Antimalarial drugs inhibit the replication of SARS-CoV-2: An in vitro evaluation.

In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. African countries see slower dynamic of COVID-19 cases and deaths. One of the assumptions that may explain this later emergence in Africa, and more particularly in malaria endemic areas, would be the use of antimalarial drugs. We investigated the in vitro antiviral activity against SARS-CoV-2 of several antimalarial drugs. Chloroquine (EC50 = 2.1 µM and EC90 = 3.8 µM), hydroxychloroquine (EC50 = 1.5 µM and EC90 = 3.0 µM), ferroquine (EC50 = 1.5 µM and EC90 = 2.4 µM), desethylamodiaquine (EC50 = 0.52 µM and EC90 = 1.9 µM), mefloquine (EC50 = 1.8 µM and EC90 = 8.1 µM), pyronaridine (EC50 = 0.72 µM and EC90 = 0.75 µM) and quinine (EC50 = 10.7 µM and EC90 = 38.8 µM) showed in vitro antiviral effective activity with IC50 and IC90 compatible with drug oral uptake at doses commonly administered in malaria treatment. The ratio Clung/EC90 ranged from 5 to 59. Lumefantrine, piperaquine and dihydroartemisinin had IC50 and IC90 too high to be compatible with expected plasma concentrations (ratio Cmax/EC90 < 0.05). Based on our results, we would expect that countries which commonly use artesunate-amodiaquine or artesunate-mefloquine report fewer cases and deaths than those using artemether-lumefantrine or dihydroartemisinin-piperaquine. It could be necessary now to compare the antimalarial use and the dynamics of COVID-19 country by country to confirm this hypothesis.

Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2.

The SARS-CoV-2 pandemic started in the end of 2019 in Wuhan, China, which highlighted the scenario of frequent cross-species transmission events. From the outbreak possibly initiated by viral spill-over into humans from an animal reservoir, now we face the human host moving globally while interacting with domesticated and peridomestic animals. The emergence of a new virus into the ecosystem leads to selecting forces and species-specific adaptations. The adaptation of SARS-CoV-2 to other animals represents a risk to controlling the dissemination of this coronavirus and the emergence of new variants. Since 2020, several mink farms in Europe and the United States have had SARS-CoV-2 outbreaks with human-mink and mink-human transmission, where the mink-selected variants possibly hold evolutionary concerning advantages. Here we investigated the permissibility of mink lung-derived cells using two cell lines, Mv-1-Lu and ENL-R, against several lineages of SARS-CoV-2, including some classified as variants of concern. The viral release rate and the infectious titers indicate that these cells support infections by different SARS-CoV-2 lineages. The viral production occurs in the first few days after infection with the low viral release by these mink cells, which is often absent for the omicron variant for lung cells. The electron microscopy reveals that during the viral replication cycle, the endomembrane system of the mink-host cell undergoes typical changes while the viral particles are produced, especially in the first days of infection. Therefore, even if limited, mink lung cells may represent a selecting source for SARS-CoV-2 variants, impacting their transmissibility and pathogenicity and making it difficult to control this new coronavirus.

SARS-CoV-2 Testing of Aircraft Wastewater Shows That Mandatory Tests and Vaccination Pass before Boarding Did Not Prevent Massive Importation of Omicron Variant into Europe.

BACKGROUND: Most new SARS-CoV-2 epidemics in France occurred following the importation from abroad of emerging viral variants. Currently, the risk of new variants being imported is controlled based on a negative screening test (PCR or antigenic) and proof of up-to-date vaccine status, such as the International Air Transport Association travel pass. METHODS: The wastewater from two planes arriving in Marseille (France) from Addis Ababa (Ethiopia) in December 2021 was tested by RT-PCR to detect SARS-CoV2 and screen for variants. These tests were carried out between landing and customs clearance and were then sequenced by MiSeq Illumina. Antigenic tests and sequencing by NovaSeq were carried out on respiratory samples collected from the 56 passengers on the second flight. RESULTS: SARS-CoV-2 RNA suspected of being from the Omicron BA.1 variant was detected in the aircraft's wastewater. SARS-CoV2 RNA was detected in 11 [20%) passengers and the Omicron BA.1 variant was identified. CONCLUSION: Our work shows the efficiency of aircraft wastewater testing to detect SARS-CoV-2 cases among travellers and to identify the viral genotype. It also highlights the low efficacy of the current control strategy for flights entering France from outside Europe, which combines a requirement to produce a vaccine pass and proof of a negative test before boarding.

