Effective inhibition of HCoV-OC43 and SARS-CoV-2 by phytochemicals in vitro and in vivo.

Several coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus OC43 (HCoV-OC43) can cause respiratory infections in humans. To address the need for reliable anti-coronavirus therapeutics, we screened 16 active phytochemicals selected from medicinal plants used in traditional applications for respiratory-related illnesses. An initial screen was completed using HCoV-OC43. The phytochemicals lycorine (LYC), capsaicin (CAP), rottlerin (RTL), piperine (PIP) and chebulinic acid (CHU) inhibited HCoV-OC43-induced cytopathic effect and reduced viral titers by up to four logs. LYC, RTL and CHU also suppressed virus replication and cell death following SARS-CoV-2 infection. In vivo, RTL significantly reduced SARS-CoV-2-induced mortality by approximately 40% in human ACE2 expressing K18 mice. Collectively, these studies indicate that RTL and other phytochemicals have therapeutic potential to reduce SARS-CoV-2 and HCoV-43 infections.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause respiratory infections with lethal pneumonia termed COVID-19 [1,2]. Approximately 2-14 days after exposure to the virus, common symptoms may appear, which include cough, cold-like symptoms, shortness of breath or difficulty breathing, repeated shaking with chills, and sore throat. Infection can progress to more severe respiratory illness including systemic hypoxemia and acute respiratory syndrome [3]. These symptoms can progress to respiratory failure and in some cases death. SARS-CoV-2 has the potential to affect all communities across the world, which may have different resources and technologies to combat the disease. Current approved therapeutics are costly and can sometimes be in short supply resulting in some COVID-19 patients being treated only with supportive care [4].
In addition to SARS-CoV-2, several other coronaviruses cause diseases in humans including multiple viruses that cause the common cold. Although not as severe as SARS-CoV-2, human coronavirus OC43 (HCoV-OC43) also causes acute respiratory tract infection [5], and Gastroenteritis (abdominal pain, diarrhea, appetite loss, nausea and vomiting) [6]. Initially, HCoV-OC43 causes mild upper respiratory tract infections but it can lead to severe lower respiratory tract illness, including bronchiolitis, asthma, and pneumonia in children, elderly, and immunecompromised adults [7]. Currently, there are no specific antiviral therapies available for HCoV-OC43. Anti-virals that inhibit HCoV-OC43 and other cold coronaviruses would be highly valuable to limit illness and prevent the development of more severe disease.
It is estimated 80% of the world's population depends on traditional medicine for their healthcare needs [8]. The various indigenous medical systems like Unani, Siddha, Ayurveda, Traditional Chinese medicine, African traditional medicine and Kampo utilize a number of medicinal plants as an abundant source of bioactive phytochemicals for ancient medical need [9].
For example, India has a large tribal population, composing 8.6% of the total population [10]. In spite of advances in modern medicine, these tribal groups largely depend upon their traditional medicaments consisting of different plant parts including root, stem, bark, leaf, flower, seeds, or whole plant, for the prevention and treatment of diverse ailments. Several of these plants are used as tool for first line treatment for respiratory and related complications [11]. To date there is no evidence of the efficacy of these medicinal plants on COVID-19, despite being the first line of treatment for many tribal communities. As numerous pharmaceuticals were initially derived from plants, analysis of traditional medications for activity against SARS-CoV-2 infection may yield beneficial cost-effective treatment for entire populations.
Both HCoV-OC43 and SARS-CoV-2 belong to the same genus of Betacoronavirus, which primarily causes respiratory infections and can be studied under biosafety level 2 (BSL2) conditions, making it an ideal virus to conduct initial screens for drug discovery against respiratory coronaviruses. In this study, we selected 14 commonly used medicinal plants from Southeast Asia belonging to the Paleotropical phytogeographic kingdom [12]. These plants have been traditionally used to treat respiratory and related illnesses ( Table 1). Each of these medicinal plants contains a wide range of bioactive phytochemicals such as alkaloids, flavonoids, steroids, tannins, polyphenol, etc. Based on literature we chose one to two key bioactive phytochemicals from each selected medicinal plant. All these phytochemicals were belonging from either different class of alkaloid or other phytochemical groups ( Table 1). A total of 16 active phytochemicals were selected to conduct a primary screen using HCoV-OC43 to identify compounds that inhibit virusinduced cytopathic effect (CPE) and cell death ( Table 1). We identified five potential compounds with efficacy against HCoV-OC43 infection, which we then tested on SARS-CoV-2. The most active compound, Rottlerin, was further validated in the K18-hACE2 mouse model and demonstrated significant reduction in mortality.

Phytochemicals:
All selected phytochemicals were procured from different pharmaceutical companies (detailed in Supplementary Table S1) and the stocks were prepared by dissolving 1 to 10 mg of each compound in 1 ml DMSO or directly in media and stored for future use.  hs. Cells were then stained using NucGreen™ Dead 488 ReadyProbes™ Reagent (ThermoFisher) dead indicator, which stains cells that have lost plasma membrane integrity. Cells nuclei were counterstained using DAPI (ThermoFisher). Cell cultures were imaged using an EVOS FL Auto 2 Cell Imaging System (ThermoFisher). Cell death was quantified by enumerating the number of NucGreen positive cells proportional to total cell nuclei per field of view [43]. be well tolerated in mice [45]. Control mice received DMSO/PBS in the same schedule, route and at the same time as RTL-treated mice.

Statistical Analysis:
All statistical analysis was performed using GraphPad Prism v.8.02 and 9.03. Specific analysis information is described in the figure legends.

