Comparative transcriptome reveals EphA2 and c-Fos as key factors driving enhanced replication in high-passage porcine deltacoronavirus strain.

Porcine deltacoronavirus (PDCoV), a cross-species transmissible enterovirus, frequently induces severe diarrhea and vomiting symptoms in piglets, which not only pose a significant menace to the global pig industry but also a potential public safety risk. In a previous study, we isolated a vaccine candidate, PDCoV CZ2020-P100, by passaging a parental PDCoV strain in vitro, exhibiting attenuated virulence and enhanced replication. However, the factors underlying these differences between primary and passaged strains remain unknown. In this study, we present the transcriptional landscapes of porcine kidney epithelial cells (LLC-PK1) cells infected with PDCoV CZ2020-P1 strain and P100 strain using the RNA-sequencing. We identified 105 differentially expressed genes (DEGs) in P1-infected cells and 295 DEGs in P100-infected cells. Enrichment analyses indicated that many DEGs showed enrichment in immune and inflammatory responses, with a more and higher upregulation of DEGs enriched in the P100-infected group. Notably, the DEGs were concentrated in the MAPK pathway within the P100-infected group, with significant upregulation in EphA2 and c-Fos. Knockdown of EphA2 and c-Fos reduced PDCoV infection and significantly impaired P100 replication compared to P1, suggesting a novel mechanism in which EphA2 and c-Fos are highly involved in passaged virus replication. Our findings illuminate the resemblances and distinctions in the gene expression patterns of host cells infected with P1 and P100, confirming that EphA2 and c-Fos play key roles in high-passage PDCoV replication. These results enhance our understanding of the changes in virulence and replication capacity during the process of passaging.

Effective inhibition of PDCoV infection in chimeric APN gene-edited neonatal pigs.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection in vitro, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals. IMPORTANCE: Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.

Transcriptomic and antiviral analyses of PoIFN-Delta5 against porcine enteric viruses in porcine intestinal epithelial cells.

The interferon-delta family was first reported in domestic pigs and belongs to the type I interferon (IFN-I) family. The enteric viruses could cause diarrhea in newborn piglets with high morbidity and mortality. We researched the function of the porcine IFN-delta (PoIFN-δ) family in the porcine intestinal epithelial cells (IPEC-J2) cells infected with porcine epidemic diarrhea virus (PEDV). Our study found that all PoIFN-δs shared a typical IFN-I signature and could be divided into five branches in the phylogenic tree. Different strains of PEDV could induce typical IFN transitorily, and the virulent strain AH2012/12 had the strongest induction of porcine IFN-δ and IFN-alpha (PoIFN-α) in the early stage of infection. In addition, it was found that PoIFN-δ5/6/9/11 and PoIFN-δ1/2 were highly expressed in the intestine. PoIFN-δ5 had a better antiviral effect on PEDV compared to PoIFN-δ1 due to its higher induction of ISGs. PoIFN-δ1 and PoIFN-δ5 also activated JAK-STAT and IRS signaling. For other enteric viruses, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV), PoIFN-δ1 and PoIFN-δ5 both showed an excellent antiviral effect. Transcriptome analyses uncovered the differences in host responses to PoIFN-α and PoIFN-δ5 and revealed thousands of differentially expressed genes were mainly enriched in the inflammatory response, antigen processing and presentation, and other immune-related pathways. PoIFN-δ5 would be a potential antiviral drug, especially against porcine enteric viruses. These studies were the first to report the antiviral function against porcine enteric viruses and broaden the new acquaintances of this type of interferon though not novelly discovered.

Research Progress on Emerging Viral Pathogens of Small Ruminants in China during the Last Decade.

China is the country with the largest number of domestic small ruminants in the world. Recently, the intensive and large-scale sheep/goat raising industry has developed rapidly, especially in nonpastoral regions. Frequent trading, allocation, and transportation result in the introduction and prevalence of new pathogens. Several new viral pathogens (peste des petits ruminants virus, caprine parainfluenza virus type 3, border disease virus, enzootic nasal tumor virus, caprine herpesvirus 1, enterovirus) have been circulating and identified in China, which has attracted extensive attention from both farmers and researchers. During the last decade, studies examining the etiology, epidemiology, pathogenesis, diagnostic methods, and vaccines for these emerging viruses have been conducted. In this review, we focus on the latest findings and research progress related to these newly identified viral pathogens in China, discuss the current situation and problems, and propose research directions and prevention strategies for different diseases in the future. Our aim is to provide comprehensive and valuable information for the prevention and control of these emerging viruses and highlight the importance of surveillance of emerging or re-emerging viruses.

Caprine parainfluenza virus type 3 N protein promotes viral replication via inducing apoptosis.