Antiviral Activity of Repurposing Ivermectin against a Panel of 30 Clinical SARS-CoV-2 Strains Belonging to 14 Variants.

Over the past two years, several variants of SARS-CoV-2 have emerged and spread all over the world. However, infectivity, clinical severity, re-infection, virulence, transmissibility, vaccine responses and escape, and epidemiological aspects have differed between SARS-CoV-2 variants. Currently, very few treatments are recommended against SARS-CoV-2. Identification of effective drugs among repurposing FDA-approved drugs is a rapid, efficient and low-cost strategy against SARS-CoV-2. One of those drugs is ivermectin. Ivermectin is an antihelminthic agent that previously showed in vitro effects against a SARS-CoV-2 isolate (Australia/VI01/2020 isolate) with an IC50 of around 2 µM. We evaluated the in vitro activity of ivermectin on Vero E6 cells infected with 30 clinically isolated SARS-CoV-2 strains belonging to 14 different variants, and particularly 17 strains belonging to six variants of concern (VOC) (variants related to Wuhan, alpha, beta, gamma, delta and omicron). The in vitro activity of ivermectin was compared to those of chloroquine and remdesivir. Unlike chloroquine (EC50 from 4.3 ± 2.5 to 29.3 ± 5.2 µM) or remdesivir (EC50 from 0.4 ± 0.3 to 25.2 ± 9.4 µM), ivermectin showed a relatively homogeneous in vitro activity against SARS-CoV-2 regardless of the strains or variants (EC50 from 5.1 ± 0.5 to 6.7 ± 0.4 µM), except for one omicron strain (EC50 = 1.3 ± 0.5 µM). Ivermectin (No. EC50 = 219, mean EC50 = 5.7 ± 1.0 µM) was, overall, more potent in vitro than chloroquine (No. EC50 = 214, mean EC50 = 16.1 ± 9.0 µM) (p = 1.3 × 10-34) and remdesivir (No. EC50 = 201, mean EC50 = 11.9 ± 10.0 µM) (p = 1.6 × 10-13). These results should be interpreted with caution regarding the potential use of ivermectin in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results into actual clinical treatment in patients.

Heterogeneity in susceptibility to hydroxychloroquine of SARS-CoV-2 isolates.

BACKGROUND: Despite the fact that the clinical efficacy of hydroxychloroquine is still controversial, it has been demonstrated in vitro to control SARS-CoV-2 multiplication on Vero E6 cells. In this study, we tested the possibility that some patients with prolonged virus excretion could be infected by less susceptible strains. METHOD: Using a high-content screening method, we screened 30 different selected isolates of SARS-CoV-2 from different patients who received azithromycin ± hydroxychloroquine. We focused on patients with viral persistence, i.e., positive virus detection in a nasopharyngeal sample ≥10 days, and who were tested during two French epidemic waves, late winter-spring of 2020 and the summer of 2020. Dose-response curves in single-molecule assays with hydroxychloroquine were created for isolates with suspected reduced susceptibility. Genome clustering was performed for all isolates. RESULTS: Of 30 tested strains, three were detected as replicating in the presence of azithromycin + hydroxychloroquine, each at 5 µM. The dose-response model showed a decrease in susceptibility of these three strains to hydroxychloroquine. Whole genome sequencing revealed that these three strains are all from the second epidemic wave and two cluster with isolates from Africa. CONCLUSIONS: Reduced susceptibility to hydroxychloroquine was not associated with viral persistence in naso-pharyngeal samples. Rather, it was associated with occurring during the second epidemic wave, which began in the summer and with strains clustering with those with a common genotype in Africa, where hydroxychloroquine was the most widely used.

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France.

The monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the "UK variant" in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest.

In Vitro Evaluation of the Antiviral Activity of Methylene Blue Alone or in Combination against SARS-CoV-2.

