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.

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.

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".

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.

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.

Detection of Pneumocystis jirovecii in Hospitalized Children Less Than 3 Years of Age.

Few data are available in the literature regarding Pneumocystis jirovecii infection in children under 3 years old. This retrospective cohort study aimed to describe medically relevant information among them. All children under 3 years old treated in the same medical units from April 2014 to August 2020 and in whom a P. jirovecii evaluation was undertaken were enrolled in the study. A positive case was defined as a child presenting at least one positive PCR for P. jirovecii in a respiratory sample. Medically relevant information such as demographical characteristics, clinical presentation, microbiological co-infections, and treatments were collected. The objectives were to describe the characteristics of these children with P. jirovecii colonization/infection to determine the key underlying diseases and risk factors, and to identify viral respiratory pathogens associated. The PCR was positive for P. jirovecii in 32 children. Cardiopulmonary pathologies (21.9%) were the most common underlying disease in them, followed by severe combined immunodeficiency (SCID) (18.8%), hyaline membrane disease (15.6%), asthma (9.4%) and acute leukaemia (6.3%). All SCID children were diagnosed with pneumocystis pneumonia. Co-infection with Pj/Rhinovirus (34.4%) was not significant. Overall mortality was 18.8%. Paediatric pneumocystis is not restricted to patients with HIV or SCID and should be considered in pneumonia in children under 3 years old.

Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2.

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of imatinib in multiple models. Our results indicated that imatinib and another TKI, the masitinib, exhibit an antiviral activity in VeroE6 cells. However, imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of imatinib and similar TKIs as antivirals in the treatment of Covid-19.

Replicative Fitness of a SARS-CoV-2 20I/501Y.V1 Variant from Lineage B.1.1.7 in Human Reconstituted Bronchial Epithelium.

Since its emergence in 2019, circulating populations of the new coronavirus (CoV) continuously acquired genetic diversity. At the end of 2020, a variant named 20I/501Y.V1 (lineage B.1.1.7) emerged and replaced other circulating strains in several regions. This phenomenon has been poorly associated with biological evidence that this variant and the original strain exhibit different phenotypic characteristics. Here, we analyze the replication ability of this new variant in different cellular models using for comparison an ancestral D614G European strain (lineage B1). Results from comparative replication kinetics experiments in vitro and in a human reconstituted bronchial epithelium showed no difference. However, when both viruses were put in competition in human reconstituted bronchial epithelium, the 20I/501Y.V1 variant outcompeted the ancestral strain. All together, these findings demonstrate that this new variant replicates more efficiently and may contribute to a better understanding of the progressive replacement of circulating strains by the severe acute respiratory CoV-2 (SARS-CoV-2) 20I/501Y.V1 variant. IMPORTANCE The emergence of several SARS-CoV-2 variants raised numerous questions concerning the future course of the pandemic. We are currently observing a replacement of the circulating viruses by the variant from the United Kingdom known as 20I/501Y.V1, from the B.1.1.7 lineage, but there is little biological evidence that this new variant exhibits a different phenotype. In the present study, we used different cellular models to assess the replication ability of the 20I/501Y.V1 variant. Our results showed that this variant replicates more efficiently in human reconstituted bronchial epithelium, which may explain why it spreads so rapidly in human populations.

Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model.

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we use a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment is initiated before or simultaneously to infection, favipiravir has a strong dose effect, leading to reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlates with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. Antiviral efficacy is achieved with plasma drug exposure comparable with those previously found during human clinical trials. Notably, the highest dose of favipiravir tested is associated with signs of toxicity in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

Development and characterization of recombinant tick-borne encephalitis virus expressing mCherry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.

The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-ß-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

Reverse Genetics of RNA Viruses: ISA-Based Approach to Control Viral Population Diversity without Modifying Virus Phenotype.

Reverse genetic systems are essential for the study of RNA viruses. Infectious clones remain the most widely used systems to manipulate viral genomes. Recently, a new PCR-based method called ISA (infectious subgenomic amplicons) has been developed. This approach has resulted in greater genetic diversity of the viral populations than that observed using infectious clone technology. However, for some studies, generation of clonal viral populations is necessary. In this study, we used the tick-borne encephalitis virus as model to demonstrate that utilization of a very high-fidelity, DNA-dependent DNA polymerase during the PCR step of the ISA procedure gives the possibility to reduce the genetic diversity of viral populations. We also concluded that the fidelity of the polymerase is not the only factor influencing this diversity. Studying the impact of genotype modification on virus phenotype is a crucial step for the development of reverse genetic methods. Here, we also demonstrated that the utilization of different PCR polymerases did not affect the phenotype (replicative fitness in cellulo and virulence in vivo) compared to the initial ISA procedure and the use of an infectious clone. In conclusion, we provide here an approach to control the genetic diversity of RNA viruses without modifying their phenotype.

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice.

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

Comparison of chikungunya viruses generated using infectious clone or the Infectious Subgenomic Amplicons (ISA) method in Aedes mosquitoes.

Reverse genetics systems provide the opportunity to manipulate viral genomes and have been widely used to study RNA viruses and to develop new antiviral compounds and vaccine strategies. The recently described method called ISA (Infectious Subgenomic Amplicons) gives the possibility to rescue RNA viruses in days. We demonstrated in cell culture that the use of the ISA method led to a higher genetic diversity of viral populations than that observed using infectious clone technology. However, no replicative fitness difference was observed. In the present study, we used the chikungunya virus as a model to compare in Aedes aegypti and Aedes albopictus mosquitoes the genotypic and phenotypic characteristics of viruses produced either from an infectious clone or using the ISA method. We confirmed the results found in cellulo corroborating that the use of the ISA method was associated with higher genetic diversity of viral populations in mosquitoes but did not affect the vector competence validating its use for in vivo experiments.

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated 'SuPReMe' (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.