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Teetat Kongratanapasert,1,* Supasek Kongsomros,2,5,* Nlin Arya,3 Kripitch Sutummaporn,3 Witthawat Wiriyarat,3 Yada Akkhawattanangkul,4 Tussapon Boonyarattanasoonthorn,5 Nithi Asavapanumas,5 Phongthon Kanjanasirirat,6 Ampa Suksatu,2 Khanit Sa-ngiamsuntorn,7 Suparerk Borwornpinyo,6,8 Pornpun Vivithanaporn,5 Somchai Chutipongtanate,5,9 Suradej Hongeng,6,9 Boonsong Ongphiphadhanakul,10 Arunee Thitithanyanont,2 Phisit Khemawoot,5 Piyamitr Sritara10 1Program in Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand; 2Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 3Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhonpathom, 73170, Thailand; 4Department of Clinical Medicine and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhonpathom, 73170, Thailand; 5Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samutprakarn, 10540, Thailand; 6Excellence Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 7Department of Biochemistry, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand; 8Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 9Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand; 10Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand*These authors contributed equally to this workCorrespondence: Phisit Khemawoot, Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, 10540, Thailand, Tel/Fax +66 28395161, Email phisit.khe@mahidol.ac.thBackground: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems.Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo.Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A.Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals.Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development.Keywords: Boesenbergia rotunda, Zingiberaceae, panduratin A, SARS-CoV-2, COVID-19, hamster

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 μg/mL for A. paniculata extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 μg/mL for A. paniculata extract and 13.2–81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

The immunopathogenesis of H5N1 virus has been studied intensively since it caused cross-species infection and induced high mortality to human. We previously observed the interaction between monocytes and B cells, which increased the susceptibility of B cell to H5N1 virus infection after a co-culture. Levels of α2,3 sialic acid (avian flu receptor) were also significantly increased on B cell surface in this co-culture model with unclear explanation. In this study, we aimed to determine the possible mechanism that responded for this increase in α2,3 sialic acid on B cells. Acquisition of α2,3 SA by B cells via cell contact-dependent trogocytosis was proposed. Results showed that the lack of α2,3 SA was detected on B cell surface, and B cells acquired membrane-bound α2,3 SA molecules from monocytes in H5N1-infected co-cultures. Occurrence of membrane exchange mainly relied on H5N1 infection and cell-cell contact as opposed to a mock infection and transwell. The increase in α2,3 SA on B cell surface mediated by trogocytosis was associated with the enhanced susceptibility to H5N1 infection. These observations thus provide the evidence that H5N1 influenza virus may utilize trogocytosis to expand its cell tropism and spread to immune cells despite the lack of avian flu receptor.

Viruses have developed direct cell-to-cell transfer strategies to enter target cells without being released to escape host immune responses and antiviral treatments. These strategies are more rapid and efficient than transmission through indirect mechanisms of viral infection between cells. Here, we demonstrate that an H5N1 influenza virus can spread via direct cell-to-cell transfer in Madin-Darby canine kidney (MDCK) cells. We compared cell-to-cell transmission of the H5N1 virus to that of a human influenza H1N1 virus. The H5N1 virus has been found to spread to recipient cells faster than the human influenza H1N1 virus. Additionally, we showed that plasma membrane exchange (trogocytosis) occurs between co-cultured infected donor cells and uninfected recipient cells early point, allowing the intercellular transfer of viral material to recipient cells. Notably, the H5N1 virus induced higher trogocytosis levels than the H1N1 virus, which could explain the faster cell-to-cell transmission rate of H5N1. Importantly, this phenomenon was also observed in A549 human lung epithelial cells, which are representative cells in the natural infection site. Altogether, our results provide evidence demonstrating that trogocytosis could be the additional mechanism utilized by the H5N1 virus for rapid and efficient cell-to-cell transmission.