Targeting IL-6 trans-signalling by sgp130Fc attenuates severity in SARS-CoV-2 -infected mice and reduces endotheliopathy.

BACKGROUND: SARS-CoV-2 infection is considered as a relapsing inflammatory process with a dysregulation of IL-6 signalling. Classic IL-6 signalling is thought to represent a defence mechanism against pathogens. In contrast, IL-6 trans-signalling has pro-inflammatory effects. In severe COVID-19, therapeutic strategies have focused on global inhibition of IL-6, with controversial results. We hypothesized that specific blockade of IL-6 trans-signalling could inhibit inflammatory response preserving the host defence activity inherent to IL-6 classic signalling. METHODS: To test the role of the specific IL-6 trans-signalling inhibition by sgp130Fc in short- and long-term consequences of COVID-19, we used the established K18-hACE2 transgenic mouse model. Histological as well as immunohistochemical analysis, and pro-inflammatory marker profiling were performed. To investigate IL-6 trans-signalling in human cells we used primary lung microvascular endothelial cells and fibroblasts in the presence/absence of sgp130Fc. FINDINGS: We report that targeting IL-6 trans-signalling by sgp130Fc attenuated SARS-CoV-2-related clinical symptoms and mortality. In surviving mice, the treatment caused a significant decrease in lung damage. In vitro, IL-6 trans-signalling induced strong and persisting JAK1/STAT3 activation in endothelial cells and lung fibroblasts with proinflammatory effects, which were attenuated by sgp130Fc. Our data also suggest that in those cells with scant amounts of IL-6R, the induction of gp130 and IL-6 by IL-6:sIL-6R complex sustains IL-6 trans-signalling. INTERPRETATION: IL-6 trans-signalling fosters progression of COVID-19, and suggests that specific blockade of this signalling mode could offer a promising alternative to mitigate both short- and long-term consequences without affecting the beneficial effects of IL-6 classic signalling. These results have implications for the development of new therapies of lung injury and endotheliopathy in COVID-19. FUNDING: The project was supported by ISCIII, Spain (COV-20/00792 to MB, PI23/01351 to MARH) and the European Commission-Next generation EU (European Union) (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global, SGL2103029 to MB). PID2019-110587RB-I00 (MB) supported by MICIN/AEI/10.13039/501100011033/and PID2022-143034OB-I00 (MB) by MICIN/AEI/10.13039/501100011033/FEDER. MAR-H acknowledges support from ISCIII, Spain and the European Commission-Next generation EU (European Union), through CSIC's Global Health PTI.

Veratramine inhibits porcine epidemic diarrhea virus entry through macropinocytosis by suppressing PI3K/Akt pathway.

Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC50) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Comparison of the Antiviral Activity of Remdesivir, Chloroquine, and Interferon-ß as Single or Dual Agents Against the Human Beta-Coronavirus OC43.

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.

Multifaceted Effects and Mechanisms of N-Acetylcysteine on Intestinal Injury in a Porcine Epidemic Diarrhea Virus-Infected Porcine Model.

SCOPE: This study investigates the potential effects of N-acetylcysteine (NAC) on intestinal injury in a porcine epidemic diarrhea virus (PEDV)-infected porcine model. METHODS AND RESULTS: Thirty-two piglets are randomly assigned to one of four groups: the control, PEDV, NAC, and NAC+PEDV. Piglets in the NAC+PEDV group are orally administrated with NAC (100 mg (kg·BW)-1  day-1 ) for 4 consecutive days after 2 days of PEDV infection. The results show that NAC administration decreases the diarrhea rate and improves intestinal morphology. The concentration of diamine oxidase and intestinal fatty-acid binding protein, as well as IL-1ß, IL-8, and TNF-α in the plasma, is decreased by NAC. Intriguingly, NAC administration significantly increases the viral load in the jejunum and ileum and down-regulates the expression of interferon-related genes. Microarray and proteomic analyses show that the differentially expressed genes/proteins between NAC+PEDV and PEDV groups are highly enriched in substance transport. Furthermore, aquaporin 8/10 expression is significantly increased by NAC upon PEDV infection. CONCLUSION: NAC administration alleviates PEDV-induced intestinal injury by inhibiting inflammatory responses and improving substance transport, but promotes viral replication by inhibiting interferon signaling. These results suggest NAC exhibits multifaceted effects upon PEDV infection, and thus caution is required when using NAC as a dietary supplement to prevent viral infection.

