Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.

Israelow B ，Song E ，Mao T ，Lu P ，Meir A ，Liu F ，Alfajaro MM ，Wei J ，Dong H ，Homer RJ ，Ring A ，Wilen CB ，Iwasaki A ... -  《-》

RETRACTED: Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial.

Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2, and reported to be efficient in Chinese COV-19 patients. We evaluate the effect of hydroxychloroquine on respiratory viral loads. French Confirmed COVID-19 patients were included in a single arm protocol from early March to March 16th, to receive 600mg of hydroxychloroquine daily and their viral load in nasopharyngeal swabs was tested daily in a hospital setting. Depending on their clinical presentation, azithromycin was added to the treatment. Untreated patients from another center and cases refusing the protocol were included as negative controls. Presence and absence of virus at Day6-post inclusion was considered the end point. Six patients were asymptomatic, 22 had upper respiratory tract infection symptoms and eight had lower respiratory tract infection symptoms. Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D6-post inclusion compared to controls, and much lower average carrying duration than reported in the litterature for untreated patients. Azithromycin added to hydroxychloroquine was significantly more efficient for virus elimination. Despite its small sample size, our survey shows that hydroxychloroquine treatment is significantly associated with viral load reduction/disappearance in COVID-19 patients and its effect is reinforced by azithromycin. This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). Concerns have been raised regarding this article, the substance of which relate to the articles' adherence to Elsevier's publishing ethics policies and the appropriate conduct of research involving human participants, as well as concerns raised by three of the authors themselves regarding the article's methodology and conclusions. Elsevier's Research Integrity and Publishing Ethics Team, in collaboration with the journal's co-owner, the International Society of Antimicrobial Chemotherapy (ISAC), and with guidance from an impartial field expert acting in the role of an independent Publishing Ethics Advisor, Dr. Jim Gray, Consultant Microbiologist at the Birmingham Children's and Women's Hospitals, U.K., conducted an investigation and determined that the below points constituted cause for retraction: • The journal has been unable to confirm whether any of the patients for this study were accrued before ethical approval had been obtained. The ethical approval dates for this article are stated as being 5th and 6th of March 2020 (ANSM and CPP respectively), while the article states that recruitment began in “early March”. The 17th author, Prof. Philippe Brouqui, has confirmed that the start date for patient accrual was 6th March 2020. The journal has not been able to establish whether all patients could have entered into the study in time for the data to have been analysed and included in the manuscript prior to its submission on the 20th March 2020, nor whether all patients were enrolled in the study upon admission as opposed to having been hospitalised for some time before starting the treatment described in the article. Additionally, the journal has not been able to establish whether there was equipoise between the study patients and the control patients. • The journal has not been able to establish whether the subjects in this study should have provided informed consent to receive azithromycin as part of the study. The journal has concluded that that there is reasonable cause to conclude that azithromycin was not considered standard care at the time of the study. The 17th author, Prof. Philippe Brouqui has attested that azithromycin treatment was not, at the time of the study, an experimental treatment but a possible treatment for, or preventative measure against, bacterial superinfections of viral pneumonia as described in section 2.4 of the article, and as such the treatment should be categorised as standard care that would not require informed consent. This does not fully address the journal's concerns around the use of azithromycin in the study. In section 3.1 of the article, it is stated that six patients received azithromycin to prevent (rather than treat) bacterial superinfection. All of these were amongst the patients who also received hydroxychloroquine (HCQ). None of the control patients are reported to have received azithromycin. This would indicate that only patients in the HCQ arm received azithromycin, all of whom were in one center. The recommendations for use of macrolides in France at the time the study was conducted indicate that azithromycin would not have been a logical agent to use as first-line prophylaxis against pneumonia due to the frequency of macrolide resistance amongst bacteria such as pneumococci. These two points suggest that azithromycin would not have been standard practice across southern France at the time the study was conducted and would have required informed consent. • Three of the authors of this article, Dr. Johan Courjon, Prof. Valérie Giordanengo, and Dr. Stéphane Honoré have contacted the journal to assert their opinion that they have concerns regarding the presentation and interpretation of results in this article and have stated they no longer wish to see their names associated with the article. • Author Prof. Valérie Giordanengo informed the journal that while the PCR tests administered in Nice were interpreted according to the recommendations of the national reference center, it is believed that those carried out in Marseille were not conducted using the same technique or not interpreted according to the same recommendations, which in her opinion would have resulted in a bias in the analysis of the data. This raises concerns as to whether the study was partially conducted counter to national guidelines at that time. The 17th author, Prof. Philippe Brouqui has attested that the PCR methodology was explained in reference 17 of the article. However, the article referred to by reference 17 describes several diagnostic approaches that were used (one PCR targeting the envelope protein only; another targeting the spike protein; and three commercially produced systems by QuantiNova, Biofire, and FTD). This reference does not clarify how the results were interpreted. It has also been noted during investigation of these concerns that only 76% (19/25) of patients were viral culture positive, resulting in uncertainty in the interpretation of PCR reports as has been raised by Prof. Giordanengo. As part of the investigation, the corresponding author was contacted and asked to provide an explanation for the above concerns. No response has been received within the deadline provided by the journal. Responses were received by the 3rd and 17th authors, Prof. Philippe Parola and Prof. Philippe Brouqui, respectively, and were reviewed as part of the investigation. These two authors, in addition to 1st author Dr. Philippe Gautret, 13th author Prof. Philippe Colson, and 15th author Prof. Bernard La Scola, disagreed with the retraction and dispute the grounds for it. Having followed due process and concluded the aforementioned investigation and based on the recommendation of Dr. Jim Gray acting in his capacity as independent Publishing Ethics Advisor, the co-owners of the journal (Elsevier and ISAC) have therefore taken the decision to retract the article.