Caprine parainfluenza virus type 3 (CPIV3) is one of the most important viral respiratory pathogens of goat. Accumulating evidence demonstrates that apoptosis is a cellular mechanism for the host response to pathogens, and it participates in regulating viral replication. However, there is little study on CPIV3-induced host cells apoptosis. In this study, primary goat tracheal epithelial (GTE) cells were established as a cellular model that is permissive to CPIV3 infection. Then, we showed that CPIV3 infection induced apoptosis in GTE cells, as determined by morphological changes, flow cytometry and TUNEL assay. Moreover, Caspase activity and the expression of pro-apoptotic genes further suggested that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. Mechanistically, the ability of CPIV3 to induce apoptosis was activated by N protein, and the viral protein increased CPIV3 replication through effecting apoptosis. Overall, our findings showed that GTE cells that will enable further analysis of CPIV3 infection and offers novel insights into the mechanisms of CPIV3-induced apoptosis in host cells.

Screening interferon antagonists from accessory proteins encoded by P gene for immune escape of Caprine parainfluenza virus 3.

The Caprine parainfluenza virus 3 (CPIV3) is a novel Paramyxovirus that is isolated from goats suffering from respiratory diseases. Presently, the pathogenesis of CPIV3 infection has not yet been fully characterized. The Type I interferon (IFN) is a key mediator of innate antiviral responses, as many viruses have developed strategies to circumvent IFN response, whether or how CPIV3 antagonizes type I IFN antiviral effects have not yet been characterized. This study observed that CPIV3 was resistant to IFN-α treatment and antagonized IFN-α antiviral responses on MDBK and goat tracheal epithelial (GTE) cell models. Western blot analysis showed that CPIV3 infection reduced STAT1 expression and phosphorylation, which inhibited IFN-α signal transduction on GTE cells. By screening and utilizing specific monoclonal antibodies (mAbs), three CPIV3 accessory proteins C, V and D were identified during the virus infection process on the GTE cell models. Accessory proteins C and V, but not protein D, was identified to antagonize IFN-α antiviral signaling. Furthermore, accessory protein C, but not protein V, reduced the level of IFN-α driven phosphorylated STAT1 (pSTAT1), and then inhibit STAT1 signaling. Genetic variation analysis to the PIV3 accessory protein C has found two highly variable regions (VR), with VR2 (31-70th aa) being involved in for the CPIV3 accessory protein C to hijack the STAT1 signaling activation. The above data indicated that CPIV3 is capable of inhibiting IFN-α signal transduction by reducing STAT1 expression and activation, and that the accessory protein C, plays vital roles in the immune escape process.

Bta-miR-98 Suppresses Replication of Caprine Parainfluenza Virus Type 3 Through Inhibiting Apoptosis by Targeting Caspase-3.

Caprine parainfluenza virus type 3 (CPIV3) is an emerging respiratory pathogen that affects the sheep and goat industry in China and possibly other countries around the world. Accumulating evidence suggests that microRNAs play important roles in regulating virus-host interactions and can suppress or facilitate viral replication. In this study, we showed that CPIV3 infection induced apoptosis in Madin-Darby bovine kidney (MDBK) cells, as determined by morphological changes and flow cytometry. Caspase activity and the expression of pro-apoptotic genes further indicated that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. We also demonstrated the involvement of bta-microRNA-98 (bta-miR-98) in regulating CPIV3-induced apoptosis. Bta-miR-98 was downregulated in MDBK cells infected with CPIV3. Overexpression of bta-miR-98 significantly decreased the activities of caspase-3, -8, and -9. Conversely, inhibition of bta-miR-98 had completely opposite effects. Furthermore, our data showed that bta-miR-98 markedly affected CPIV3 replication by regulating apoptosis. Importantly, we found that bta-miR-98 modulated CPIV3-induced apoptosis by targeting caspase-3, an effector of apoptosis. Collectively, our results may suggest that CPIV3 infection induced apoptosis and downregulated the levels of bta-miR-98, and this miRNA regulated viral replication through effected apoptosis. This study contributes to our understanding of the molecular mechanisms underlying CPIV3 pathogenesis.

Cholesterol-rich lipid rafts both in cellular and viral membrane are critical for caprine parainfluenza virus type3 entry and infection in host cells.

