Antiviral effects of ergosterol peroxide in a pig model of porcine deltacoronavirus (PDCoV) infection involves modulation of apoptosis and tight junction in the small intestine.

Porcine deltacoronavirus (PDCoV) is a newly discovered swine enteropathogenic coronavirus with worldwide distribution. However, efficient strategies to prevent or treat the infection remain elusive. Our in vitro study revealed that ergosterol peroxide (EP) from the mushroom Cryptoporus volvatus has efficient anti-PDCoV properties. The aim of this study is to evaluate the potential of EP as a treatment for PDCoV in vivo and elucidate the possible mechanisms. Seven-day-old piglets were infected with PDCoV by oral administration in the presence or absence of EP. Piglets infected with PDCoV were most affected, whereas administration of EP reduced diarrhea incidence, alleviated intestinal lesion, and decreased viral load in feces and tissues. EP reduced PDCoV-induced apoptosis and enhanced tight junction protein expressions in the small intestine, maintaining the integrity of the intestinal barrier. EP showed immunomodulatory effect by suppressing PDCoV-induced pro-inflammatory cytokines and the activation of IκBα and NF-κB p65, and upregulating IFN-I expression. Knockdown of p38 inhibited PDCoV replication and alleviated PDCoV-induced apoptosis, implying that EP inhibited PDCoV replication and alleviated PDCoV-induced apoptosis via p38/MAPK signaling pathway. Collectively, ergosterol peroxide can protect piglets from PDCoV, revealing the potential of EP for development as a promising strategy for treating and controlling the infection of PDCoV.

Porcine deltacoronavirus infection is inhibited by Griffithsin in cell culture.

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus that causes severe diarrhea in piglets and results in serious economic losses. There are no effective vaccines and antiviral drugs to prevent and treat PDCoV infection currently. Griffithsin (GRFT) is a lectin with potent antiviral activity against enveloped viruses because of its ability to specifically bind N-linked high-mannose oligosaccharides. GRFT has been reported to possess antiviral activity against severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and porcine epidemic diarrhea virus (PEDV). Here, we first confirmed the antiviral activity of GRFT against PDCoV in vitro. The infected cells (%) and virus titers were significantly decreased at concentration 1 µg/mL or above of GRFT. Time-course experiments revealed that GRFT inhibits PDCoV infection at the adsorption and penetration step. GRFT binding to PDCoV spike (S) protein on the surface wraps the virus and blocks its entry. The outstanding antiviral potency indicates that GRFT has the potential value as a candidate drug for the prevention and treatment of PDCoV infection.

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.

Bile acids LCA and CDCA inhibited porcine deltacoronavirus replication in vitro.

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes gastroenteritis in pigs and no vaccines or antiviral drugs are available. Bile acids are active factors in intestines and influence the replication of enteric viruses. Currently, the role of bile acids on PDCoV replication is unknown. In this study, we tested the effects of different types of bile acids on the replication of PDCoV in cell culture. We found that physiological concentrations of bile acids chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) had antiviral activity against PDCoV in porcine kidney cell line (LLC-PK1) and porcine small intestinal epithelial cell line (IPEC-J2). In IPEC-J2 cells, CDCA and LCA inhibited PDCoV replication at post-entry stages by inducing the production of interferon (IFN)-λ3 and IFN-stimulated gene 15 (ISG15) via G protein-coupled receptor (GPCR). In summary, bile acids CDCA and LCA restricted PDCoV infection and LCA functioned through a GPCR-IFN-λ3-ISG15 signaling axis in IPEC-J2 cells. Our results may open new avenues for the development of antiviral drugs to treat PDCoV infection in pigs.

Ergosterol peroxide suppresses porcine deltacoronavirus (PDCoV)-induced autophagy to inhibit virus replication via p38 signaling pathway.

Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus (CoV) that continues to spread globally, placing strain on economic and public health. Currently, the pathogenic mechanism of PDCoV remains largely unclear, and effective strategies to prevent or treat PDCoV infection are still limited. In this study, the interaction between autophagy and PDCoV replication in LLC-PK1 cells was investigated. We demonstrated that PDCoV infection induced a complete autophagy process. Pharmacologically induced autophagy with rapamycin increased the expression of PDCoV N, while pharmacologically inhibited autophagy with wortmannin decreased the expression of PDCoV N, suggesting that PDCoV-induced autophagy facilitates virus replication. Further experiments showed that PDCoV infection activated p38 signaling pathway to trigger autophagy. Besides, ergosterol peroxide (EP) alleviated PDCoV-induced activation of p38 to suppress autophagy, thus exerting its antiviral effects. Finally, we employed a piglet model of PDCoV infection to demonstrate that EP prevented PDCoV infection by suppressing PDCoV-induced autophagy via p38 signaling pathway in vivo. Collectively, these findings accelerate the understanding of the pathogenesis of PDCoV infection and provide new insights for the development of EP as an effective therapeutic strategy for PDCoV.

Ergosterol peroxide exhibits antiviral and immunomodulatory abilities against porcine deltacoronavirus (PDCoV) via suppression of NF-κB and p38/MAPK signaling pathways in vitro.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that poses economic and public health burdens. Currently, there are no effective antiviral agents against PDCoV. Cryptoporus volvatus often serves as an antimicrobial agent in Traditional Chinese Medicines. This study aimed to evaluate the antiviral activities of ergosterol peroxide (EP) from C. volvatus against PDCoV infection. The inhibitory activity of EP against PDCoV was assessed by using virus titration and performing Quantitative Reverse transcription PCR (RT-qPCR), Western blotting and immunofluorescence assays in LLC-PK1 cells. The mechanism of EP against PDCoV was analyzed by flow cytometry, RT-qPCR and Western blotting. We found that EP treatment inhibited PDCoV infection in LLC-PK1 cells in a dose-dependent manner. Subsequently, we demonstrated that EP blocked virus attachment and entry using RT-qPCR. Time-of-addition assays indicated that EP mainly exerted its inhibitory effect at the early and middle stages in the PDCoV replication cycle. EP also inactivated PDCoV infectivity directly as well as suppressed PDCoV-induced apoptosis. Furthermore, EP treatment decreased the phosphorylation of IκBα and p38 MAPK induced by PDCoV infection as well as the mRNA levels of cytokines (IL-1ß, IL-6, IL-12, TNF-α, IFN-α, IFN-ß, Mx1 and PKR). These results imply that EP can inhibit PDCoV infection and regulate host immune responses by downregulating the activation of the NF-κB and p38/MAPK signaling pathways in vitro. EP can be used as a potential candidate for the development of a new anti-PDCoV therapy.

Rhodanine derivative LJ001 inhibits TGEV and PDCoV replication in vitro.

Transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are members of the family coronaviridae and mainly cause acute diarrhea/vomiting, dehydration and mortality in piglets, which lead to huge economic losses to the swine industry. Rhodanine derivative LJ001 has been verified to be effective against some enveloped virus infections in vitro. In this study, we evaluated the antiviral activity of LJ001 towards TGEV and PDCoV replication on swine testicular(ST) cells. Our results showed the 50 % cellular cytotoxicity (CC50) value of LJ001 was 146.4 µM on ST cell. The virus titers of TGEV and PDCoV were obviously decreased in the presence of LJ001 with the concentrations of 3.125 and 12.5 µM, and LJ001 potently inhibited TGEV and PDCoV infection at the replication stages of viral life cycle. Further study indicated that LJ001 inhibited TGEV and PDCoV replication by inhibition of viral RNA and protein synthesis, and reducing virus yields at 12 and 24 h post-inoculation. These data indicated that LJ001 had antiviral activities on TGEV and PDCoV replications in vitro, which may serve as a new candidate for treatment of coronaviruses infections.