ZBP1 inhibits the replication of Senecavirus A by enhancing NF-κB signaling pathway mediated antiviral response in porcine alveolar macrophage 3D4/21 cells.

BACKGROUND: Senecavirus A (SVA) caused porcine idiopathic vesicular disease (PIVD) showing worldwide spread with economic losses in swine industry. Although some progress has been made on host factors regulating the replication of SVA, the role of Z-DNA binding protein 1 (ZBP1) remains unclear. METHODS: The expression of ZBP1 in SVA-infected 3D/421 cells was analyzed by quantitative real-time PCR (qRT-PCR) and western blot. Western blot and qRT-PCR were used to detect the effects of over and interference expression of ZBP1 on SVA VP2 gene and protein. Viral growth curves were prepared to measure the viral proliferation. The effect on type I interferons (IFNs), interferon-stimulated genes (ISGs), and pro-inflammatory cytokines in SVA infection was analyzed by qRT-PCR. Western blot was used to analysis the effect of ZBP1 on NF-κB signaling pathway and inhibitor are used to confirm. RESULTS: ZBP1 is shown to inhibit the replication of SVA by enhancing NF-κB signaling pathway mediated antiviral response. SVA infection significantly up-regulated the expression of ZBP1 in 3D4/21 cells. Infection of cells with overexpression of ZBP1 showed that the replication of SVA was inhibited with the enhanced expression of IFNs (IFN-α, IFN-ß), ISGs (ISG15, PKR, and IFIT1) and pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), while, infected-cells with interference expression of ZBP1 showed opposite effects. Further results showed that antiviral effect of ZBP1 is achieved by activation the NF-κB signaling pathway and specific inhibitor of NF-κB also confirmed this. CONCLUSIONS: ZBP1 is an important host antiviral factor in SVA infection and indicates that ZBP1 may be a novel target against SVA.

IFITM1 and IFITM2 inhibit the replication of senecavirus A by positive feedback with RIG-I signaling pathway.

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-ß), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus.

CD46 inhibits the replication of swine influenza viruses by promoting the production of type I IFNs in PK-15 cells.

Swine flu caused by swine influenza A virus (swIAV) is an acute respiratory viral disease that is spreading in swine herds worldwide. Although the effect of some host factors on replication of swIAV has been identified, the role of CD46 in this process is unclear. Here, we report that CD46 inhibits the replication of swIAV by promoting the production of type I interferons (IFNs) in porcine kidney (PK-15) cells. CD46 knockout (CD46-KO) and stably expressing (CD46-overexpression) PK-15 cells were prepared using lentivirus-mediated CRISPR/Cas9 gene editing and seamless cloning technology. The results of virus infection in CD46-overexpression PK-15 cells showed that the replication of H1N1 and H3N2 swIAVs were inhibited, and the production of type I IFNs (IFN-α, IFN-ß), interferon regulatory factor (IRF) 3, and mitochondrial antiviral-signaling protein (MAVS) was enhanced. Virus infection in CD46-KO PK-15 cells showed the opposite results. Further results showed that CD46-KO PK-15 cells have a favorable ability to proliferate influenza viruses compared to Madin-Darby canine kidney (MDCK) and PK-15 cells. These findings indicate that CD46 acts as promising target regulating the replication of swIAV, and help to develop new agents against infection and replication of the virus.

Recombinant Orf virus induced antibody production against capsid protein of porcine circovirus type 3 in mice.

