression of duTRIM32 significantly activated the IFN-ß promoter and upregulated the mRNA levels of IFN-ß, IRF7, and Mx, which indicates that duTRIM32 is involved in the type I IFN pathway. Furthermore, duTRIM32 was found to directly interact with duck STING (duSTING) and to contribute to the expression of IFN-ß mediated by duSTING. The mRNA level of duTRIM32 was significantly upregulated in the lungs and spleens of H5N6 highly pathogenic avian influenza virus (HPAIV) infected ducks 3 days post-infection (DPI). Furthermore, overexpression of duTRIM32 could inhibit the replication of H5N6 HPAIV in duck embryo fibroblasts (DEFs). Therefore, these results indicate that duTRIM32 is involved in the type I IFN pathway and exhibit an antiviral effect against H5N6 HPAIV infection.

Duck PIAS2 negatively regulates RIG-I mediated IFN-ß production by interacting with IRF7.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-ß expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-ß production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-ß promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-ß promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-ß production induced by duck IRF7.

Molecular characterization of a feline calicivirus isolated from tiger and its pathogenesis in cats.

Feline calicivirus (FCV) is a virus that causes respiratory disease in cats. In this study, the FCV TIG-1 was isolated from Siberian tiger feces collected in 2014 in Heilongjiang Province, China. Phylogenetic analysis among TIG-1 and other FCVs showed that TIG-1 does not share the same lineage with other FCV isolates from Heilongjiang or other regions in China but is located in the same cluster with the FCV strain Urbana, which was isolated from the United States. The growth kinetics in vitro and the pathogenicity in cats between TIG-1 and the domestic cat-origin FCV strain F9 (vaccine strain) and strain 2280 were compared. We found that the growth kinetics of strains TIG-1 and 2280 were faster than that of strain F9 from 12h to 36h post-infection, indicating that strains TIG-1 and 2280 produce infectious virions and reach peak yields earlier. Challenge experiments in cats showed that TIG-1 grew faster than the other two strains in the lungs of cats and that TIG-1 is a virulent FCV with 100% morbidity and lethality. In addition, the histopathological results showed that the virulent TIG-1 strain directly led to severe lung tissue damage and indirectly led to intestinal damage. The results presented here show that a tiger-origin FCV exhibits high virulence in cats.

N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression.

In vitro antiviral effect of germacrone on feline calicivirus.

Feline calicivirus (FCV) often causes respiratory tract and oral disease in cats and is a highly contagious virus. Widespread vaccination does not prevent the spread of FCV. Furthermore, the low fidelity of the RNA-dependent RNA polymerase of FCV leads to the emergence of new variants, some of which show increased virulence. Currently, few effective anti-FCV drugs are available. Here, we found that germacrone, one of the main constituents of volatile oil from rhizoma curcuma, was able to effectively reduce the growth of FCV strain F9 in vitro. This compound exhibited a strong anti-FCV effect mainly in the early phase of the viral life cycle. The antiviral effect depended on the concentration of the drug. In addition, germacrone treatment had a significant inhibitory effect against two other reference strains, 2280 and Bolin, and resulted in a significant reduction in the replication of strains WZ-1 and HRB-SS, which were recently isolated in China. This is the first report of antiviral effects of germacrone against a calicivirus, and extensive in vivo research is needed to evaluate this drug as an antiviral therapeutic agent for FCV.

The function of feline stimulator of interferon gene (STING) is evolutionarily conserved.

Stimulator of interferon gene (STING) mediates the induction of type I IFN responses. In this study, feline STING was cloned. Full-length STING contains 1134bp and encodes a 377 amino acid product that shares the highest similarity with bovine STING. STING is primarily expressed in the spleen, lungs and lymph nodes. An examination of its cellular localization indicated that STING is localized in the endoplasmic reticulum (ER) and contains two ER retention motifs, RPR and KKNF. Overexpressing STING induced the IFN response via the IRF3, NF-κB and AP-1 pathways. Moreover, the C-terminus of STING was required for the activation of IRF3 and AP-1. Knockdown of STING impaired the IFN-ß response triggered by poly(dA:dT), poly(I:C) or SeV. Finally, STING activated the ISRE promoter and increased the expression of ISG15 and viperin. Collectively, our findings indicate that STING is involved in the regulation of the IFN-ß pathway in felines.