A new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19), which emerged in Wuhan, China in December 2019, has spread worldwide. Currently, very few treatments are officially recommended against SARS-CoV-2. Identifying effective, low-cost antiviral drugs with limited side effects that are affordable immediately is urgently needed. Methylene blue, a synthesized thiazine dye, may be a potential antiviral drug. Antiviral activity of methylene blue used alone or in combination with several antimalarial drugs or remdesivir was assessed against infected Vero E6 cells infected with two clinically isolated SARS-CoV-2 strains (IHUMI-3 and IHUMI-6). Effects both on viral entry in the cell and on post-entry were also investigated. After 48 h post-infection, the viral replication was estimated by RT-PCR. The median effective concentration (EC50) and 90% effective concentration (EC90) of methylene blue against IHUMI-3 were 0.41 ± 0.34 µM and 1.85 ± 1.41 µM, respectively; 1.06 ± 0.46 µM and 5.68 ± 1.83 µM against IHUMI-6. Methylene blue interacted at both entry and post-entry stages of SARS-CoV-2 infection in Vero E6 cells as retrieved for hydroxychloroquine. The effects of methylene blue were additive with those of quinine, mefloquine and pyronaridine. The combinations of methylene blue with chloroquine, hydroxychloroquine, desethylamodiaquine, piperaquine, lumefantrine, ferroquine, dihydroartemisinin and remdesivir were antagonist. These results support the potential interest of methylene blue to treat COVID-19.

Viral RNA in City Wastewater as a Key Indicator of COVID-19 Recrudescence and Containment Measures Effectiveness.

In recent years, and more specifically at the beginning of the COVID-19 crisis, wastewater surveillance has been proposed as a tool to monitor the epidemiology of human viral infections. In the present work, from July to December 2020, the number of copies of SARS-CoV-2 RNA in Marseille's wastewater was correlated with the number of new positive cases diagnosed in our Institute of Infectious Disease, which tested about 20% of the city's population. Number of positive cases and number of copies of SARS-CoV-2 RNA in wastewater were significantly correlated (p = 0.013). During the great epidemic peak, from October to December 2020, the curves of virus in the sewers and the curves of positive diagnoses were perfectly superposed. During the summer period, the superposition of curves was less evident as subject to many confounding factors that were discussed. We also tried to correlate the effect of viral circulation in wastewater with containment measures, probably the most unbiased correlation on their potential inflection effect of epidemic curves. Not only is this correlation not obvious, but it also clearly appears that the drop in cases as well as the drop in the viral load in the sewers occur before the containment measures. In fact, this suggests that there are factors that initiate the end of the epidemic peak independently of the containment measure. These factors will therefore need to be explored more deeply in the future.

Culture of SARS-CoV-2 in a panel of laboratory cell lines, permissivity, and differences in growth profile.

The emergence of COVID-19 disease due to SARS-CoV-2 at the end of 2019 was rapidly associated with the isolation of the strain from co-culture onto VERO cells. These isolations quickly made it possible to carry out the first tests for antiviral agents' susceptibility and drug repurposing. However, it seems important to make an inventory of all the cells that can support the growth of this virus and evaluate possible differences between isolates. In the present work, we tested 4 strains of SARS-CoV-2 locally isolated on a panel of 34 cell lines present in our laboratory and commonly used for the isolation of human pathogenic microorganism. After inoculation, cells were observed for cytopathic effects and quantitative real-time polymerase reaction was used to measure the virus replication on the cells. We were able to obtain growth on 7 cell lines, 6 simian, and the human Caco-2. The cytopathogenic effects are variable, ranging from lysis of the cell monolayer in 48-72 h to no cytopathic effect in spite of intense multiplication, as in Caco-2 cells. Interestingly, effect and multiplication varied widely according to the strain tested. In this paper, we explored the species specificity and tissue tropism of SARS-CoV-2 in vitro on a panel of cells available in our laboratory and identified human and animal cell lines susceptible to support SARS-CoV-2 replication. Our work highlights the importance of testing multiple strains when testing antiviral molecules and performing patho-physiological analyzes.

In Vitro Antiviral Activity of Doxycycline against SARS-CoV-2

In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), emerged in Wuhan, China. Despite containment measures, SARS-CoV-2 spread in Asia, Southern Europe, then in America and currently in Africa. Identifying effective antiviral drugs is urgently needed. An efficient approach to drug discovery is to evaluate whether existing approved drugs can be efficient against SARS-CoV-2. Doxycycline, which is a second-generation tetracycline with broad-spectrum antimicrobial, antimalarial and anti-inflammatory activities, showed in vitro activity on Vero E6 cells infected with a clinically isolated SARS-CoV-2 strain (IHUMI-3) with median effective concentration (EC50) of 4.5 ±2.9 µM, compatible with oral uptake and intravenous administrations. Doxycycline interacted both on SARS-CoV-2 entry and in replication after virus entry. Besides its in vitro antiviral activity against SARS-CoV-2, doxycycline has anti-inflammatory effects by decreasing the expression of various pro-inflammatory cytokines and could prevent co-infections and superinfections due to broad-spectrum antimicrobial activity. Therefore, doxycycline could be a potential partner of COVID-19 therapies. However, these results must be taken with caution regarding the potential use in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results to actual clinical treatment in patients. In vivo evaluation in animal experimental models is required to confirm the antiviral effects of doxycycline on SARS-CoV-2 and more trials of high-risk patients with moderate to severe COVID-19 infections must be initiated.