The emerging worldwide distributed porcine circovirus type 3 (PCV3) infection poses a serious threat to swine herds. An important means of preventing and controlling PCV3 infection is the development of the vaccine, while, the inability to cultivate in vitro has become the biggest obstacle. Orf virus (ORFV), the prototypic member of the Parapoxviridae, has been proven to be a novel valid vaccine vector for preparing various candidate vaccines. Here, recombinant ORFV expressing capsid protein (Cap) of PCV3 was obtained and proved its favorable immunogenicity inducing antibody against Cap in BALB/c mice. Based on the enhanced green fluorescent protein (EGFP) as a selectable marker, the recombinant rORFVΔ132-PCV3Cap-EGFP was generated. Then, recombinant ORFV expressing Cap only, rORFVΔ132-PCV3Cap, was obtained based on rORFVΔ132-PCV3Cap-EGFP using a double homologous recombination method by screening single non-fluorescent virus plaque. Results of the western blot showed that the Cap can be detected in rORFVΔ132-PCV3Cap infected OFTu cells. The results of immune experiments in BALB/c mice indicated that a specific antibody against Cap of PCV3 in serum was induced by rORFVΔ132-PCV3Cap infection. The results presented here provide a candidate vaccine against PCV3 and a feasible technical platform for vaccine development based on ORFV.

Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I.

Senecavirus A (SVA)-induced porcine idiopathic vesicular disease has caused huge economic losses worldwide. Glucose metabolism in the host cell is essential for SVA proliferation; however, the impact of the virus on glucose metabolism in host cells and the subsequent effects are still unknown. Here, glycolysis induced by SVA is shown to facilitate virus replication by promoting lactate production, which then attenuates the interaction between the mitochondrial antiviral-signaling protein (MAVS) and retinoic acid-inducible gene I (RIG-I). SVA induces glycolysis in PK-15 cells, as indicated by significantly increased expression of hexokinase 2 (HK2), 6-phosphofructokinase (PFKM), pyruvate kinase M (PKM), phosphoglycerate kinase 1 (PGK1), hypoxia-inducible factor-1 alpha (HIF-1α), and superoxide dismutase-2 (SOD2) in a dose-and replication-dependent manner, and enhanced lactate production, while reducing ATP generation. Overexpression of PKM, PGK1, HIF-1α, and PDK3 in PK-15 cells and high glucose concentrations promote SVA replication, while glycolytic inhibitors decrease it. Inhibition of RLR signaling allowed better replication of SVA by promoting lactate production to attenuate the interaction between MAVS and RIG-I, and regulatory effect of glycolysis on replication of SVA was mainly via RIG-I signaling. SVA infection in mice increased expression of PKM and PGK1 in tissues and serum yields of lactate. Mice treated with high glucose and administered sodium lactate showed elevated lactate levels and better SVA replication, as well as suppressed induction of RIG-I, interferon beta (IFNß), IFNα, interferon-stimulated gene 15 (ISG15), and interleukin 6 (IL-6). The inhibitory effect on interferons was lower in mice administered sodium oxamate and low glucose compared to the high glucose, indicating that RLR signaling was inhibited by SVA infection through lactate in vitro and in vivo. These results provide a new perspective on the relationship between metabolism and innate immunity of the host in SVA infection, suggesting that glycolysis or lactate may be new targets against the virus.

H3N2 canine influenza virus NS1 protein inhibits canine NLRP3 inflammasome activation.

Inflammation is an innate immune response of the body against pathogens and other irritants. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is a major player in the inflammatory response against pathogenic microorganisms. In this study, we analyzed the relationship between the NLRP3 inflammasome and the influenza virus NS1 protein, which is involved in host immune escape. The canine influenza virus NS1 protein transcriptionally attenuated proinflammatory cytokines by inhibiting the nuclear factor-κB (NF-κB) activator. NS1 also directly interacted with NLRP3 and blocked ASC (Apoptosis-associated speck-like protein containing CARD) oligomerization, which deactivated the NLRP3 inflammasome. In addition, NS1 inhibited pro-caspase 1 cleavage into caspase-1, which prevents maturation of IL-1ß and IL-18 from their respective precursors, eventually reducing the inflammatory response. Taken together, the influenza NS1 protein evades host immunity, and our findings provide a theoretical basis for the prevention and treatment of canine influenza.

