Porcine delta coronavirus inhibits NHE3 activity of porcine intestinal epithelial cells through miR-361-3p/NHE3 regulatory axis.

Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.

Identification of a receptor tyrosine kinase inhibitor CP-724714 inhibits SADS-CoV related swine diarrhea coronaviruses infection in vitro.

The outbreak of the COVID-19 epidemic in 2020 has caused unprecedented panic among all mankind, pointing the major importance of effective treatment. Since the emergence of the swine acute diarrhea syndrome coronavirus (SADS-CoV) at the end of 2017, multiple reports have indicated that the bat-related SADS-CoV possesses a potential threat for cross-species transmission. Vaccines and antiviral drugs development deserve more attention. In this study, we found that the HER2 phosphorylation inhibitor (CP-724714) inhibited SADS-CoV infection in a dose-dependent manner. Further validation demonstrated that CP-724714 affected at the post-entry stage of SADS-CoV infection cycle. Also, efficient SADS-CoV infection required the activation of HER2 and its cascade Ras-Raf-Mek-Erk signaling pathway. In addition, CP-724714 has a broad-spectrum anti-swine diarrhea coronaviruses activity, and can dose-dependently combat SADS-CoV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) infection in vitro with a specificity index of greater than 21.98, 9.38, 95.23 and 31.62, respectively. These results highlight the potential utility of CP-724714 or antiviral drugs targeting with HER2 and its cascade Ras-Raf-Mek-Erk signaling pathway as host-targeted SADS-CoV and other related coronaviruses therapeutics.

Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Introduction: Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Methods: Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). Results and discussion: The newly developed system was shown to be sensitive with detection limits of 101 copies/µl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/µl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

Combined Analysis of the Whole Transcriptome of Piglets Infected with SADS-CoV Virulent and Avirulent Strains.

When piglets are infected by virulent and avirulent strains of swine acute diarrhea syndrome coronavirus (SADS-CoV), there are obvious differences in their clinical symptoms; however, the specific mechanisms of pathogenicity and the immune regulation of highly pathogenic and low pathogenic strains are unknown. We collected intestinal tissues from SADS-CoV-infected piglets, performed a whole transcriptome sequencing analysis, including mRNA, miRNA, lncRNA, cicrRNA, and TUCP, and performed functional and correlation analyses of differentially expressed RNAs. Our results showed that the differentially expressed RNAs in group A versus group B (AvsB), group A versus group C (AvsC), and group B versus group C (BvsC) were relevant to immune and disease-related signaling pathways that participate in the organisms' viral infection and immune regulation. Furthermore, data obtained from the HAllA analysis suggested that there was a strong correlation between the differentially expressed RNAs. Specifically, LNC_011487 in the P set was significantly negatively correlated with ssc-miR-215, and LNC_011487 was positively correlated with PI3. Moreover, we also constructed a differentially expressed RNA association network map. This study provides a valuable resource for studying the SADS-CoV transcriptome and pathogenic mechanism from the perspective of RNA to understand the differences in and consistency of the interaction between virulent and attenuated SADS-CoV strains and hosts.

Early life dynamics of ARG and MGE associated with intestinal virome in neonatal piglets.

The pre- and post-weaning stages for piglets are critical periods for the maturation of intestinal functions and contamination with antibiotic resistant bacterial pathogens will threaten their intestinal health. The presence of bacteriophage can also alter bacterial populations in the intestine but whether transmission of antibiotic resistance genes (ARG) is affected by phage during maturation of the neonatal piglet intestine is not known. We therefore identified the intestinal virome along with ARGs and mobile genetic elements (MGE) from piglet fecal samples collected from 3 to 28 days representing the different growth stages. We found wide fluctuations for the intestinal virome of weaning piglets and most virus - related antibiotic resistance was derived from temperate phage suggesting a reservoir of multidrug resistance was present in the neonatal porcine gut. Our results provide a comprehensive understanding of ARGs associated with the intestinal virome that therefore represents a potential risk for horizontal ARG transfer to pathogenic bacteria.

Swine Acute Diarrhea Syndrome Coronavirus Nucleocapsid Protein Antagonizes Interferon-ß Production via Blocking the Interaction Between TRAF3 and TBK1.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-ß promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-ß production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.

Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 101 copies/µL, 102 copies/µL and 102 copies/µL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s<5%) and specificities (100%), and possessed sensitivities of 92.24%, 92.19% and 91.23% for PEDV, PDCoV and SADS-CoV respectively. In summary, the established microfluidic-RT-LAMP chip detection system could satisfy the demanding in field diagnoses, which was suitable for promotion in remote areas due to its fast, portable and cost-effective characters.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) antagonizes interferon-ß production via blocking IPS-1 and RIG-I.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly emerging enteric coronavirus, is considered to be associated with swine acute diarrhea syndrome (SADS) which has caused significantly economic losses to the porcine industry. Interactions between SADS-CoV and the host innate immune response is unclear yet. In this study, we used IPEC-J2 cells as a model to explore potential evasion strategies employed by SADS-CoV. Our results showed that SADS-CoV infection failed to induce IFN-ß production, and inhibited poly (I:C) and Sendai virus (SeV)-triggered IFN-ß expression. SADS-CoV also blocked poly (I:C)-induced phosphorylation and nuclear translocation of IRF-3 and NF-κB. Furthermore, SADS-CoV did not interfere with the activity of IFN-ß promoter stimulated by IRF3, TBK1 and IKKε, but counteracted its activation induced by IPS-1 and RIG-I. Collectively, this study is the first investigation that shows interactions between SADS-CoV and the host innate immunity, which provides information of the molecular mechanisms underlying SASD-CoV infection.

Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. METHODS: B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. RESULTS: We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. CONCLUSION: The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.

A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs.

PEDV variants, causing severe diarrhea in neonatal suckling piglets with a mortality rate up to 100%, have being epidemic since late 2010 in china. To meet the pressing need of safe and cost-efficient PED maternal vaccines against PEDV variant, we vaccinated growing piglets with a flagellin-adjuvanted PED vaccine rSF-COE-3D by injection at Houhai acupoint. The vaccination not only enhanced the antibody responses of serum IgG/IgA, mucosal IgA and serum neutralizing antibody, but also improved the production of IFN-γ and IL-4. Moreover, rSF-COE-3D could provide a better protection against the challenge of a high pathogenic PEDV variant, with less diarrhea pigs, less pigs with detectable PEDV shed, lower rank values of feces and less apparent lesions and inflammation in duodenum of the PEDV infected pigs. The improved protective efficiency of rSF-COE-3D compared with COE was mostly benefited from the enhanced production of serum IgA, mucosal IgA in feces and serum neutralizing antibody titers. Taken together, our data suggest that rSF-COE-3D would be a novel efficient PED vaccine.

PED subunit vaccine based on COE domain replacement of flagellin domain D3 improved specific humoral and mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is an important re-emergent infectious disease and inflicts huge economic losses to the swine industry worldwide. To meet the pressing need of developing a safe and cost-efficient PED maternal vaccine, we generated three PED subunit vaccine candidates, using recombined Salmonella flagellin (rSF) as a mucosal molecular adjuvant. Domain D3 in rSF was replaced with COE domain of PEDV to generate rSF-COE-3D. COE fused to the flanking C'/N' terminal of rSF yielded rSF-COE-C and rSF-COE-N. As a result, rSF-COE-3D could significantly improve COE specific antibody production including serum IgG, serum IgA, mucosal IgA and PEDV neutralizing antibody. Furthermore, rSF-COE-3D elicited more CD3+CD8+ T cell and cytokine production of IFN-γ and IL-4 in mouse splenocytes. In summary, our data showed that rSF-COE-3D could improve specific humoral and mucosal immunity in mice, thus suggesting that rSF-COE-3D could be applied as a novel efficient maternal PED vaccine.

Rapid quantitation of porcine epidemic diarrhea virus (PEDV) by Virus Counter.

Virus Counter was applied for the quantitation of porcine epidemic diarrhea virus (PEDV); the working range and reproducibility of the quantification were characterized, and the performance of Virus Counter was evaluated using the plaque assay as the gold standard. In this study, the single burst curves of PEDV were first constructed with the Virus Counter or plaque assay respectively, and the results from Virus Counter and plaque assay showed similar high correlation for interval of 12-24 h (r=0.9581, p<0.05). Then three virus batches from independent virus propagations were harvested at interval of 16-20 h and serial-diluted; The correlation between the calculated titer and the results obtained by Virus Counter were better than those obtained by using the plaque assay. The comparison between the plaque assay data and the Virus Counter results revealed a linear relationship (slope=1.155±0.46, R(2)=0.9665) with a significant Pearson correlation (r=0.9809, p<0.0001). The results of this study demonstrated that Virus Counter is a simple and reliable method to quantify the viral particles of PEDV.