In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2.

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 µM, EC90 = 0.75 ± 0.01 µM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 µM, EC90 = 0.57 ± 0.21 µM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 µM, EC90 = 0.81 ± 0.20 µM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 µM and EC50 = 0.042 ± 0.007 µM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 µg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 µg/mL (0.042 µM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).

Alpiniae oxyphyllae fructus polysaccharide 3 inhibits porcine epidemic diarrhea virus entry into IPEC-J2 cells.

Porcine epidemic diarrhea virus (PEDV) is deadly for suckling piglets and is a significant threat to most pig farms. Alpiniae oxyphyllae fructus polysaccharide 3 (AOFP3) shows antiviral activity against PEDV. However, the anti-PEDV mechanism of AOFP3 is unknown. Entering the host cell is important for viral infection, and many drugs play antiviral roles by inhibiting this process. To understand the antiviral mechanism of AOFP3 against PEDV, the effect of AOFP3 on PEDV entering IPEC-J2 cells was investigated in the present study. Real-time PCR and immunofluorescence were used to study the effect of AOFP3 on PEDV binding and penetrating IPEC-J2 cells. The effect of PEDV on AOFP3 attachment to IPEC-J2 cells was also investigated. Afterward, the effect of AOFP3 on PEDV spike (S) protein binding to porcine aminopeptidase was tested by using coimmunoprecipitation, and the effect of AOFP3 on the cholesterol level of IPEC-J2 cells was detected. The results showed that AOFP3 competitively inhibited PEDV adsorption on IPEC-J2 cells by blocking PEDV S protein binding to porcine aminopeptidase in IPEC-J2 cells. Furthermore, AOFP3 decreased PEDV penetration into host cells by decreasing the cholesterol level in IPEC-J2 cells.

Remdesivir-induced emergence of SARS-CoV2 variants in patients with prolonged infection.

We here investigate the impact of antiviral treatments such as remdesivir on intra-host genomic diversity and emergence of SARS-CoV2 variants in patients with a prolonged course of infection. Sequencing and variant analysis performed in 112 longitudinal respiratory samples from 14 SARS-CoV2-infected patients with severe disease progression show that major frequency variants do not generally arise during prolonged infection. However, remdesivir treatment can increase intra-host genomic diversity and result in the emergence of novel major variant species harboring fixed mutations. This is particularly evident in a patient with B cell depletion who rapidly developed mutations in the RNA-dependent RNA polymerase gene following remdesivir treatment. Remdesivir treatment-associated emergence of novel variants is of great interest in light of current treatment guidelines for hospitalized patients suffering from severe SARS-CoV2 disease, as well as the potential use of remdesivir to preventively treat non-hospitalized patients at high risk for severe disease progression.

Long-term survival benefit of male and multimorbid COVID-19 patients with 5-day remdesivir treatment.