Suppressor of cytokine signaling 1 (SOCS1) inhibits antiviral responses to facilitate Senecavirus A infection by regulating the NF-κB signaling pathway.

Senecavirus A (SVA) is a new virus inducing porcine idiopathic vesicular disease that causes significant economic losses. Although some progress has been made in etiological research, the role of host factors in SVA infection remains unclear. This study investigated the role of the host factor, suppressor of cytokine signaling 1 (SOCS1), in SVA infection. The expression of SOCS1 was significantly upregulated with infection of SVA in a dose-dependent manner, and SOCS1 inhibited the expression of type I interferons (IFN-α, IFN-ß) and the production of interferon stimulating genes (ISGs) (ISG56, ISG54, PKR), thereby facilitating viral replication. Further results showed that inhibition of antiviral responses of SOCS1 was achieved by regulating the NF-κB signaling pathway, which attenuates the production of IFNs and pro-inflammatory cytokines. These findings provide a new perspective of SVA pathogenesis and may partially explain the persistence of this infection. Moreover, the data indicate that targeting SOCS1 can help in developing new agents against SVA infection.

Negative Regulators of Inflammation Response to the Dynamic Expression of Cytokines in DF-1 and MDCK Cells Infected by Avian Influenza Viruses.

The H5N1 and H9N2 avian influenza viruses (AIVs) seriously endanger the poultry industry and threaten human health. Characteristic inflammatory responses caused by H5N1 and H9N2 AIVs in birds and mammals result in unique clinical manifestations. The role of anti-inflammatory regulators, PTX3, Del-1, and GDF-15, in H5N1 and H9N2-AIV-mediated inflammation in birds and mammals has not yet been verified. Here, the expression of PTX3, Del-1, and GDF-15 in DF-1 and MDCK cells infected with H5N1 and H9N2 AIVs and their effect on inflammatory cytokines were analyzed. Infection with both AIVs increased PTX3, Del-1, and GDF-15 expression in DF-1 and MDCK cells. Infection with H9N2 or H5N1 AIV in DF-1 and MDCK cells with overexpression of all three factors, either alone or in combination, inhibited the expression of tested inflammatory cytokines. Furthermore, co-expression of PTX3, Del-1, and GDF-15 enhanced the inhibition, irrespective of the cell line. The findings from this study offer insight into the pathogenic differences between H5N1 and H9N2 AIVs in varied hosts. Moreover, our findings can be used to help screen for host-specific anti-inflammatory agents.

Phylogenetic and codon usage analysis for replicase and capsid genes of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is a highly contagious virus belonging to the family Circoviridae that causes the severe dermatitis and nephropathy syndrome. To date, PCV3 has a worldwide distribution and bring huge economic losses to swine industry. Replicase (Rep) and capsid (Cap) are two major coded proteins of PCV3. Considering the large number of new PCV3 isolates were reported in the past few years and the research for the codon usage pattern of Rep and Cap genes was still a gap, phylogenetic and codon usage analysis of these two genes was performed. Phylogenetic analyses showed that Rep genes in PCV3a were dispersed with no clear clusters while corresponding sequences in PCV3b clustered into two groups and Cap genes clustered into distinct clades according to different genotypes. Relative synonymous codon usage (RSCU) analysis revealed that the codon usage bias existed and effective number of codon (ENC) analysis showed that the bias was slight low. ENC-GC3s plot indicated that mutational pressure and other factors both played a role in PCV3 codon usage and neutrality plot analysis showed that natural selection was the main force influencing the codon usage pattern. The results presented here provided the important basic data on codon usage pattern of Rep and Cap genes, and a better understanding of the evolution and potential origin of PCV3.

Vaccination with virus-like particles of atypical porcine pestivirus inhibits virus replication in tissues of BALB/c mice.

