Zhenhui Song

Feline infectious peritonitis virus (FIPV), a highly pathogenic subtype of feline coronavirus, is characterized by antigenic variability and immune evasion, resulting in limited efficacy of existing clinical therapeutic regimens. In recent years, ursodeoxycholic acid (UDCA), a hydrophilic bile acid derivative, has been found to have broad antiviral activity. Based on this, this study significantly improved the replication efficiency of FIPV by constructing a CRFK-PHBLV-N cell line that can stably express the FIPV N protein; UDCA had obvious antiviral effects, and signaling pathways related to UDCA’s anti-FIPV activity were screened by transcriptome sequencing, and it was found that UDCA could significantly promote secretion of interferon-β by CRFK-PHBLV-N cells, activating the JAK-STAT signaling pathway and upregulating the expression of interferon-stimulated genes. This study revealed that FIPV escapes host immune response by inhibiting the JAK-STAT pathway, and UDCA was able to reverse this inhibition and enhance the expression of antiviral proteins, thus effectively inhibiting the replication and spread of FIPV. Furthermore, UDCA can also directly disrupt the envelope components of FIPV, inducing the disintegration of the viral structure. This study not only provides a new strategy for the efficient in vitro culture of FIPV, but also lays a theoretical foundation for the development of anti-FIPV drugs targeting the JAK-STAT pathway. IMPORTANCE: In this study, through the establishment of a complete research chain of “virus titer-enhanced cell model–drug effect evaluation–signaling pathway analysis”, we confirmed that ursodeoxycholic acid (UDCA) can inhibit feline infectious peritonitis virus (FIPV) infection and revealed that UDCA can activate the JAK-STAT signaling pathway by promoting the production of large amounts of interferon-β in host cells to protect against FIPV infection. It not only provides a new experimental tool (CRFK-PHBLV-N cell line) and drug candidate (UDCA) for FIP prevention and control, but also lays a theoretical foundation for the development of novel therapeutic strategies targeting the host antiviral pathway through the elucidation of the JAK-STAT pathway mechanism.

Viruses rely on energy and biosynthetic materials of host cell from glucose metabolism to support their replication, and glucose plays a crucial role in viral infection. In this study, we found that porcine epidemic diarrhea virus (PEDV) infection significantly increased cellular glucose uptake and stimulated the production of the glycometabolite lactate. Exogenous supplementation of glucose or L-lactate confirmed that it significantly promoted PEDV proliferation, indicating that replication of PEDV was enhanced by regulating host glucose metabolism, particularly reprogramming glycolysis. Based on these findings, we explored the potential antiviral approach targeting the virus through regulating glycolytic processes. Metformin hydrochloride (MH) is a well-known hypoglycemic agent, which has shown notable anti-PEDV activity. After MH treatment, the transcriptome analysis showed the differential genes were mainly enriched in PI3K-AKT signaling pathway, and the expression levels of its downstream molecule GSK3B and MYC were significantly upregulated and downregulated, respectively. The gene expression related to glycolysis was also significantly inhibited. Further experiments showed that MH significantly inhibited the phosphorylation of AKT and its translocation to plasma membrane, while reducing the phosphorylation level of GSK3B. MH maintained GSK3B in a non-phosphorylated state by blocking the activation of the EGFR/PI3K/AKT/GSK3B pathway, mediated the degradation of c-MYC through phosphorylation, inhibited the glycolysis process, reduced the production of lactic acid, and finally exerted its antiviral effect. This study demonstrated that PEDV infection could induce glycolysis through metabolic reprogramming, thereby promoting viral replication; whereas, MH was able to effectively reverse this process, significantly inhibiting the virus-induced glycolysis pathway and exhibiting antiviral activity. IMPORTANCE: This study aims to elucidate the antiviral effects and molecular mechanisms of MH against PEDV. The results show that MH can inhibit the activation of the PI3K-AKT signaling pathway induced by PEDV infection, thereby suppressing the production of the glycolytic product L-lactic acid and ultimately resisting PEDV infection. This research provides new insights into the prevention and control of PEDV and offers scientific evidence for the application of MH in veterinary medicine.

