Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 μg/mL (CC50 = 28.06 µg/mL) and 0.81 μΜ (CC50 = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50 = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

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 25TCID 50 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 IC 50 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 CC 50 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.

Antibodies were prepared by immunizing mice with empty, immature particles of human enterovirus 71 (EV71), a picornavirus that causes severe neurological disease in young children. The capsid structure of these empty particles is different from that of the mature virus and is similar to "A" particles encountered when picornaviruses recognize a potential host cell before genome release. The monoclonal antibody E18, generated by this immunization, induced a conformational change when incubated at temperatures between 4°C and 37°C with mature virus, transforming infectious virions into A particles. The resultant loss of genome that was observed by cryo-EM and a fluorescent SYBR Green dye assay inactivated the virus, establishing the mechanism by which the virus is inactivated and demonstrating that the E18 antibody has potential as an anti-EV71 therapy. The antibody-mediated virus neutralization by the induction of genome release has not been previously demonstrated. Furthermore, the present results indicate that antibodies with genome-release activity could also be produced for other picornaviruses by immunization with immature particles.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 μg/mL (CC50 = 28.06 µg/mL) and 0.81 μΜ (CC50=14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50=43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

The coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem affecting more than fifty million cases with over one million deaths globally. The 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 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. Post-infection treatment of A. paniculata and andrographolide in SARS-CoV–2 infected Calu-3 cells significantly inhibited the production of infectious virions with the IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by 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 the CC50 of >100 μg/mL for A. paniculata extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to the selectivity index 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.

Avian influenza viruses are a possible threat to human health as they may cause an influenza pandemic.Asian open-bill storks are migratory birds that brought H5N1 viruses into Thailand during the 2004-2005 epidemic. However, to date, there are no reports of direct transmission of stork-derived H5N1 viruses to Thais. Therefore, we questioned whether or not H5N1 viruses secreted in the feces of infected storks could directly infect cells derived from the human respiratory tract. To answer this question, we used primary NHBE cells as a model. We found that H5N1 viruses from two of the three cloacal swabs rapidly replicated and caused severe structural damage to the infected NHBE cells within the early phase of infection. Viruses from the remaining swab replicated poorly and caused no damage to the infected cells. The rapid-replicating viruses were able to replicate efficiently even in the presence of a high level of type I IFN production and stimulated a high level of IL-6 production but not the immunosuppressive cytokine, IL-10. The genotypic study revealed that the major genotypes of the two rapid-replicating viruses present in stork feces were the best-fit genotypes for replication in the primary NHBE cells. In contrast, the major NA-based genotype found in the cloacal swab containing slow-replicating viruses could not survive in the primary NHBE cells. Altogether, the data suggested that those stork-derived H5N1 viruses that preferentially replicated in human airway epithelial cells may exist in nature, and may not require additional mutations in order to defeat the species barrier.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, and scientists around the world are currently studying the virus intensively in order to fight against the on-going pandemic of the virus. To do so, SARS-CoV-2 is typically grown in the lab to generate viral stocks for various kinds of experimental investigations. However, accumulating evidence suggests that such viruses often undergo cell culture adaptation. Here, we systematically explored cell culture adaptation of two SARS-CoV-2 variants, namely the B.1.36.16 variant and the AY.30 variant, a sub lineage of the B.1.617.2 (Delta) variant, propagated in three different cell lines, including Vero E6, Vero E6/TMPRSS2, and Calu-3 cells. Our analyses detected numerous potential cell culture adaptation changes scattering across the entire virus genome, many of which could be found in naturally circulating isolates. Notable ones included mutations around the spike glycoprotein's multibasic cleavage site, and the Omicron-defining H655Y mutation on the spike glycoprotein, as well as mutations in the nucleocapsid protein's linker region, all of which were found to be Vero E6-specific. Our analyses also identified deletion mutations on the non-structural protein 1 and membrane glycoprotein as potential Calu-3-specific adaptation changes. S848C mutation on the non-structural protein 3, located to the protein's papain-like protease domain, was also identified as a potential adaptation change, found in viruses propagated in all three cell lines. Our results highlight SARS-CoV-2 high adaptability, emphasize the need to deep-sequence cultured viral samples when used in intricate and sensitive biological experiments, and illustrate the power of experimental evolutionary study in shedding lights on the virus evolutionary landscape.