Congenital tremor (CT) type A-II in piglets is a worldwide disease caused by an emerging atypical porcine pestivirus (APPV). Preparation and evaluation of vaccines in laboratory animals is an important preliminary step toward prevention and control of the disease. Here, virus-like particles (VLPs) of APPV were prepared and VLPs vaccine was evaluated in BALB/c mice. Purified Erns and E2 proteins expressed in E. coli were allowed to self-assemble into VLPs, which had the appearance of hollow spherical particles with a diameter of about 100 nm by transmission electron microscopy (TEM). The VLPs induced strong antibody responses and reduced the viral load in tissues of BALB/c mice. The data from animal challenge experiments, RT-PCR, and immunohistochemical analysis demonstrated that BALB/c mice are an appropriate laboratory model for APPV. These results suggest the feasibility of using VLPs as a vaccine for the prevention and control of APPV and provide useful information for further study of APPV in laboratory animals.

Comprehensive Analysis of Synonymous Codon Usage Bias for Complete Genomes and E2 Gene of Atypical Porcine Pestivirus.

Atypical porcine pestivirus (APPV) is an emerging novel pestivirus causing the congenital tremor (CT) in piglets. The worldwide distribution characteristic of APPV make it a threat to global swine health. E2 is the major envelope glycoprotein of APPV and the crucial target for vaccine development. Considering the genetic variability of APPV complete genomes and its E2 gene as well as gaps for codon analysis, a comprehensive analysis of codon usage patterns was performed. Relative synonymous codon usage (RSCU) and effective number of codon (ENC) analyses showed that a relatively instable change existed and a slight low codon usage bias (CUB) were displayed in APPV genomes. ENC-plot analysis and correlation analyses of nucleotide compositions and ENC showed that mutation pressure and natural selection both affected the codon usage bias of the APPV and natural selection had a more obvious influence for E2 gene compared with complete genomes. Principal component analysis (PCA) and correlation analyses confirmed the above results. Correlation analyses between Gravy and Aromaticity values and the codon bias showed that natural selection played an important role in shaping the synonymous codon bias. Furthermore, neutrality plot analysis showed that natural selection was the main force while mutation pressure was a minor force influencing the codon usage pattern of the APPV E2 gene and complete genomes. The results could illustrate the codon usage patterns of APPV genomes and provided valuable basic data for further fundamental research of evolution of APPV.

Comprehensive analysis of synonymous codon usage patterns and influencing factors of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen belonging to the family Coronaviridae that causes the porcine epidemic diarrhea, a highly contagious disease with high mortality in piglets and symptoms that include dehydration and severe diarrhea. Considering the high frequency of genetic mutations in PEDV and its potential for interspecies transmission, as it can infect and replicate in bat and human cells, a comprehensive analysis of its codon usage bias was performed. The effective number of codons (ENC) and the relative synonymous codon usage (RSCU) were determined, revealing codon usage bias in the PEDV genome. Principal component analysis (PCA), an ENC plot, and a parity rule 2 (PR2) plot showed that mutation pressure and natural selection have influenced the codon usage bias of the PEDV genomes. Correlation analysis with GRAVY and aromaticity values and neutrality plot analysis indicated that natural selection was the main force influencing the codon usage pattern, while mutation pressure played a minor role. This study provides valuable basic data for further fundamental research on evolution of PEDV.

Genomic diversity and recombination of Seneca Valley viruses emerged in pig herds in Guangdong Province during 2019.

Seneca Valley virus (SVV) is an emerging global picornavirus that causes porcine idiopathic vesicular disease. We characterized the genome and conducted evolutionary and recombination analyses of four newly identified SVV strains which were CH-GDZS-2019, CH-GDMZ-2019, CH-GDHZ01-2019, and CH-GDHZ02-2019. Sequence alignment and phylogenetic analysis showed that strains circulating in swine herds in China were genetically diverse and complex. Recombination analyses indicated that strain CH-GDZS-2019 was derived from strains USA-IA44662-2015-P1 and USA-GBI29-2015, which were both isolated in the USA in 2015, while CH-GDMZ-2019 was derived from the Chinese field strains 1-2018-BH-China and CH-GDQC-2017. Our results provided important insights into the molecular characterization of the SVV strains co-circulating in Guangdong Province in China in 2019 and demonstrated the importance of additional SVV surveillance in China.