Porcine Respiratory Coronavirus (PRCV) is a genetic variant of Transmissible Gastroenteritis Virus (TGEV). It is only pathogenic to the respiratory tract, mainly manifesting as atypical interstitial pneumonia and mild subclinical symptoms. In addition, PRCV can also serve as a potential animal respiratory coronavirus model for the study of human respiratory coronaviruses. Developing effective PRCV antagonists is of great significance for the prevention and control of this disease. In this study, we found that Ursodeoxycholic acid (UDCA) can significantly inhibit the infection of PRCV in Porcine respiratory epithelial cells (NPTR). Further studies have shown that UDCA inhibits PRCV mainly through two mechanisms: First, UDCA can directly disrupt the components of the viral envelope and induce the disintegration of viral structure; Second, UDCA can significantly promote the secretion of IFN-β in NPTR cells, enhance the phosphorylation and nuclear translocation of STAT1, and up-regulate the expression of the interferon-stimulated genes ISG15 and MX1. Molecular dynamics simulations showed that UDCA can be embedded into the hydrophobic pocket of the TLR4 dimerization domain, thereby activating the TLR4-IRF3 signaling pathway, inducing the production of IFN-β, and inhibiting PRCV infection. Schaftoside, an inhibitor of the TLR4 signaling pathway, can effectively reverse the anti-PRCV activity of UDCA. Finally, an ex vivo lung tissue slice model of piglets was established to verify that UDCA can effectively reduce the PRCV viral load and inflammatory response in lung tissues. The results of this study provide a scientific basis for the development of antiviral drugs against PRCV and offer new insights into the research on human respiratory coronaviruses.

Hepatitis E virus (HEV) is one of the pathogens that cause viral hepatitis, and its clinical symptoms can manifest as acute, chronic viral hepatitis, or asymptomatic infection. Among them, swines are the main animal source of HEV. Open reading frame 3 (ORF3) is a multifunctional protein essential for swine hepatitis E virus (SHEV) infection and release, involved in biological processes such as intracellular signal transduction regulation. In our preliminary research, we utilized adenovirus-mediated overexpression of type IV SHEV ORF3 in HepG2 cells, extracted total RNA, and performed high-throughput long non coding RNAs (lncRNAs) and transcriptome sequencing. In this study, we screened and analyzed lncRNAs involved in the GO pathway: viral process (GO: 0016032), and combined them with differentially expressed mRNAs for target gene prediction. We identified two lncRNAs—lncRNA AL137002 (MSTRG. 7478) and lncRNA AL049840 (MSTRG. 8427)—that are associated with viral progression and have p ≤ 0.05 in HepG2 cells expressing ORF3 of porcine hepatitis E virus type IV. We predicted their five lncRNA-mRNA networks, which are lncRNA AL137002 (MSTRG. 7478)-ENST0000375440, lncRNA AL137002 (MSTRG. 7478)-ENST0000375441, lncRNA AL049840(MSTRG. 8427)-ENST0000246489, lncRNA AL049840 (MSTRG. 8427)-ENST0000554280 and lncRNA AL049840 (MSTRG. 8427)-ENST0000452929, and were used to predict their lncRNA mRNA binding sites and construct relevant molecular models. This will lay a solid foundation for further revealing the function of SHEV ORF3 and elucidating the mechanism of SHEV infection.