Background: Treatment of the coronavirus disease (COVID-19) is still challenging due to the lack of evidence-based treatment protocols and continuously changing epidemiological situations and vaccinations. Remdesivir (RDV) is among the few antiviral medications with confirmed efficacy for specific patient groups. However, real-world data on long-term outcomes for a short treatment course are scarce. Methods: This retrospective observational cohort study included real-life data collected during the second and third wave of the COVID-19 pandemic in Hungary (September 1, 2020-April 30, 2021) from inpatients at a University Center (n = 947). Participants consisted of two propensity score-matched cohorts (370/370 cases): Group RDV including patients receiving RDV and supplementary oxygen and Group standard of care (SOC) as control. The primary outcome was the effect of 5-day RDV treatment on 30- and 60-day all-cause mortality. Multivariate analyses were performed to assess the effect of RDV by different covariates. Results: Group RDV included significantly more patients from the alpha variant wave, with greater frequency of comorbidities diabetes and anemia, and larger degree of parenchymal involvement. All-cause mortality at 30- and 60-day were significantly lower in Group RDV compared to Group SOC. Significant risk reduction of 60-day all-cause mortality was observed for RDV treatment in men and patients with COPD or multiple comorbidities. Conclusions: Hospitalized COVID-19 patients with 5-day RDV treatment had significantly lower 30- and 60-day all-cause mortality, despite their more severe clinical condition. Men and patients with multiple comorbidities, including COPD, profited the most from RDV treatment in the long term. Due to the ongoing COVID-19 pandemic, effective treatment regimens are needed for hospitalized patients.

Nirmatrelvir-remdesivir association for non-hospitalized adults with COVID-19, point of view.

The efforts of the scientific world directed to identifying new antiviral drugs and therapies effective against SARS-CoV-2 continue. New oral antivirals against SARS-CoV-2 such as paxlovid have recently authorized. Evidence shows that these antivirals have good efficacy in reducing the risk of hospitalization in COVID-19 positive patients. Remdesivir is an authorized antiviral for the treatment of SARS-CoV-2 infection. To date, there are still few data in the literature on the safety profile and the risk of generating antiviral-resistant SARS-CoV-2 drug variants. In this manuscript we describe the evidence in the literature on the monotherapy use of paxlovid and monotherapy use of remdesivir, and the scientific hypothesis of using nirmatrelvir and remdesivir in association with the aim of increasing treatment efficacy, reducing the risk of adverse reactions and generating antiviral drug-resistant variants.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus In Vitro and In Vivo.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the family Coronaviridae that causes severe diarrhea and high mortality in neonatal suckling piglets. Currently, there is no effective medication against this pathogen. Cepharanthine (CEP), tetrandrine (TET), and fangchinoline (FAN) are natural bis-benzylisoquinoline alkaloids with anti-inflammatory, antitumor, and antiviral properties. Here, we first found that CEP, TET, and FAN had anti-PEDV activity with IC50 values of 2.53, 3.50, and 6.69 µM, respectively. The compounds could block all the processes of viral cycles, but early application of the compounds before or during virus infection was advantageous over application at a late stage of virus replication. FAN performed inhibitory function more efficiently through interfering with the virus entry and attachment processes or through attenuating the virus directly. CEP had a more notable effect on virus entry. With the highest SI index of 11.8 among the three compounds, CEP was chosen to carry out animal experiments. CEP in a safe dosage of 11.1 mg/kg of body weight could reduce viral load and pathological change of piglet intestinal tracts caused by PEDV field strain challenge, indicating that CEP efficiently inhibited PEDV infection in vivo. All of these results demonstrated that the compounds of bis-benzylisoquinoline alkaloids could inhibit PEDV proliferation efficiently and had the potential of being developed for PED prevention and treatment.

Ongoing Living Update of COVID-19 Therapeutic Options: Summary of Evidence. Rapid Review, 4 May 2022

