In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. To a lesser extent, these tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals.We previously developed the 'Infectious Subgenomic Amplicons' (ISA) method, which enables rescue of single-stranded positive-sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly to animals. Almost all animals were infected when quantities of DNA inoculated were at least 20µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2-fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses.This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Generation and evaluation of protease inhibitor-resistant SARS-CoV-2 strains.

Since the start of the SARS-CoV-2 pandemic, the search for antiviral therapies has been at the forefront of medical research. To date, the 3CLpro inhibitor nirmatrelvir (Paxlovid®) has shown the best results in clinical trials and the greatest robustness against variants. A second SARS-CoV-2 protease inhibitor, ensitrelvir (Xocova®), has been developed. Ensitrelvir, currently in Phase 3, was approved in Japan under the emergency regulatory approval procedure in November 2022, and is available since March 31, 2023. One of the limitations for the use of antiviral monotherapies is the emergence of resistance mutations. Here, we experimentally generated mutants resistant to nirmatrelvir and ensitrelvir in vitro following repeating passages of SARS-CoV-2 in the presence of both antivirals. For both molecules, we demonstrated a loss of sensitivity for resistance mutants in vitro. Using a Syrian golden hamster infection model, we showed that the ensitrelvir M49L mutation, in the multi-passage strain, confers a high level of in vivo resistance. Finally, we identified a recent increase in the prevalence of M49L-carrying sequences, which appears to be associated with multiple repeated emergence events in Japan and may be related to the use of Xocova® in the country since November 2022. These results highlight the strategic importance of genetic monitoring of circulating SARS-CoV-2 strains to ensure that treatments administered retain their full effectiveness.

Sotrovimab retains activity against SARS-CoV-2 omicron variant BQ.1.1 in a non-human primate model.

The SARS-CoV2 Omicron variants have acquired new Spike mutations leading to escape from the most of the currently available monoclonal antibody treatments reducing the options for patients suffering from severe Covid-19. Recently, both in vitro and in vivo data have suggested that Sotrovimab could retain partial activity against recent omicron sub-lineage such as BA.5 variants, including BQ.1.1. Here we report full efficacy of Sotrovimab against BQ.1.1 viral replication as measure by RT-qPCR in a non-human primate challengemodel.

Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.

Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, is currently being evaluated in preclinical and clinical studies for the treatment of various infectious diseases including COVID-19. We developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of favipiravir and its hydroxide metabolite (M1), in human and hamster biological matrices. Analytes were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) after a simple protein precipitation with acetonitrile. The mobile phase consisted of water and methanol, each containing 0.05% formic acid. Experiments were performed using electrospray ionization in the positive and negative ion mode, with protonated molecules used as the precursor ion and a total run time of 6 min. The MS/MS response was linear over the concentration ranges from 0.5-100 µg/ml for favipiravir and 0.25-30 µg/ml for M1. Intra- and inter-day accuracy and precision were within the recommended limits of the European Medicines Agency guidelines. No significant matrix effect was observed, and the method was successfully applied to inform favipiravir dose adjustments in six immunocompromised children with severe RNA viral infections. In conclusion, the UPLC-MS/MS assay is suitable for quantification of favipiravir over a wide range of dosing regimens, and can easily be adapted to other matrices and species.

Activity of Sotrovimab against BQ.1.1 and XBB.1 Omicron sublineages in a hamster model.

The successive emergence of SARS-CoV-2 Omicron variants has completely changed the modalities of use of therapeutic monoclonal antibodies. Recent in vitro studies indicated that only Sotrovimab has maintained partial activity against BQ.1.1 and XBB.1. In the present study, we used the hamster model to determine whether Sotrovimab retains antiviral activity against these Omicron variants in vivo. Our results show that at exposures consistent with those observed in humans, Sotrovimab remains active against BQ.1.1 and XBB.1, although for BQ.1.1 the efficacy is lower than that observed against the first globally dominant Omicron sublineages BA.1 and BA.2.

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains.

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3'UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3'UTR regions are important in Cx. pipiens infection and possibly in vector competence.

Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates.

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19.

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform.

Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2.

BACKGROUND: To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. METHODS: In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. FINDINGS: First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. INTERPRETATION: These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19. FUNDING: This work was supported by the Fondation de France "call FLASH COVID-19", project TAMAC, by "Institut national de la santé et de la recherche médicale" through the REACTing (REsearch and ACTion targeting emerging infectious diseases), by REACTING/ANRS MIE under the agreement No. 21180 ('Activité des molécules antivirales dans le modèle hamster'), by European Virus Archive Global (EVA 213 GLOBAL) funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 871029 and DNDi under support by the Wellcome Trust Grant ref: 222489/Z/21/Z through the COVID-19 Therapeutics Accelerator".

In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5.

The replacement of the Omicron BA.1 variant of SARS-CoV-2 by the BA.2 and the rapid growth of the BA.5 sub lineage, which have both different sets of mutations in the spike glycoprotein, alters the spectrum of activity of therapeutic antibodies currently licensed in the European Union. Using clinical strains of the Omicron BA.2 and BA.5 variants, we compared the neutralising power of monoclonal antibodies against the Omicron BA.1, BA.2 and BA.5 variants, using an ancestral strain (lineage B.1, D614G) and a Delta variant strain as reference. Sotrovimab/Vir-7831 is less active against BA.2 than against BA.1 (fold change reduction ~ 1,4) and even less active against BA.5 (fold change reduction ~ 2.7). Within the Evusheld /AZD7442 cocktail, Cilgavimab/AZD1061 is more active against BA.2 and BA.5 than against BA.1 (fold change increase ~ 32), whilst the very low activity of Tixagevimab/AZD8895 against BA.1 is not enhanced against BA.2 nor BA.5. In total, compared to BA.1, the activity of the Evusheld/AZD7442 is significantly improved against BA.2 while BA.5 is intermediate but closer to BA.2.

A One-Health Approach to Investigating an Outbreak of Alimentary Tick-Borne Encephalitis in a Non-endemic Area in France (Ain, Eastern France): A Longitudinal Serological Study in Livestock, Detection in Ticks, and the First Tick-Borne Encephalitis Virus Isolation and Molecular Characterisation.

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

The SARS-CoV-2 Alpha variant exhibits comparable fitness to the D614G strain in a Syrian hamster model.

Late 2020, SARS-CoV-2 Alpha variant emerged in United Kingdom and gradually replaced G614 strains initially involved in the global spread of the pandemic. In this study, we use a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeed to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicate to almost the same level and induced a comparable disease and immune response. A slight fitness advantage is noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium. This study highlights the need to combine data from different laboratories using various animal models to decipher the biological properties of newly emerging SARS-CoV-2 variants.

A simple reverse genetics method to generate recombinant coronaviruses.

Engineering recombinant viruses is a pre-eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on "infectious subgenomic amplicons" (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS-CoV-2 and also to the feline enteric coronavirus. In both cases we rescue wild-type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS-CoV-2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS-CoV-2 variants, and to accelerate the development of effective therapeutic reagents.

Hydroxychloroquine and azithromycin used alone or combined are not effective against SARS-CoV-2 ex vivo and in a hamster model.

Drug repositioning has been used extensively since the beginning of the COVID-19 pandemic in an attempt to identify antiviral molecules for use in human therapeutics. Hydroxychloroquine and azithromycin have shown inhibitory activity against SARS-CoV-2 replication in different cell lines. Based on such in vitro data and despite the weakness of preclinical assessment, many clinical trials were set up using these molecules. In the present study, we show that hydroxychloroquine and azithromycin alone or combined does not block SARS-CoV-2 replication in human bronchial airway epithelia. When tested in a Syrian hamster model, hydroxychloroquine and azithromycin administrated alone or combined displayed no significant effect on viral replication, clinical course of the disease and lung impairments, even at high doses. Hydroxychloroquine quantification in lung tissues confirmed strong exposure to the drug, above in vitro inhibitory concentrations. Overall, this study does not support the use of hydroxychloroquine and azithromycin as antiviral drugs for the treatment of SARS-CoV-2 infections.

Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the Alpha (B.1.1.7), Beta (B.1.351) and Delta variant (B.1.617.2).

SARS-CoV-2 variants are emerging with potential increased transmissibility highlighting the great unmet medical need for new therapies. Niclosamide is a potent anti-SARS-CoV-2 agent that has advanced in clinical development. We validate the potent antiviral efficacy of niclosamide in a SARS-CoV-2 human airway model. Furthermore, niclosamide remains its potency against the D614G, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants. Our data further support the potent anti-SARS-CoV-2 properties of niclosamide and highlights its great potential as a therapeutic agent for COVID-19.

Favipiravir Inhibits Mayaro Virus Infection in Mice.

Mayaro virus (MAYV) is an emergent alphavirus that causes MAYV fever. It is often associated with debilitating symptoms, particularly arthralgia and myalgia. MAYV infection is becoming a considerable health issue that, unfortunately, lacks a specific antiviral treatment. Favipiravir, a broad-spectrum antiviral drug, has recently been shown to exert anti-MAYV activity in vitro. In the present study, the potential of Favipiravir to inhibit MAYV replication in an in vivo model was evaluated. Immunocompetent mice were orally administrated 300 mg/kg/dose of Favipiravir at pre-, concurrent-, or post-MAYV infection. The results showed a significant reduction in infectious viral particles and viral RNA transcripts in the tissues and blood of the pre- and concurrently treated infected mice. A significant reduction in the presence of both viral RNA transcript and infectious viral particles in the tissue and blood of pre- and concurrently treated infected mice was observed. By contrast, Favipiravir treatment post-MAYV infection did not result in a reduction in viral replication. Interestingly, Favipiravir strongly decreased the blood levels of the liver disease markers aspartate- and alanine aminotransferase in the pre- and concurrently treated MAYV-infected mice. Taken together, these results suggest that Favipiravir is a potent antiviral drug when administered in a timely manner.

Investigation of Bufavirus and Parvovirus 4 in Patients with Gastro-Enteritis from the South-East of France.

Bufavirus (BuV) and human parvovirus 4 (PARV4) belong to the Parvoviridae family. We assessed BuV and PARV4 DNA presence by real-time PCR analysis in stool, blood and respiratory samples collected in patients from Marseille and Nice, two large cities in the South-East of France. Bu-V DNA was detected in diarrheic stool samples from 92 patients (3.6% of 2583 patients), particularly men and adults, and patients from the nephrology and the infectious disease departments. Among the patients with a BuV-positive stool sample and for whom at least one blood sample was available (n = 30 patients), BuV DNA was detected also in 3 blood samples. In contrast, BuV DNA was not detected in any of the respiratory samples from 23 patients with BuV-positive stool. BuV detection rate was comparable in stool samples from patients with and without diarrhea. We did not detect PARV4 DNA in any of the stool specimens (n = 2583 patients). Our results suggest that PARV4 fecal-oral transmission is rare or non-existent in the South-East of France while BuV circulates with a relatively high rate in this area.

Rapid reconstruction of porcine reproductive and respiratory syndrome virus using synthetic DNA fragments.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most challenging infectious disease of pig populations causing devastating economic loss to swine industry. Reverse genetics allow to engineer modified viruses such attenuated strains for vaccine development. Some reverse genetic systems were described for PRRSVs but, due to genome complexity of PRRSVs, construction and modification of such systems remain laborious and time-consuming. In this study, we described a reverse genetics approach based on the "Infectious-Subgenomic Amplicons" (ISA) method to rescue infectious PRRSV particles. Permissive cells were transfected with 4 overlapping synthetic DNA fragments covering the entire genome of PRRSV which allowed the rapid reconstruction of the complete virus genome and the subsequent generation of infectious wild-type particles within days. The ISA method represent a rapid alternative of conventional reverse genetic systems. This method will help to generate genetically modified and attenuated strains for the development of sanitary countermeasures in the future.