Stratification of viral shedding patterns in saliva of COVID-19 patients (preprint)

Living with COVID-19 requires continued vigilance against the spread and emergence of variants of concern (VOCs). Rapid and accurate saliva diagnostic testing, alongside basic public health responses, is a viable option contributing to effective transmission control. Nevertheless, our knowledge regarding the dynamics of SARS-CoV-2 infection in saliva is not as advanced as our understanding of the respiratory tract. Here we analyzed longitudinal viral load data of SARS-CoV-2 in saliva samples from 144 patients with mild COVID-19 (a combination of our collected data and published data). Using a mathematical model, we successfully stratified infection dynamics into three distinct groups with clear patterns of viral shedding: viral shedding durations in the three groups were 11.5 days (95% CI: 10.6 to 12.4), 17.4 days (16.6 to 18.2), and 30.0 days (28.1 to 31.8), respectively. Surprisingly, this stratified grouping remained unexplained despite our analysis of 47 types of clinical data, including basic demographic information, clinical symptoms, results of blood tests, and vital signs. Additionally, we quantified the expression levels of 92 micro-RNAs in a subset of saliva samples, but these also failed to explain the observed stratification, although the mir-1846 level may have been weakly correlated with peak viral load. Our study provides insights into SARS-CoV-2 infection dynamics in saliva, highlighting the challenges in predicting the duration of viral shedding without indicators that directly reflect an individual's immune response, such as antibody induction. Given the significant individual heterogeneity in the kinetics of saliva viral shedding, identifying biomarker(s) for viral shedding patterns will be crucial for improving public health interventions in the era of living with COVID-19.

An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity (preprint)

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2 (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into the impact of variant emergence on choosing optimal drug treatment.

Dasabuvir inhibits human norovirus infection in human intestinal enteroids (preprint)

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, with a human intestinal enteroid (HIE) culture system where HuNoVs are able to replicate reproducibly, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity. Dasabuvir, which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human A rotavirus (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation.

Circadian regulation of SARS-CoV-2 infection in lung epithelial cells (preprint)

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via binding human angiotensin-converting enzyme (ACE2), and infection can result in pneumonia and acute respiratory distress syndrome. Circadian rhythms coordinate an organisms response to its environment and recent studies report a role for the circadian clock to regulate host susceptibility to virus infection. Influenza A infection of arhythmic mice, lacking the circadian component BMAL1, results in higher viral replication and elevated inflammatory responses leading to more severe bronchitis, highlighting the impact of circadian pathways in respiratory function. We demonstrate circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with the synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and RNA replication. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Our study suggests new approaches to understand and improve therapeutic targeting of COVID-19.

Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro (preprint)

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22( R )-hydroxycholesterol, 24( S )-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

Anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) potency of Mefloquine as an entry inhibitor in vitro (preprint)

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 M, IC90 = 2.31 M, and IC99 = 4.39 M in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

Hypoxic and Pharmacological Activation of HIF Inhibits SARS-CoV-2 Infection of Lung Epithelial Cells (preprint)

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 1 million fatalities to date. Understanding how host factors modify the viral life cycle could inform susceptibility to viral infection and the design of new therapies. Viral replication is shaped by the cellular microenvironment and one important factor is local oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent signalling pathway. Further, hypoxia and Roxadustat inhibit viral replication in SARS-CoV-2 infected cells, showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of hypoxia and HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention and/or treatment of COVID-19.Funding: The McKeating laboratory is funded by a Wellcome Investigator Award (IA) 200838/Z/16/Z, UK Medical Research Council (MRC) project grant MR/R022011/1 and Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002). The Ratcliffe laboratory is funded by the Oxford Branch of the Ludwig Institute for Cancer Research; Wellcome IA 106241/Z/14/Z; the Francis Crick Institute, which receives core funding from Cancer Research UK (FC001501), UK MRC (FC001501) and Wellcome (FC001501); the Paradifference Foundation. PJR, EJH and TB are additionally funded by the COVID-19 Research Response Fund, University of Oxford. SK is funded by the Clarendon Scholarships Fund and the Christopher Welch Trust. The Davis laboratory is funded by Wellcome IA 209412/Z/17/Z and Wellcome Strategic Awards 091911/B/10/Z and 107457/Z/15/Z. JYL is funded by the Medial Sciences Graduate Studentship, University of Oxford. The Hinks laboratory is funded by grants from the Wellcome (104553/z/14/z, 211050/Z/18/z) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre; the views expressed are those of the authors and not those of the NHS or NIHR. Conflict of Interest: EJH is employed under the Cambridge Experimental Medicine Initiative, which is partly funded by AstraZeneca although they have not been involved in this project. The other authors declare no financial interests.Ethical Approval: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).

Identification of Anti-COVID-19 Agents, Cepharanthine and Nelfinavir, and Their Potential Usage for Combination Treatment (preprint)

Antiviral treatments targeting the coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine: the anti-inflammatory drug Cepharanthine and HIV protease inhibitor Nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry into cells, whilst Nelfinavir inhibited the catalytic activity of viral main protease to suppress viral replication. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will shorten the period until viral clearance by 5.5-days and the combining Cepharanthine/Nelfinavir enhanced their predicted efficacy to control viral proliferation. In summary, this study identifies a new multidrug combination treatment for COVID-19.Funding: This work was supported by The Agency for Medical Research and Development (AMED) emerging/re-emerging infectious diseases project (JP19fk0108111, JP19fk0108110, JP20fk0108104); the AMED Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS, JP19am0101114, JP19am0101069, JP19am0101111) program; The Japan Society for the Promotion of Science 260 KAKENHI (JP17H04085, JP20H03499, JP15H05707, 19H04839); The JST MIRAI program; and Wellcome Trust funded Investigator award (200838/Z/16/Z). Conflict of Interest: None.