Background: The urgent need for evidence on measures to respond to the COVID-19 pandemic had led to a rapid escalation in numbers of studies testing potential therapeutic options. The vast amount of data generated by these studies must be interpreted quickly so that physicians have the information to make optimal treatment decisions and manufacturers can scale-up production and bolster supply chains. Moreover, obtaining a quick answer to the question of whether or not a particular intervention is effective can help investigators involved in the many ongoing clinical trials to change focus and pivot to more promising alternatives. It is crucial for healthcare workers to have access to the most up-to-date research evidence to inform their treatment decisions. To address this evidence gap, we compiled the following database of evidence on potential therapeutic options for COVID-19. We hope this information will help investigators, policy makers, and prescribers navigate the flood of relevant data to ensure that management of COVID19, at both individual and population levels, is based on the best available knowledge. We will endeavor to continually update this resource as more research is released into the public space. Summary of evidence: Tables 1 and 2, which divide the total group of identified studies into randomized (Table 1) and non-randomized (Table 2) designs, indicate the primary outcome measures used for each investigation and the level of certainty. Table 3 summarizes the status of evidence for the 193 potential therapeutic options for COVID-19 for which studies were identified through our systematic review.

Peptide candidates for the development of therapeutics and vaccines against ß-coronavirus infection.

Betacoronaviruses (ß-CoVs) have caused major viral outbreaks in the last two decades in the world. The mutation and recombination abilities in ß-CoVs resulted in zoonotic diseases in humans. Proteins responsible for viral attachment and replication are highly conserved in ß-CoVs. These conserved proteins have been extensively studied as targets for preventing infection and the spread of ß-CoVs. Peptides are among the most promising candidates for developing vaccines and therapeutics against viral pathogens. The immunostimulatory and viral inhibitory potential of natural and synthetic peptides has been extensively studied since the SARS-CoV outbreak. Food-derived peptides demonstrating high antiviral activity can be used to develop effective therapeutics against ß-CoVs. Specificity, tolerability, and customizability of peptides can be explored to develop potent drugs against ß-CoVs. However, the proteolytic susceptibility and low bioavailability of peptides pose challenges for the development of therapeutics. This review illustrates the potential role of peptides in eliciting an adaptive immune response and inhibiting different stages of the ß-CoV life cycle. Further, the challenges and future directions associated with developing peptide-based therapeutics and vaccines against existing and future ß-CoV pathogens have been discussed.

Peptide-Based Dual HIV and Coronavirus Entry Inhibitors.

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins.

Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.

Porcine Epidemic Diarrhea: Causative Agent, Epidemiology, Clinical Characteristics, and Treatment Strategy Targeting Main Protease.

AIMS: This aimed to study the causative agent, epidemiology, clinical characteristics, and treatment strategy targeting the main protease in porcine epidemic diarrhea. BACKGROUND: Porcine epidemic diarrhea (PED) is a contagious intestinal viral infection causing severe diarrhea, vomiting, and dehydration in pigs. High rates of mortalities and severe morbidities, approaching 100%, are reported in piglets infected with PEDV. In recent years, PED has been observed to influence the swine-farming nations in Europe, Asia, the USA, South Korea, and Canada. The PED virus (PEDV) transmission takes place through a faecal-oral route. OBJECTIVE: The objective is to review the characteristics of PEDV and its role in the disease. In addition, we aim to outline some possible methods to combat PED infection, including targeting the main protease of coronavirus and their future perspectives. METHODS: This study is a review of literature on the PED virus. RESULTS: Apart from symptomatic treatment and supportive care, there is no available specific treatment for PEDV. Appropriate disinfectants and cleaning are pivotal for the control of PEDV. To date, apart from anti-PEDV inhibitors, there are no specific drugs available commercially to treat the disease. Therefore, 3C-like protease (3CLpro) in PEDV that has highly conserved structure and catalytic mechanism serves as an alluring drug as it plays a vital role during viral polyprotein processing at the time of infection. CONCLUSION: A well synchronized and collective effort of scientists, swine veterinarians, pork industry experts, and associated authorities is essential for the accomplishment of proper execution of these required measures.

Ergosterol Peroxide Inhibits Porcine Epidemic Diarrhea Virus Infection in Vero Cells by Suppressing ROS Generation and p53 Activation.