Cholesterol-rich lipid rafts have been shown to play important roles in the life cycle of various non-enveloped and enveloped viruses. Deletion of cholesterol from lipid rafts could influence different steps of viral replication cycle including entry, infection, assembly and release. Caprine parainfluenza virus type3 (CPIV3) is a newly identified member of Paramyxoviridae family. CPIV3 is highly prevalence and threatened the goat industry in China. The infection mechanism of CPIV3 is under exploring and still not fully understood, the roles of cholesterol and lipid rafts for CPIV3 infection remains unclear. In this study, we investigated the association of cholesterol and lipid rafts with CPIV3 during the different viral replication stages (binding, entry and infection) in two cells [MDBK and goat bronchial epithelial (GBE) cells]. Methyl-ß- cyclodextrin (MßCD) was used to deplete cholesterol from cell and viral membranes. The results showed that MßCD treatment significantly inhibited CPIV3 entry and infection in these two cells with a dose-dependent manner, but didn't impair the binding of CPIV3. Addition of exogenous cholesterol to the cells after MßCD treatment restored the viral infection. In addition, treatment of MßCD only before virus-entry showed inhibitory effect in MDBK cells. Depletion of cholesterol from virion envelop also decreased the entry and infection of CPIV3 in the two cells. Furthermore, lipid rafts isolation test indicated that viral proteins (HN and N) co-localized with lipid rafts during infection in MDBK and GBE cells. Viral N protein co-localized with caveolin-1 (the marker of lipid rafts) in these two cells both at the entry and infection steps, as detected by con-focal laser scanning microscopy test. In conclusion, the results presented here demonstrated that cholesterol rich lipid rafts play an important role in CPIV3 life cycle. The findings give new insights on understanding of the mechanism of CPIV3 infection and provide a new anti-CPIV3 strategy.

Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells.

Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.

Epidemiological investigation and genomic characterization of Caprine herpesvirus 1 from goats in China.

Caprine herpesvirus 1 (CpHV-1) is a member of the alpha subfamily of herpersviruses, and is responsible for genital lesions and latent infections in goat population worldwide. Here, we describe goats suffered severe respiratory diseases caused by alphaherpesvirus during 2013 to 2014 in Jiangsu province of China. CpHV-1 was detected out by PCR with a prevalence of 21.1% (40/190), among which three novel CpHV-1 strains were firstly identified and isolated in China. Phylogenetic analysis of glycoprotein B (gB) gene revealed that these new viruses were closely clustered with CpHV-1 strain E/CH. The isolate JSHA1405 was further studied by transmission electron microscopy, and displayed typical herpesvirus morphology. Then, for the first time, complete viral genome of JSHA1405 was sequenced by Illumina Hiseq and third-generation sequencing technology. The viral genome is 134,617 bp in length and the genome characteristics were deeply analyzed. 69 open reading frames were predicted and annotated, which was less than that of BoHV-1. Phylogenetic analysis of the complete genome revealed that JSHA1405 was classified into the same branch with previous CpHV-1 strains as well. Moreover, the pathogenicity test is further evidence that JSHA1405 strain induced obvious symptoms of high fever and nasal discharge in infected goats, consistent with clinical manifestations. This is the first report about isolation and identification of CpHV-1 in China and the first characterization of CpHV-1 genome structure. The research also provides a basis for understanding the characteristics, viral genome and pathogenicity of the virus.

Pathogenicity and horizontal transmission studies of caprine parainfluenza virus type 3 JS2013 strain in goats.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. In 2014, one novel PIV3 strain was first isolated from goats suffered respiratory diseases in Jiangsu and Anhui provinces of eastern China and named as caprine PIV3 (CPIV3) JS2013. In order to systematically evaluate the pathogenicity and horizontal transmission ability of this new virus, experimental infection of goats with the CPIV3 strain was done. The virus-inoculated goats (challenge control (CC) group) displayed coughing and nasal discharges from 3days post infection (dpi) and lasted for about 2 weeks. Two goats in group CC showed fever between 7 and 12dpi. As detected by a TaqMan real time quantitative RT-PCR (qRT-PCR), viremia was detected during 3-11dpi, peaked at 6dpi; and virus shedding from nasal discharge and faeces were confirmed during 3-21dpi and 4-21dpi, respectively. Virus-specific HI antibodies and neutralizing antibodies (NAs) became positive since 7dpi and 14dpi; peaked at 14dpi and 28dpi, respectively; and lasted at least 70days. Pathological lesions were mainly found on the lungs and tracheas. In addition, viruses were also detected in part of the tracheal secretion and lung samples, and the viral load in tracheal secretion was higher than that in lungs. Goats in horizontal infected group (hCC, kept in different cages in the same house with CC group) showed to be horizontally infected, with slightly milder clinical signs and pathological changes; and slightly shorter period of viremia and virus shedding. This was the first report of the detailed pathogenicity characterization of the novel CPIV3 and demonstrated its horizontal transmission ability. The results would be helpful for further studies on the preventive and control strategies for CPIV3 infections.