Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro-resolving mediators, have previously demonstrated anti-influenza activity as well as anti-inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir- and baloxavir-resistant variants. In ST6GalI-MDCK cells, the PDX analog AN-137B reduced viral replication in a dose-dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 mu M for susceptible and resistant A(H1N1)pdm09 viruses. In MTS-based cell viability experiments, AN-137B showed a 50% cellular cytotoxicity (CC50) of 638.7 mu M with a resulting selectivity index of 26.8. Of greater importance, the combination of AN-137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)-stimulated macrophages with AN-137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti-inflammatory effect. In conclusion, our results indicate that the protectin analog AN-137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models.

The fitness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants was determined in human epithelial and continuous cells of the respiratory and gastrointestinal tracts. Competition experiments over 4 days were performed followed by quantification of variant ratios by reverse transcription-droplet digital PCR. These quantitative data were correlated with whole-genome sequencing. In competition experiments of 2 subvariants, the more recent XBB.1 subvariant outcompeted the BA.1.15 subvariant at early time points in the upper respiratory tract epithelium. No difference in replication was observed between the 2 subvariants in the lower respiratory tract. Furthermore, XBB.1 predominated over BA.1.15 and JN.1.1 subvariants in the gastrointestinal tract.

Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmacological measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-α/β/λ1/λ2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-α/β/λ2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 did not significantly interfere with A/H3N2 or A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. SARS-CoV-2 induced a marginal IFN production and reduced the IFN response during coinfections with influenza. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen.

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.

Objective: To develop a new method for reliable and rapid determination of the fitness of SARS-CoV-2 variants of concern. Methods: Competition experiments between two SARS-CoV-2 variants were performed in cells of the upper (nasal human airway epithelium) and lower (Calu-3 cells) respiratory tracts followed by quantification of variant ratios by droplet digital reverse transcription (ddRT)-PCR.Results: In competition experiments, the delta variant outcompeted the alpha variant in both cells of the upper and lower respiratory tracts. A 50/50% mixture of delta and omicron variants indicated a predominance of omicron in the upper respiratory tract whereas delta predominated in the lower respiratory tract. There was no evidence of recombination events between variants in competition as assessed by whole gene sequencing.Conclusion: Differential replication kinetics were shown between variants of concern which may explain, at least partly, the emergence and disease severity associated with new SARS-CoV-2 variants.

Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant human pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin, like the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin, reduced SARS-CoV-2 replication in vitro (EC50 of 2–5 μM). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells, suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index of <10. Thus, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index.

The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC50 of 1-3 μM). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC50 of 2-5 μM). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se , due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID19, a new emerging pandemic affecting humans. Here, single viruses were analyze by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess infectious virus morphology in its native conformation, or upon inactivation treatments, at the nanoscale level and in 3D. High resolution AFM reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory, as revealed by combining AFM and plaque assays, to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-atomic force microscopy is a remarkable toolbox for rapid and direct virus identification and characterization.