Development of the reverse genetics system for emerging atypical porcine pestivirus using in vitro and intracellular transcription systems.

Atypical porcine pestivirus (APPV) is a novel pestivirus causing congenital tremor (CT) type AII in piglets and exhibiting a broad geographical distribution. Lack of an operating system for the viral genome is one of bottlenecks which restrict further research on pathogenesis and gene functions of APPV. Reverse genetics system (RGS) is a feasible solution to this bottleneck problem, but, to-date, no RGSs have been developed for APPV. Here, for the first time, recombinant APPV CH-GD2017 were rescued using in vitro and intracellular transcription systems and the virons were observed via transmission electron microscopy. As the process of in vitro transcription is time-consuming and inefficient, a full-length cDNA clone in an intracellular transcription was further constructed using an RNA polymerase II system. Then, the rescued virus was identified via RT-PCR detection, indirect immunofluorescent assay, and transmission electron microscopy. Development of the RGS for APPV will provide an important tool for further research on this newly emerging pathogen.

Newcastle disease virus RNA-induced IL-1ß expression via the NLRP3/caspase-1 inflammasome.

Newcastle disease virus (NDV) infection causes severe inflammation and is a highly contagious disease in poultry. Virulent NDV strains (GM) induce large quantities of interleukin-1ß (IL-1ß), which is the central mediator of the inflammatory reaction. Excessive expression of IL-1ß exacerbates inflammatory damage. Therefore, exploring the mechanisms underlying NDV-induced IL-1ß expression can aid in further understanding the pathogenesis of Newcastle disease. Here, we showed that anti-IL-1ß neutralizing antibody treatment decreased body temperature and mortality following infection with virulent NDV. We further explored the primary molecules involved in NDV-induced IL-1ß expression from the perspective of both the host and virus. This study showed that overexpression of NLRP3 resulted in increased IL-1ß expression, whereas inhibition of NLRP3 or caspase-1 caused a significant reduction in IL-1ß expression, indicating that the NLRP3/caspase-1 axis is involved in NDV-induced IL-1ß expression. Moreover, ultraviolet-inactivated GM (chicken/Guangdong/GM/2014) NDV failed to induce the expression of IL-1ß. We then collected virus from GM-infected cell culture supernatant using ultracentrifugation, extracted the viral RNA, and stimulated the cells further with GM RNA. The results revealed that RNA alone was capable of inducing IL-1ß expression. Moreover, NLRP3/caspase-1 was involved in GM RNA-induced IL-1ß expression. Thus, our study elucidated the critical role of IL-1ß in the pathogenesis of Newcastle disease while also demonstrating that inhibition of IL-1ß via anti-IL-1ß neutralizing antibodies decreased the damage associated with NDV infection; furthermore, GM RNA induced IL-1ß expression via NLRP3/caspase-1.

Emergence of spontaneously occurring neoplastic disease caused by reticuloendotheliosis virus in breeding Muscovy ducks in China, 2019.

Reticuloendotheliosis virus (REV) has a wide range of avian hosts leading to reticuloendotheliosis, and its characteristic of vertical transmission makes it to be one of the most important diseases in breeder avian populations. Up to date, reports on neoplastic disease caused by REV in breeding ducks are few. Here, spontaneously occurring neoplastic disease caused by REV in breeding Muscovy ducks was reported in Guangdong province, China. The most significant gross lesions of sick ducks were tumour-bearing liver and enlarged spleen. Histopathological examination found proliferation of malignant lymphoreticular cells in the liver and reticuloendothelial cells in the spleen. REV strain, CH-GD2019, was successfully isolated using DF-1 cells, and the presence of REV was confirmed by PCR detection and transmission electron microscopy. The length of complete proviral genome is 8,238 nucleotides. Genetic and phylogenetic analyses revealed that CH-GD2019 was closely related to chicken-origin REV strains circulating in China. The results will provide a basic data for better understanding of REV in breeding ducks and suggest that REV from chickens may be a threat to ducks.

