Porcine deltacoronavirus NS7a antagonizes JAK/STAT pathway by inhibiting the interferon-stimulated gene factor 3 (ISGF3) formation.

The accessory proteins of coronaviruses play a crucial role in facilitating virus-host interactions and modulating host immune responses. Previous study demonstrated that the NS7a protein of porcine deltacoronavirus (PDCoV) partially hindered the host immune response by impeding the induction of IFN-α/ß. However, the potential additional functions of NS7a protein in evading innate immunity have yet to be elucidated. This study aimed to investigate the mechanism of PDCoV NS7a protein regulating the JAK/STAT signaling pathway. We presented evidence that NS7a effectively inhibited ISRE promoter activity and ISGs transcription. NS7a hindered STAT1 phosphorylation, interacted with STAT2 and IRF9, and further impeded the formation and nuclear accumulation of ISGF3. Furthermore, comparative analysis of NS7a across different PDCoV strains revealed that the mutation of Leu4 to Pro4 led to an increase in the molecular weights of NS7a and disrupted its inhibition on the JAK/STAT signaling pathway. This finding implied that NS7a with key amino acids may be an indicator of virulence for PDCoV strains. Taken together, this study revealed a novel role of NS7a in antagonizing the IFN-I signaling pathway.

Coding-Complete Genome Sequences of Two Atypical Porcine Pestivirus Strains from Anhui Province, China.

In 2021, two atypical porcine pestivirus (APPV) strains, AH06/2021 and AH22/2021, were identified from suckling piglets showing congenital tremor in Anhui Province, China. Genome sequence analysis indicated that the two strains shared 81.19% to 93.98% nucleotide identities with other APPV strains.

Red recombination enables a wide variety of markerless manipulation of porcine epidemic diarrhea virus genome to generate recombinant virus.

Porcine epidemic diarrhea virus (PEDV) is a member of the genera Alphacoronavirus that has been associated with acute watery diarrhea and vomiting in swine. Unfortunately, no effective vaccines and antiviral drugs for PEDV are currently available. Reverse genetics systems are crucial tools for these researches. Here, a PEDV full-length cDNA clone was constructed. Furtherly, three PEDV reporter virus plasmids containing red fluorescent protein (RFP), Nano luciferase (Nluc), or green fluorescence protein (GFP) were generated using Red recombination with the GS1783 E. coli strain. These reporter-expressing recombinant (r) PEDVs showed similar growth properties to the rPEDV, and the foreign genes were stable to culture up to P9 in Vero cells. Using the Nluc-expressing rPEDV, the replication of PEDV was easily quantified, and a platform for rapid anti-PEDV drug screening was constructed. Among the three drugs, Bergenin, Umifenovir hydrochloride (Arbidol), and Ganoderma lucidum triterpenoids (GLTs), we found that GLTs inhibited PEDV replication mainly after the stage of virus "Entry". Overall, this study will broaden insight into the method for manipulating the PEDV genome and provide a powerful tool for screening anti-PEDV agents.

Porcine sapelovirus 2A protein induces mitochondrial-dependent apoptosis.

Porcine sapelovirus (PSV) is an emerging pathogen associated with symptoms of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in swine, resulting in significant economic losses. Although PSV is reported to trigger cell apoptosis, its specific molecular mechanism is unclear. In this research, the cell apoptosis induced by PSV infection and its underlying mechanisms were investigated. The morphologic features of apoptosis include nuclear condensation and fragmentation, were observed after PSV infection. The cell apoptosis was confirmed by analyzing the apoptotic rates, caspase activation, and PARP1 cleavage. Caspase inhibitors inhibited the PSV-induced intrinsic apoptosis pathway and reduced viral replication. Among the proteins encoded by PSV, 2A is an important factor in inducing the mitochondrial apoptotic pathway. The conserved residues H48, D91, and C164 related to protease activity in PSV 2A were crucial for 2A-induced apoptosis. In conclusion, our results provide insights into how PSV induces host cell apoptosis.

Disruption of interferon-ß production by the Npro of atypical porcine pestivirus.