Estimation of the incubation period of COVID-19 using viral load data (preprint)

The incubation period, or the time from infection to symptom onset of COVID-19 has been usually estimated using data collected through interviews with cases and their contacts. However, this estimation is influenced by uncertainty in recalling effort of exposure time. We propose a novel method that uses viral load data collected over time since hospitalization, hindcasting the timing of infection with a mathematical model for viral dynamics. As an example, we used the reported viral load data from multiple countries (Singapore, China, Germany, France, and Korea) and estimated the incubation period. The median, 2.5, and 97.5 percentiles of the incubation period were 5.23 days (95% CI: 5.17, 5.25), 3.29 days (3.25, 3.37), and 8.22 days (8.02, 8.46), respectively, which are comparable to the values estimated in previous studies. Using viral load to estimate the incubation period might be a useful approach especially when impractical to directly observe the infection event.

Rethinking antiviral effects for COVID-19 in clinical studies: early initiation is key to successful treatment (preprint)

Development of an effective antiviral drug for COVID-19 is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence for effective drugs from clinical studies is limited. The lack of evidence could be in part due to heterogeneity of virus dynamics among patients and late initiation of treatment. We first quantified the heterogeneity of viral dynamics which could be a confounder in compassionate use programs. Second, we demonstrated that an antiviral drug is unlikely to be effective if initiated after a short period following symptom onset. For accurate evaluation of the efficacy of an antiviral drug for COVID-19, antiviral treatment should be initiated before or soon after symptom onset in randomized clinical trials.

Multidrug treatment with nelfinavir and cepharanthine against COVID-19 (preprint)

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

Inferring Timing of Infection Using Within-host SARS-CoV-2 Infection Dynamics Model: Are "Imported Cases" Truly Imported? (preprint)

Importance: Although the COVID-19 epidemic in some countries such as China are in the last phase by large effort for containment of the disease, another outbreaks can occur because huge susceptible population remains. Further, there remain countries in the early phase of outbreak with zero or limited number of cases in southern hemisphere countries. In those countries at risk of future outbreak, ascertaining whether cases are imported or the result of local secondary transmission is important for government to shape appropriate public health strategies. Objective: To develop a method to estimate timing of infection establishment, which helps differentiate imported and autochthonous cases. Design, Setting and Participants: Of the first 18 cases reported in Singapore, 12 were used in our study (1 case with insufficient data and 5 on anti-viral treatment were excluded from the analysis). The viral load data from these initial cases considered imported due to their travel history to Wuhan were analyzed. Another viral load data from 3 cases reported from Zhuhai, China, for whom exposed day were known, were also analyzed to determine the viral load threshold for infection establishment. Exposures: SARS-CoV-2 infection confirmed by the polymerase-chain-reaction (PCR) test. Main Outcomes and Measures: The timing of infection establishment of each case was assessed by analysing viral load data after symptom onset using a within-host viral dynamics model for SARS-CoV-2. Estimated timing of infection will indicate whether cases are imported or autochthonous transmission within Singapore. Results: Six among the 12 cases were clearly imported cases, whereas we could not rule out the possibility of secondary transmission for the rest of 6 cases, which collectively evidenced ongoing transmission in Singapore. For the 6 cases who could be the results of secondary transmission, further investigation to identify the source of infection within Singapore should be warranted (i.e., contact tracing). Conclusions and Relevance: In an early phase of outbreak due to entrance or re-entrance of the virus to countries/communities, collecting viral load data over time from cases from symptom onset is highly recommended, because viral load data are valuable to infer the timing of infection and distinguish between imported cases and ongoing local transmission.

Modelling SARS-CoV-2 Dynamics: Implications for Therapy (preprint)

The scientific community is focussed on developing antiviral therapies to mitigate the impacts of the ongoing novel coronavirus disease (COVID-19) outbreak. This will be facilitated by improved understanding of viral dynamics within infected hosts. Here, using a mathematical model in combination with published viral load data collected from the same specimen (throat / nasal swabs or nasopharyngeal / sputum / tracheal aspirate), we compare within-host dynamics for patients infected in the current outbreak with analogous dynamics for MERS-CoV and SARS-CoV infections. Our quantitative analyses revealed that SARS-CoV-2 infection dynamics are more severe than those for mild cases of MERS-CoV, but are similar to severe cases, and that the viral dynamics of SARS-CoV infection are similar to those of MERS-CoV in mild cases but not in severe case. Consequently, SARS-CoV-2 generates infection dynamics that are more severe than SARS-CoV. Furthermore, we used our viral dynamics model to predict the effectiveness of unlicensed drugs that have different methods of action. The effectiveness was measured by AUC of viral load. Our results indicated that therapies that block de novo infections or virus production are most likely to be effective if initiated before the peak viral load (which occurs around three days after symptom onset on average), but therapies that promote cytotoxicity are likely to have only limited effects. Our unique mathematical approach provides insights into the pathogenesis of SARS-CoV-2 in humans, which are useful for development of antiviral therapies.