Viral load and histological distribution of atypical porcine pestivirus in different tissues of naturally infected piglets.

A newly identified atypical porcine pestivirus (APPV) associated with congenital tremors in newborn piglets has been shown to have a worldwide geographic distribution. In view of the function of Erns in pestivirus infection and replication, the viral load and histological distribution of APPV in different tissues of naturally infected piglets were analyzed by quantitative RT-PCR and immunohistochemical detection using Erns as the target. The results showed that the viral copy number was higher in the cerebellum, submandibular lymph nodes, and thymus than in other tissues, indicating that these are important target organs of APPV. The histological distribution of APPV was mainly in the matrix and nerve fiber in nervous tissues, endothelial cells in lymphoid tissues, and epithelial cells in other tissues, suggesting that these cells were target cells of APPV. The results will provide basic data for elucidating the pathogenesis and deepening the understanding of this newly discovered pathogen.

Comparative Analysis of Whole-Transcriptome RNA Expression in MDCK Cells Infected With the H3N2 and H5N1 Canine Influenza Viruses.

This study aimed to detect changes in the complete transcriptome of MDCK cells after infection with the H5N1 and H3N2 canine influenza viruses using high-throughput sequencing, search for differentially expressed RNAs in the transcriptome of MDCK cells infected with H5N1 and H3N2 using comparative analysis, and explain the differences in the pathogenicity of H5N1 and H3N2 at the transcriptome level. Based on the results of our comparative analysis, significantly different levels of expression were found for 2,464 mRNAs, 16 miRNAs, 181 lncRNAs, and 262 circRNAs in the H3N2 infection group and 448 mRNAs, 12 miRNAs, 77 lncRNAs, and 189 circRNAs in the H5N1 infection group. Potential functions were predicted by performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the target genes of miRNAs, lncRNAs and circRNAs, and the ncRNA-mRNA regulatory network was constructed based on differentially expressed RNAs. A greater number of pathways regulating immune metabolism were altered in the H3N2 infection group than in the H5N1 infection group, which may be one reason why the H3N2 virus is less pathogenic than is the H5N1 virus. This study provides detailed data on the production of ncRNAs during infection of MDCK cells by the canine influenza viruses H3N2 and H5N1, analyzed differences in the total transcriptomes between H3N2- and H5N1-infected MDCK cells, and explained these differences with regard to the pathogenicity of H3N2 and H5N1 at the transcriptional level.

Altered Expression of Zinc Transporter ZIP12 in Broilers of Ascites Syndrome Induced by Intravenous Cellulose Microparticle Injection.

Ascites syndrome (AS) is a harmful disease in fast-growing broilers characterized by heart failure and serious fluid accumulation in the abdominal cavity. One of the known functions of zinc transporter ZIP12 is an important regulator in pulmonary hypertension (PH) in rat. Whether chicken ZIP12 is involved in the process of AS need to be explored. Here, chicken ZIP12 was sequenced and expression pattern and histological distribution were detected in broilers of AS induced by intravenous cellulose microparticle injection. Phylogenetic analysis showed that ZIP12 was significantly different between chicken and mammalian. The relative mRNA expression level of ZIP12 in the liver and lung in AS and pre-ascites (PAS) groups were significantly higher (P < 0.01) than that in control. The immunohistological staining using rabbit anti-chicken ZIP12 IgG and integrated optical density analysis showed the positive cells of ZIP12 distributed in detected tissues and the expression level of ZIP12 protein increased in AS and PAS groups compared to control. The results will provide the basic data of ZIP12 in the pathological process of AS in broiler chickens and offer an important reference for prevention and control of the disease.