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes severe watery diarrhea in piglets with high morbidity and mortality, resulting in serious economic losses to the farming industry. Ergosterol peroxide (EP) is a sterol with diverse biological activities including antiviral activity. In this study, we explored whether EP extracted from the fruiting body of the mushroom Cryptoporus volvatus had the potential to inhibit PEDV infection in Vero cells. The results revealed that EP had a remarkable inhibitory effect on PEDV infection. It could significantly inhibit multiple stages of the PEDV life cycle, including internalization, replication and release, and could directly inactivate PDCoV infectivity. However, it did not affect PEDV attachment. Furthermore, EP alleviated PEDV-induced apoptosis and mitigated the decrease in mitochondrial membrane potential caused by PEDV infection. It suppressed ROS generation and p53 activation caused by PEDV infection. The ROS scavenger N-acetyl-l-cysteine (NAC) and the p53 specific inhibitor Pifithrin-α (PFT-α) suppressed PEDV-induced apoptosis and impeded viral replication, suggesting that ROS and p53 play an important role in PEDV-induced apoptosis and viral replication. Collectively, EP can prevent PEDV internalization, replication and release, possesses the ability to directly inactivate PEDV, and can inhibit PEDV-induced apoptosis by interfering with PEDV-induced ROS production and p53 activation. These findings highlight the therapeutic potential of EP against PEDV infection.

Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

Cangma Huadu granules, a new drug with great potential to treat coronavirus and influenza infections, exert its efficacy through anti-inflammatory and immune regulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Coronavirus and influenza virus infection seriously threaten human health. Cangma Huadu Granules (CMHD) is an in-hospital preparation composed of eight traditional Chinese medicines (TCM), which has been clinically used against COVID-19 in China and may be a promising candidate for the treatment of influenza. However, the role of its treatment urgently needs to be studied. AIM OF THE STUDY: To evaluate the therapeutic effects of CMHD on pneumonia induced by coronavirus (HCoV-229E) and influenza A virus (H1N1/FM1) in mice and explore its mechanism of anti-infection. MATERIALS AND METHODS: Mice were infected with HCoV-229E or H1N1/FM1 virus through the nasal cavity. CMHD (12.1, 6.05 and 3.03 g/kg/d) or the positive control drugs were administered intragastrically. The lung index and histopathological changes were used to evaluate the therapeutic effect of CMHD. The expression of TNF-α, IL-1ß, IL-6 and IL-4 in Serum and the proportion of CD4+ and CD8+ T lymphocytes in peripheral blood were detected to evaluate the anti-inflammatory and immune regulation effects of CMHD, respectively. Furthermore, the levels of p-NF-κBp65/ NF-κB p65, which was the key targets of the NF-κB pathway was analyzed. RESULTS: In HCoV-229E-induced pneumonia, the lung index was markedly reduced, and lung pathology was improved in mice that treated with CMHD (12.1, 6.05 g/kg/d). Meanwhile, the expression of TNF-α, IL-6 were obviously inhibited, but the expression of IL-4 was significantly increased in CMHD groups. Compared with the model group, CMHD could also markedly upregulate the level of CD4+ and CD8+. Furthermore, CMHD has a markedly effect on inhibit the expression of p-NF-κB p65/NF-κB p65 in the lung. In H1N1-induced pneumonia, the lung index of mice in the CMHD (12.1 g/kg/d) treatment group was lower than that in the model group, and less inflammatory infiltration could be seen in the lung pathological. Moreover, CMHD could also obviously decrease the expression of TNF-α, IL-1ß, IL-6, but significantly increase the expression of IL-4. Except for that, CMHD could also markedly downregulate the level of CD4+ and upregulate the level of CD8+ compared with the model group. In addition, CMHD has a markedly effect on inhibit the expression of p-NF-κB p65/NF-κB p65 in the lung. CONCLUSION: CMHD can significantly combats viral infections caused by HCoV-229E and H1N1, and the mechanism may be related to its multiple functions of anti-inflammatory, immunity regulating and inhibiting NF-κB signal transduction pathway.