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.

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.

Circulation of enterovirus A71 during 2019-2020, Marseille, France.

Enteroviruses A71 (EVs-A71) are known to cause serious neurological infections, especially in the pediatric population. We report here eight cases of EV-A71 infection diagnosed in Marseille over the past 2 years (seven cases in 2019 and one case in 2020). Only children under 5 years of age were affected, including one case of acute flaccid paralysis. Viral RNA was detected by RT-PCR in peripheral samples for all cases (feces and upper respiratory samples). Phylogenetic analyses based on VP1 and 2C3C coding regions revealed that all these cases of EV-A71 infection were caused by viruses belonging to the subgenogroup C1 that currently circulates in Europe and that these viruses are genetically closed to other EVs-A71 recently detected in European countries. These data therefore reinforce the usefulness of the enterovirus surveillance network and the need for systematic screening for EV-A71 in case of an enteroviral infection. This study therefore suggests that the systematic screening for EV-A71 in case of enteroviral infection could provide additional data for enterovirus surveillance networks.

Fatal underhanded chronic enterovirus infection associated with anti-CD20 monotherapy for central nervous system demyelinating disease.

We report a fatal case of coxsackievirus B4 chronic infection in a 30-year-old woman with a diagnosis of myelin oligodendrocyte glycoprotein antibody-associated disorder controlled by rituximab monotherapy for 3 years. Initially presenting as self-limited meningitis, the infection remained silent for 8 months before the sudden onset of fulminant myocarditis. Analysis of the complete genome showed that the same virus was responsible for both episodes.

Dengue Virus Type 1 Infection in Traveler Returning from Benin to France, 2019.

We investigated a case of dengue virus type 1 infection acquired in Benin. Phylogenetic analysis revealed the strain belongs to genotype V but clusters with Asian, rather than with known African, strains. Our finding suggests the introduction of Asian dengue virus in West Africa.

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.

Microcephaly Caused by Lymphocytic Choriomeningitis Virus.

We report congenital microencephaly caused by infection with lymphocytic choriomeningitis virus in the fetus of a 29-year-old pregnant women at 23 weeks' gestation. The diagnosis was made by ultrasonography and negative results for other agents and confirmed by a positive PCR result for lymphocytic choriomeningitis virus in an amniotic fluid sample.

Attenuation of tick-borne encephalitis virus using large-scale random codon re-encoding.

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5-10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.

Air pollution and children's asthma-related emergency hospital visits in southeastern France.

Children's asthma is multifactorial. Environmental factors like air pollution exposure, meteorological conditions, allergens, and viral infections are strongly implicated. However, place of residence has rarely been investigated in connection with these factors. The primary aim of our study was to measure the impact of particulate matter (PM), assessed close to the children's homes, on asthma-related pediatric emergency hospital visits within the Bouches-du-Rhône area in 2013. In a nested case-control study on 3- to 18-year-old children, each control was randomly matched on the emergency room visit day, regardless of hospital. Each asthmatic child was compared to 15 controls. PM10 and PM2.5, meteorological conditions, pollens, and viral data were linked to ZIP code and analyzed by purpose of emergency visit. A total of 68,897 visits were recorded in children, 1182 concerning asthma. Short-term exposure to PM10 measured near children's homes was associated with excess risk of asthma emergency visits (adjusted odds ratio 1.02 (95% CI 1.01-1.04; p = 0.02)). Male gender, young age, and temperature were other risk factors. Conversely, wind speed was a protective factor. CONCLUSION: PM10 and certain meteorological conditions near children's homes increased the risk of emergency asthma-related hospital visits in 3- to 18-year-old children in Bouches-du-Rhône. What is Known: • A relationship between short-term exposure to air pollution and increase in emergency room visits or hospital admissions as a result of increased pollution levels has already been demonstrated. What is New: • This study confirms these results but took into account confounding factors (viral data, pollens, and meteorological conditions) and is based on estimated pollution levels assessed close to the children's homes, rather than those recorded at the hospital. • The study area, the Mediterranean, is favorable to creation of secondary pollutants in these sunny and dry seasons.

Identification of dengue type 2 virus in febrile travellers returning from Burkina Faso to France, related to an ongoing outbreak, October to November 2016.

Dengue fever is rarely reported in travellers returning from Africa. We report two cases of dengue fever in travellers returning from Burkina Faso to France. One of them presented a severe dengue fever with ALT > 1,000 IU/L and pericarditis. Serotype 2 was identified. The cases reflect a large ongoing outbreak with over 1,000 reported cases between August and November in the capital city. Clinicians should consider dengue fever in malaria-negative febrile travellers returning from Africa.

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Respiratory viruses and bacteria among pilgrims during the 2013 Hajj.