Gautret P ，Lagier JC ，Parola P ，Hoang VT ，Meddeb L ，Mailhe M ，Doudier B ，Courjon J ，Giordanengo V ，Vieira VE ，Tissot Dupont H ，Honoré S ，Colson P ，Chabrière E ，La Scola B ，Rolain JM ，Brouqui P ，Raoult D ... -  《-》

A BSL-2 compliant mouse model of SARS-CoV-2 infection for efficient and convenient antiviral evaluation.

Chen Z ，Cui Q ，Ran Y ，Achi JG ，Chen Z ，Rong L ，Du R ... -  《-》

The flavonoid quercetin decreases ACE2 and TMPRSS2 expression but not SARS-CoV-2 infection in cultured human lung cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells, via its spike protein, and transmembrane protease, serine 2 (TMPRSS2) cleaves the spike-ACE2 complex to facilitate virus entry. As rate-limiting steps for virus entry, modulation of ACE2 and/or TMPRSS2 may decrease SARS-CoV-2 infectivity and COVID-19 severity. In silico modeling suggested the natural bioactive flavonoid quercetin can bind to ACE2 and a recent randomized clinical trial demonstrated that oral supplementation with quercetin increased COVID-19 recovery. A range of cultured human cells were assessed for co-expression of ACE2 and TMPRSS2. Immortalized Calu-3 lung cells, cultured and matured at an air-liquid interface (Calu-3-ALIs), were established as the most appropriate. Primary bronchial epithelial cells (PBECs) were obtained from healthy adult males (N = 6) and cultured under submerged conditions to corroborate the outcomes. Upon maturation or reaching 80% confluence, respectively, the Calu-3-ALIs and PBECs were treated with quercetin, and mRNA and protein expression were assessed by droplet digital PCR and ELISA, respectively. SARS-CoV-2 infectivity, and the effects of pre- and co-treatment with quercetin, was assessed by median tissue culture infectious dose assay. Quercetin dose-dependently decreased ACE2 and TMPRSS2 mRNA and protein in both Calu-3-ALIs and PBECs after 4 h, while TMPRSS2 remained suppressed in response to prolonged treatment with lower doses (twice daily for 3 days). Quercetin also acutely decreased ADAM17 mRNA, but not ACE, in Calu-3-ALIs, and this warrants further investigation. Calu-3-ALIs, but not PBECs, were successfully infected with SARS-CoV-2; however, quercetin had no antiviral effect, neither directly nor indirectly through downregulation of ACE2 and TMPRSS2. Calu-3-ALIs were reaffirmed to be an optimal cell model for research into the regulation of ACE2 and TMPRSS2, without the need for prior genetic modification, and will prove valuable in future coronavirus and respiratory infectious disease work. However, our data demonstrate that a significant decrease in the expression of ACE2 and TMPRSS2 by a promising prophylactic candidate may not translate to infection prevention.

Houghton MJ ，Balland E ，Gartner MJ ，Thomas BJ ，Subbarao K ，Williamson G ... -  《-》

Broad Anti-coronavirus Activity of Food and Drug Administration-Approved Drugs against SARS-CoV-2 In Vitro and SARS-CoV In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease.IMPORTANCE There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV in vitro We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine in vivo using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.

Weston S ，Coleman CM ，Haupt R ，Logue J ，Matthews K ，Li Y ，Reyes HM ，Weiss SR ，Frieman MB ... -  《-》