Atypical porcine pestivirus (APPV) is an emerging porcine virus that threatens global swine production. Pestiviruses can prevent interferon (IFN) production to avoid the host innate immune response, and the Npro viral protein can play a critical role. Knowledge of the host immune response to APPV infection is limited. Here, we showed that the IFN-ß production was suppressed by APPV-Npro and the IFN regulatory factor 3 (IRF3) promoter activity stimulated by adaptor molecules of the IFN-ß signaling pathway was also inhibited in the APPV-Npro-expressed cells. The APPV-Npro was able to interact with IRF3 and interfere the phosphorylation of IRF3, indicated that the IFN-ß antagonism of APPV-Npro mainly depended on blocking IRF3 activity. To identify the functional region of APPV-Npro, a panel of truncated APPV-Npro was constructed, and its influence on the IRF3 activation was investigated. The results showed that the N-terminal 31-51 amino acids of APPV-Npro were mainly associated with inhibition of the IFN-ß response. Taken together, this is the first study focusing on elucidating the function of APPV protein by revealing a novel mechanism of Npro in disruption of host IFN-ß production, which will enlighten future study in addressing APPV pathogenesis and immune evasion.

Transcriptome analysis of senecavirus A-infected cells: Type I interferon is a critical anti-viral factor.

Senecavirus A (SVA)-associated vesicular disease (SAVD) has extensively been present in the swine industry during the past years. The mechanisms of SVA-host interactions at the molecular level, subsequent to SVA infection, are unclear. We studied the gene expression profiles of LLC-PK1 cells, with or without SVA infection, for 6 h and 12 h using an RNA-seq technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on differentially expressed genes (DEGs). Immune-related genes and pathways were significantly modified after SVA infection. To confirm the RNA-seq data, 28 important DEGs were selected for RT-qPCR assays. All DEGs exhibited expression patterns consistent with the RNA-seq results. Among them, type I IFNs (including IFN-α and IFN-ß) showed the largest upregulation, followed by RSAD2, DDX58, MX1 and the 17 other DEGs. In contrary, ID2 and another 5 DEGs were down-regulated or unchanged. These results indicated that type I IFNs play a critical role in host immune responses against SVA infection at early stage, while other immune-regulated genes directly or indirectly participate in the host immune responses.

Phylogenetic and codon usage analysis of atypical porcine pestivirus (APPV).

Atypical porcine pestivirus (APPV) has been identified as the main causative agent for congenital tremor (CT) type A-II in piglets, which is threatening the health of the global swine herd. However, the evolution of APPV remains largely unknown. In this study, phylogenetic analysis showed that APPV could be divided into three phylogroups (I, II, and III). Phylogroups I and II included viral strains from China, while phylogroup III contained strains from Europe, North America, and Asia. Phylogroups I and II are tentatively thought to be of Chinese origin. Next, compositional property analysis revealed that a high frequency of nucleotide A and A-end codons was used in the APPV genome. Intriguingly, the analysis of preferred codons revealed that the AGA[Arg] and AGG[Arg] were overrepresented. Dinucleotide CC was found to be overrepresented, and dinucleotide CG was underrepresented. Furthermore, it was found that the weak codon usage bias of APPV was mainly dominated by selection pressures versus mutational forces. The codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses showed that the codon usage patterns of phylogroup II and III were more similar to the one of a pig than phylogroup I, suggesting that phylogroup II and III may be more adaptive to pigs. Overall, this study provides insights into APPV evolution through phylogeny and codon usage pattern analysis.

Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) contains two major open reading frames (ORFs) and the ORF2 gene encodes the major structural capsid protein. In this study, nuclear localization of ORF2 was demonstrated by fluorescence observation and subcellular fractionation assays in ORF2-transfected PK-15 cells. The subcellular localization of truncated ORF2 indicated that the 38 N-terminal amino acids were responsible for the nuclear localization of ORF2. The truncated and site-directed mutagenesis of this domain were constructed, and the results demonstrated that the basic amino acid residues at positions 8-32 were essential for the strict nuclear localization. The basic motifs 8RRR-R-RRR16 and 16RRRHRRR22 were further shown to be the key functional nucleolar localization signals that guide PCV3 ORF2 into nucleoli. Furthermore, sequence analysis showed that the amino acids of PCV3 nuclear localization signals were highly conserved. Overall, this study provides insight into the biological and functional characteristics of the PCV3 ORF2 protein.