Methylene blue inhibits replication of SARS-CoV-2 in vitro.

In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. Currently there is no antiviral treatment recommended against SARS-CoV-2. Identifying effective antiviral drugs is urgently required. Methylene blue has already demonstrated in vitro antiviral activity in photodynamic therapy as well as antibacterial, antifungal and antiparasitic activities in non-photodynamic assays. In this study. non-photoactivated methylene blue showed in vitro activity at very low micromolar range with an EC50 (median effective concentration) of 0.30 ± 0.03 µM and an EC90 (90% effective concentration) of 0.75 ± 0.21 µM at a multiplicity of infection (MOI) of 0.25 against SARS-CoV-2 (strain IHUMI-3). The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine (1.5 µM and 3.0 µM) and azithromycin (20.1 µM and 41.9 µM). The ratios Cmax/EC50 and Cmax/EC90 in blood for methylene blue were estimated at 10.1 and 4.0, respectively, following oral administration and 33.3 and 13.3 following intravenous administration. Methylene blue EC50 and EC90 values are consistent with concentrations observed in human blood. We propose that methylene blue is a promising drug for treatment of COVID-19. In vivo evaluation in animal experimental models is now required to confirm its antiviral effects on SARS-CoV-2. The potential interest of methylene blue to treat COVID-19 needs to be confirmed by prospective comparative clinical studies.

High-speed large-scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled to high-content screening.

OBJECTIVES: A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2. METHODS: We used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared with the classical isolation procedure. RESULTS: The isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared with only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture. CONCLUSIONS: This system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.

Antimalarial artemisinin-based combination therapies (ACT) and COVID-19 in Africa: In vitro inhibition of SARS-CoV-2 replication by mefloquine-artesunate.

OBJECTIVES: At the end of November 2019, a novel coronavirus responsible for respiratory tract infections (COVID-19) emerged in China. Despite drastic containment measures, this virus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread in Asia and Europe. The pandemic is ongoing with a particular hotspot in Southern Europe and America; many studies predicted a similar epidemic in Africa, as is currently seen in Europe and the United States of America. However, reported data have not confirmed these predictions. One of the hypotheses that could explain the later emergence and spread of COVID-19 pandemic in African countries is the use of antimalarial drugs to treat malaria, and specifically, artemisinin-based combination therapy (ACT). METHODS: The antiviral activity of fixed concentrations of ACT at concentrations consistent with those observed in human plasma when ACT is administered at oral doses for uncomplicated malaria treatment was evaluatedin vitro against a clinically isolated SARS-CoV-2 strain (IHUMI-3) in Vero E6 cells. RESULTS: Mefloquine-artesunate exerted the highest antiviral activity with % inhibition of 72.1 ± 18.3 % at expected maximum blood concentration (Cmax) for each ACT drug at doses commonly administered in malaria treatment. All the other combinations, artesunate-amodiaquine, artemether-lumefantrine, artesunate-pyronaridine, or dihydroartemisinin-piperaquine, showed antiviral inhibition in the same ranges (27.1 to 34.1 %). CONCLUSIONS: Antimalarial drugs for which concentration data in the lungs are available are concentrated from 10 to 160 fold more in the lungs than in blood. Thesein vitro results reinforce the hypothesis that antimalarial drugs could be effective as an anti-COVID-19 treatment.

In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect.

Human coronaviruses SARS-CoV-2 appeared at the end of 2019 and led to a pandemic with high morbidity and mortality. As there are currently no effective drugs targeting this virus, drug repurposing represents a short-term strategy to treat millions of infected patients at low costs. Hydroxychloroquine showed an antiviral effect in vitro. In vivo it showed efficacy, especially when combined with azithromycin in a preliminary clinical trial. Here we demonstrate that the combination of hydroxychloroquine and azithromycin has a synergistic effect in vitro on SARS-CoV-2 at concentrations compatible with that obtained in human lung.