Selection of cell lines for analysis of HCoV-OC43 infection:
To screen the phytochemicals for their ability to inhibit coronavirus replication and cell death, we first identified relevant cell lines that were susceptible to HCoV-OC43 infection and cell death. We chose five different cell lines representative of lungs and intestines for initial screening, as these are the main tissues targeted during HCoV-OC43 and SARS-CoV-2 infections (Fig. 1A). Neither cytopathic effect (CPE) nor cell death was induced by HCoV-OC43 in NCI-H292, SW-1573, or Caco-2 cells.
HCoV-OC43 also induced cell death in about 40% of HCT-8 cells and 90% of MRC-5 cells by 6 dpi (Fig. 1D). Virus titers in cell supernatants of both HCT-8 and MRC-5 cells increased by up to 3 logs from 1 to 6 dpi. (Fig. 1E). Thus, we used both HCT-8 and MRC-5 for screening of selected phytochemicals. Previously, Favipiravir (T-705) was reported to have antiviral potential against SARS-CoV-2 infection [46]. We examined T-705 as a positive control for the current study and found reduced CPE and virus titer in cell supernatants following HCoV-OC43 infection of HCT-8 and MRC-5 cells (Fig. 2A).  Figure S1 and Table 1). Another four compounds: vasicine, chebulagic acid, gallic acid, and homoherrintonin inhibited virus induced cell death and CPE lightly ( Table 1). The remaining compounds had little to no effect on CPE ( Table 1). The five compounds (CAP, LYC, RTL, PIP, CHU) that inhibited CPE promisingly were further analyzed for ability to suppress virus production by measuring infectious virus titers in cells supernatants. All five active components showed significant inhibition of virus production in culture supernatants (Fig. 2).

Effect of selected phytochemicals on SARS-CoV-2 induced cell death:
Of the five active components were examined against HCoV-OC43, four of them (CAP, LYC, RTL and CHU) reduced HCoV-OC43 replication by greater than 2 logs (Fig. 2). Based on these finding, these four active components were further validated for their ability to inhibit SARS-CoV-2 in vitro. Virus-induced cell death, and the potential protective effects of compound treatment, were assessed in human ACE2-expressing A549 cells. Initially, cell death was assessed using extracellular lactate dehydrogenase (LDH) activity as a surrogate marker of cell lysis. However, since LDH can be subject to metabolic dysregulation, we additionally validated cell death using immunofluorescent microscopy and staining using a live/dead indicator. For each compound, a dose-dependent curve in the absence and presence of active components treatment were plotted (Fig. 3). Of the four active components examined, LYC and RTL consistently inhibited virusinduced cell death (Fig. 3C to 3F). CHU significantly inhibited virus-induced death at higher concentrations using LDH assay (Fig. 3G) and at all concentrations by Live/Dead Microscopy assay (Fig. 3H). CAP showed significant effect at 40 µg/ml concentration using LDH assay ( Fig.   3A) but was not effective by microscopy cell death analysis (Fig. 3B).

LYC, RTL, and CHU effectively reduce SARS-CoV-2 titers but not CAP: Virus titers
in the supernatants were analyzed to measure how phytochemical treatment affected release of infectious virus. LYC and RTL treatment significantly reduced virus levels in culture supernatant by up to 4 logs as measured in A549 cells ( Fig. 4B and 4C). CHU treatment significantly reduced virus titer by up to 2 logs at higher concentration but not at lower concentrations (Fig. 4D).
Unfortunately, CAP did not reduce replicating virus (Fig. 4A). Thus, only LYC, RTL and CHU treatment significantly reduced SARS-CoV-2 induced cell death, which was associated with suppression of replicating virus. increased the survival by approximately 40% (Fig. 5).

Discussion
The recent SARS-CoV-2 outbreak has shown the need to develop antivirals that can be readily used to suppress viral infections. Traditionally used ethnomedicinal plants are a great source of structural ranges and widespread bioactivities which might serve as a source of possible broad-spectrum antivirals [47]. In the current study, traditionally used medicinal plants which have been reported to treat respiratory and related illnesses ( Table 1)  The leaves and flowers of this plant are traditionally used for respiratory-related illnesses like flu, bronchitis, cough, and throat complaints [27]. Interestingly, previous studies showed that RTL had a strong potential to inhibit the entry of different DNA viral infections including Kaposi's sarcomaassociated herpesvirus [50] and vaccinia virus [51]. Several RNA virus including La Crosse [52], Zika [40], Rabies [53], Porcine Reproductive and Respiratory Syndrome (PRRSV) [54] and Rift Valley Fever [55] virus were also reported to be inhibited by RTL treatment but the mechanisms of inhibition appear to be different. RTL inhibits LaCrosse virus by blocking virus release from the Golgi body [52], whereas in PRRSV [54] and Rabies virus, RTL prevented endocytosis through improper delivery of viral particles in the cytoplasm [53] most likely by decreasing intracellular ATP concentration and inhibition of mitochondrial respiration. The differing mechanism by which RTL inhibits virus replication suggests that inhibiting vesicle transport may be a key mechanism by which RTL inhibits different types of virus replication.
Traditionally Terminalia chebula Retz. was used to treat asthma, hiccough, respiratory tract infections, cough, and sore throat [34]. These phytochemicals had the therapeutic potential to reduce SARS-CoV-2 infection. However, further studies to determine the effectiveness of these small molecules against in vivo animal efficacies are required.
CAP (8-  Used for bronchitis, asthma, fever, cold, cough, eczema, irritation in the throat and help loosen phlegm deposits in the airway [13].

Vasicine (Quinazoline alkaloid)
Molecular docking study suggest that vasicine could be used to treat of COVID-19 [14]. The leaves and roots are used to cure bronchitis, whooping cough, chest complaints, eczema and asthma [15]. Leaves and bark are used to treat asthma, cough, flu, lung problems [16].