Pathogenicity of two Chinese Seneca Valley virus (SVV) strains in pigs.

Seneca Valley virus (SVV) has been identified as the causative agent of SVV-associated vesicular disease (SAVD). To investigate the pathogenicity of two newly isolated SVV strains (GD-S5/2018 and GD04/2017) in China, experimental infections of pigs were performed. In pig experiments, both SVV strains successfully infected all animals, evidenced by presence of virus shedding and robust protective antibody responses. SVV GD-S5/2018 infection resulted in characteristic clinical signs, and ulcerative lesions on the tongue and gums. However, SVV GD04/2017 did not cause any clinical symptoms except depression in pigs during the experiment. Taken together, these results demonstrate that SVV GD-S5/2018 is a virulent strain for pigs, whereas SVV GD04/2017 is nearly avirulent. The established animal models for SVV infection will be utilized to dissect the immunity and pathogenesis, and develop vaccines and antivirals.

Molecular evolution and characterization of novel Seneca Valley virus (SVV) strains in South China.

Seneca Valley virus (SVV) is an emerging swine virus associated with porcine vesicular disease. From June to the end of 2017, five SVV strains were isolated from the swine herds in Guangdong, China. Complete genomic sequences of these newly discovered SVV strains were analyzed by genomic and phylogenetic analysis. The results revealed that these SVV strains could be grouped into five genetic branches together with most of other Chinese strains. Interestingly, it is for the first time that a stretch of 11-nucleotide insertion was found in the 5' UTR region of SVV GD04/2017 strain. Additionally, phylogenetic analysis based on the ORF and VP1 genes showed that the SVV GD06/2017 strain is significantly distinct from all previous Chinese SVV strains. Furthermore, the five SVV strains displayed similar growth kinetics in the ST-R cell line, while the SVV GD04/2017 and GD06/2017 strains presented relatively slower rates than the GD01/2017 and GD03/2017 strains. The findings of this study indicate the emergence of novel SVV strains in China, which would increase the knowledge about SVV genetic diversity and reinforce the importance of SVV surveillance.

Molecular characterization of two novel atypical porcine pestivirus (APPV) strains from piglets with congenital tremor in China.

As one of emerging porcine viruses, atypical porcine pestivirus (APPV) was found in three continents since it emerged in 2015. It is now thought as the causative agent for congenital tremor type A-II in piglets. At the end of 2017, two APPV strains were identified from piglets with congenital tremor in Guangxi and Yunnan, China. The genome of APPV GX04/2017 strain was so far determined to be 11,534 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a polyprotein comprising 3,635 amino acids. Comparative analysis of ORF, Npro , E2, and NS3 gene sequences revealed that the APPV GX04/2017 strain shares nucleotide sequence identities of 82.8%-92.8% with other APPV strains, while YN01/2017 strain is 79.4%-97.4% homology to the others. Phylogenetic analysis showed that the APPV GX04/2017 and YN01/2017 are two novel APPV strains with the highest homology to each other, and relative high similarity to the APPV 000515 and JX-JM01 strains in genome sequence. The current findings provide updated information about APPV epidemiology and divergence in China, which would certainly help to establish reliable diagnosis and surveillance programs for APPV.

An emerging novel virus: Atypical porcine pestivirus (APPV).

Emerging porcine pestivirus diseases frequently challenge prevention and control strategies in the swine industry. Over the past decade, a few novel pestiviruses have been identified in pigs. This article focuses on the recently emerging atypical porcine pestivirus (APPV) that potentially threatens global swine herd health security. The virus was first identified in 2016, in the United States and thereafter, accumulated evidence shows that it is currently distributed in three continents. The clinical presentation of APPV-infected pigs is characterized by congenital tremor (CT) type A-II in piglets, while adult pigs may become persistent carriers and shedders. Here, a literature review is conducted to summarize the published findings in the virus genomic biology, transmission, epidemiology, pathogenesis, and diagnosis, which would shed light on acceleration of development of anti-APPV strategies.