Pilgrims returning from the Hajj might contribute to international spreading of respiratory pathogens. Nasal and throat swab specimens were obtained from 129 pilgrims in 2013 before they departed from France and before they left Saudi Arabia, and tested by PCR for respiratory viruses and bacteria. Overall, 21.5% and 38.8% of pre-Hajj and post-Hajj specimens, respectively, were positive for ≥1 virus (p = 0.003). One third (29.8%) of the participants acquired ≥1 virus, particularly rhinovirus (14.0%), coronavirus E229 (12.4%), and influenza A(H3N2) virus (6.2%) while in Saudi Arabia. None of the participants were positive for the Middle East respiratory syndrome coronavirus. In addition, 50.0% and 62.0% of pre-Hajj and post-Hajj specimens, respectively, were positive for Streptococcus pneumoniae (p = 0.053). One third (36.3%) of the participants had acquired S. pneumoniae during their stay. Our results confirm high acquisition rates of rhinovirus and S. pneumoniae in pilgrims and highlight the acquisition of coronavirus E229.

Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons.

Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. 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 the 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. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. 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 20 ng 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.

Broad-spectrum dengue virus detection using the commercial RealStar dengue RT-PCR kit 3.0 (Altona) and an in-house combined real-time RT-PCR assay.

In endemic areas, the genetic diversity among co-circulating dengue virus (DENV) strains is considerable and new, highly divergent strains are identified on a regular basis. It is thus critical to ensure that molecular diagnostic tools effectively detect virus genomes even in case of important genetic variation. Here, we tested both the pan-DENV detection capacity and the limit of detection of two real-time RT-PCR assays: (i) the commercial RealStar Altona 3.0 system and (ii) a laboratory developed test (LDT) combining two RT-PCR systems in a single reaction tube (DenAllDUO). We used a panel of DENV strains representative of the genetic diversity within DENV species, combined with three in vitro transcribed RNAs as surrogates for unavailable strains corresponding to recently discovered strains with substantial genetic divergence: DENV serotype 1 (DENV-1) Brun2014, DENV-2 QML22 and DENV-4 DKE121. Both systems (i) targeted the genome 3' untranslated region, (ii) displayed a broad detection spectrum, encompassing most of DENV species diversity, and (iii) detected the three aforementioned divergent strains. DenAllDUO detected all the strains tested, whereas the RealStar system failed to detect strains from DENV-4 genotype III. Altogether, our findings support the value of these two RT-PCR systems as part of the Dengue diagnostic arsenal.

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.

Further preclinical characterization of molnupiravir against SARS-CoV-2: Antiviral activity determinants and viral genome alteration patterns.

The SARS-CoV-2 pandemic has highlighted the need for broad-spectrum antiviral drugs to respond promptly to viral emergence. We conducted a preclinical study of molnupiravir (MOV) against SARS-CoV-2 to fully characterise its antiviral properties and mode of action. The antiviral activity of different concentrations of MOV was evaluated ex vivo on human airway epithelium (HAE) and in vivo in a hamster model at three escalating doses (150, 300 and 400 mg/kg/day) according to three different regimens (preventive, pre-emptive and curative). We assessed viral loads and infectious titres at the apical pole of HAE and in hamster lungs, and MOV trough concentration in plasma and lungs. To explore the mode of action of the MOV, the entire genomes of the collected viruses were deep-sequenced. MOV effectively reduced viral titres in HAE and in the lungs of treated animals. Early treatment after infection was a key factor in efficacy, probably associated with high lung concentrations of MOV, suggesting good accumulation in the lung. MOV induced genomic alteration in viral genomes with an increase in the number of minority variants, and predominant G to A transitions. The observed reduction in viral replication and its mechanism of action leading to lethal mutagenesis, supported by clinical trials showing antiviral action in humans, provide a convincing basis for further research as an additional means in the fight against COVID-19 and other RNA viruses.

Preclinical in vivo assessment of the activity of AZD7442 anti-SARS-CoV-2 monoclonal antibodies against Omicron sublineages.

Therapeutic monoclonal antibodies have been successful in protecting vulnerable populations against SARS-CoV-2. However, their effectiveness has been hampered by the emergence of new variants. To adapt the therapeutic landscape, health authorities have based their recommendations mostly on in vitro neutralization tests. However, these do not provide a reliable understanding of the changes in the dose-effect relationship and how they may translate into clinical efficacy. Taking the example of EvusheldTM (AZD7442), we aimed to investigate how in vivo data can provide critical quantitative results and project clinical effectiveness. We used the Golden Syrian hamster model to estimate 90 % effective concentrations (EC90) of AZD7442 in vivo against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5 variants. While our in vivo results confirmed the partial loss of AZD7442 activity for BA.1 and BA.2, they showed a much greater loss of efficacy against BA.5 than that obtained in vitro. We analyzed in vivo EC90s in perspective with antibody levels measured in a cohort of immunocompromised patients who received 300 mg of AZD7442. We found that a substantial proportion of patients had serum levels of anti-SARS-CoV-2 spike protein IgG above the estimated in vivo EC90 for BA.1 and BA.2 (21 % and 92 % after 1 month, respectively), but not for BA.5. These findings suggest that AZD7442 is likely to retain clinical efficacy against BA.2 and BA.1, but not against BA.5. Overall, the present study illustrates the importance of complementing in vitro investigations by preclinical studies in animal models to help predict the efficacy of monoclonal antibodies in humans.