In recent years, the prevalence and incidence of porcine epidemic diarrhea virus (PEDV) infection have been on the rise. The occurrence of multiviral infections and recombination mutations has led to accelerated viral evolution and reduced vaccine efficacy. In the present study, a PEDV strain was isolated from a pig farm (Chongqing Province, China) with an outbreak of porcine diarrhea, and its S gene was found to be recombinant. The optimal trypsin concentration for blind passage of PEDV in Vero cells was determined to be 7.5 µg/mL. Following two blind passages of the virus in Vero cells, the virus was unable to adapt to the cells. Therefore, PEDV was blindly passaged in IPEC-J2 cells using the optimal concentration of trypsin (5 µg/mL). Next, a series of characterization experiments were performed. Recombination analyses of the isolates using software revealed that the S gene of strain CHCQ-2023 was derived from the primary parent strain PEDV-1 C and secondary parent strain SQ2014, with recombination occurring at a 3152 bp breakpoint. Furthermore, a specific B-cell antigenic epitope was predicted on the S2 subunit of the S protein. A PEDV strain was isolated and characterized, and its S gene was characterized. The findings provide a bioinformatic basis for the study of PEDV strain variation due to genetic recombination.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes high mortality in piglets, thus posing a serious threat to the world pig industry. Porcine epidemic diarrhea (PED) is related to the imbalance of sodium absorption by small intestinal epithelial cells; however, the etiology of sodium imbalanced diarrhea caused by PEDV remains unclear. Herein, we first proved that PEDV can cause a significant decrease in Na ⁺ /H ⁺ exchanger 3 (NHE3) expression on the cell membrane, in a viral dose-dependent manner. Further study showed that the PEDV nucleocapsid (N) protein participates in the regulation of NHE3 activity through interacting with Ezrin. Flame atomic absorption spectroscopy results indicated a serious imbalance in Na ⁺ concentration inside and outside cells following overexpression of PEDV N. Meanwhile, molecular docking technology identified that the small molecule drug Pemetrexed acts on the PEDV N-Ezrin interaction region. It was confirmed that Pemetrexed can alleviate the imbalanced Na ⁺ concentration in IPEC-J2 cells and the diarrhea symptoms of Rongchang pigs caused by PEDV infection. Overall, our data suggest that the interaction between PEDV N and Ezrin reduces the level of phosphorylated Ezrin, resulting in a decrease in the amount of NHE3 protein on the cell membrane. This leads to an imbalance of intracellular and extracellular Na ⁺ , which causes diarrhea symptoms in piglets. Pemetrexed is effective in relieving diarrhea caused by PEDV. Our results provide a reference to screen for anti-PEDV targets and to develop drugs to prevent PED. IMPORTANCE: Porcine epidemic diarrhea (PED) has caused significant economic losses to the pig industry since its initial outbreak, and the pathogenic mechanism of porcine epidemic diarrhea virus (PEDV) is still under investigation. Herein, we found that the PEDV nucleocapsid protein interacts with Ezrin to regulate Na ⁺ /H ⁺ exchanger 3 activity. In addition, we screened out Pemetrexed, a small molecule drug, which can effectively alleviate pig diarrhea caused by PEDV. These results provide support for further exploration of the pathogenesis of PEDV and the development of drugs to prevent PED.

As part of the genus Enteropathogenic Coronaviruses, Porcine Epidemic Diarrhea Virus (PEDV) is an important cause of early diarrhea and death in piglets, and one of the most difficult swine diseases to prevent and control in the pig industry. Previously, we found that PEDV can block Na⁺ absorption and induce diarrhea in piglets by inhibiting the activity of the sodium-hydrogen ion transporter NHE3 in pig intestinal epithelial cells, but the mechanism needs to be further explored. The epidermal growth factor receptor (EGFR) has been proved to be one of the co-receptors involved in many viral infections and a key protein involved in the regulation of NHE3 activity in response to various pathological stimuli. Based on this, our study used porcine intestinal epithelial cells (IPEC-J2) as an infection model to investigate the role of EGFR in regulating NHE3 activity after PEDV infection. The results showed that EGFR mediated viral invasion by interacting with PEDV S1, and activated EGFR regulated the downstream EGFR/ERK signaling pathway, resulting in decreased expression of NHE3 and reduced NHE3 mobility at the plasma membrane, which ultimately led to decreased NHE3 activity. The low level of NHE3 expression in intestinal epithelial cells may be a key factor leading to PEDV-induced diarrhea in newborn piglets. This study reveals the importance of EGFR in the regulation of NHE3 activity by PEDV and provides new targets and clues for the prevention and treatment of PEDV-induced diarrhea in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly invades the small intestine and promotes an inflammatory response, eventually leading to severe diarrhea, vomiting, dehydration, and even death of piglets, which seriously threatens the economic development of pig farming. In recent years, researchers have found that probiotics can improve the intestinal microenvironment and reduce diarrhea. At the same time, certain probiotics have been shown to have antiviral effects; however, their mechanisms are different. Herein, we aimed to investigate the inhibitory effect of Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism. We used IPEC-J2 cells as a model to assess the inhibitory effect of LP-1S on PEDV and to further investigate the relationship between LP-1S, Ca²⁺, and PEDV. The results showed that a divalent cation chelating agent (EGTA) and calcium channel inhibitors (Bepridil hydrochloride and BAPTA-acetoxymethylate) could inhibit PEDV proliferation while effectively reducing the intracellular Ca²⁺ concentration. Furthermore, LP-1S could reduce PEDV-induced loss of calcium channel proteins (TRPV6 and PMCA1b), alleviate intracellular Ca²⁺ accumulation caused by PEDV infection, and promote the balance of intra- and extracellular Ca²⁺ concentrations, thereby inhibiting PEDV proliferation. In summary, we found that LP-1S has potential therapeutic value against PEDV, which is realized by modulating Ca²⁺. This provides a potential new drug to treat PEDV infection.