Biology subjects

de Lamballerie, X.

Publications and source records attributed to de Lamballerie, X..

26 records · Page 2Linked to original sources

bioRxiv2020

Favipiravir and severe acute respiratory syndrome coronavirus 2 in 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 used a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment was initiated before or simultaneously to infection, favipiravir had a strong dose effect, leading to dramatic reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlated with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. The antiviral efficacy observed in this study was achieved with plasma drug exposure comparable with those previously found during human clinical trials and was associated with weight losses in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

microbiology↗

bioRxiv2020

Evaluation of heating and chemical protocols for inactivating SARS-CoV-2

Clinical samples collected in COVID-19 patients are commonly manipulated in BSL-2 laboratories for diagnostic purpose. We used the French norm NF-EN-14476+A2 derived from the European standard EN-14885. To avoid the risk of exposure of laboratory workers, we showed that Triton-X100 must be added to guanidinium thiocyanate-lysis buffers to obtain a 6-log reduction of infectious virus. Although heating protocol consisting of 92{degrees}C-15min was more effective rather than 56{degrees}C-30min and 60{degrees}C-60min to achieve 6-log reduction, it is not amenable for molecular detection on respiratory specimens because of important decrease of detectable RNA copies in the treated sample vs untreated sample. The 56{degrees}C-30min and 60{degrees}C-60min should be used for inactivation of serum / plasma samples for serology because of the 5log10 reduction of infectivity and low viral loads in blood specimens.

microbiology↗

bioRxiv2020

In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication

A novel coronavirus, named SARS-CoV-2, emerged in 2019 from Hubei region in China and rapidly spread worldwide. As no approved therapeutics exists to treat Covid-19, the disease associated to SARS-Cov-2, there is an urgent need to propose molecules that could quickly enter into clinics. Repurposing of approved drugs is a strategy that can bypass the time consuming stages of drug development. In this study, we screened the Prestwick Chemical Library(R) composed of 1,520 approved drugs in an infected cell-based assay. 90 compounds were identified. The robustness of the screen was assessed by the identification of drugs, such as Chloroquine derivatives and protease inhibitors, already in clinical trials. The hits were sorted according to their chemical composition and their known therapeutic effect, then EC50 and CC50 were determined for a subset of compounds. Several drugs, such as Azithromycine, Opipramol, Quinidine or Omeprazol present antiviral potency with 2<EC50<20{micro}M. By providing new information on molecules inhibiting SARS-CoV-2 replication in vitro, this study could contribute to the short-term repurposing of drugs against Covid-19.

microbiology↗

bioRxiv2019

A new high-throughput tool to screen mosquito-borne viruses in Zika virus endemic/epidemic areas

Mosquitoes are vectors of arboviruses affecting animal and human health. Arboviruses circulate primarily within an enzootic cycle and recurrent spillovers contribute to the emergence of human-adapted viruses able to initiate an urban cycle involving anthropophilic mosquitoes. The increasing volume of travel and trade offers multiple opportunities for arbovirus introduction in new regions. This scenario has been exemplified recently with the Zika pandemic. To incriminate a mosquito as vector of a pathogen, several criteria are required such as the detection of natural infections in mosquitoes. In this study, we used a high-throughput chip based on the BioMarkTM Dynamic arrays system capable of detecting 64 arboviruses in a single experiment. A total of 17,958 mosquitoes collected in Zika-endemic/epidemic countries (Brazil, French Guiana, Guadeloupe, Suriname, Senegal, and Cambodia) were analyzed. Here we show that this new tool can detect endemic and epidemic viruses in different mosquito species in an epidemic context. Thus, this fast and low-cost method can be suggested as a novel epidemiological surveillance tool to identify circulating arboviruses.

molecular biology↗

bioRxiv2019

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 virus infection. 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. Stochastic molecular simulations indicate that the steric freedom caused by the E460D substitution increases close electrostatic interactions between the inhibitor and the interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an incorrect/modified nucleotide. 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 only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. 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. ImportanceTick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in large areas of 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 virus infection, 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 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.

microbiology↗

bioRxiv2018

Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared different constructs and confirmed the need to modify the cleavage site between the pre-peptide and prM protein. Genotypic characterization of the chimeras indicates that emergence of compensatory mutations in the E protein is required to restore virus replicative fitness. Using an immunocompromised mouse model, we demonstrate that mice infected with the chimeric virus produced levels of neutralizing antibodies close to those observed following infection with ZIKV and that pre-immunized mice were protected against viscerotropic and neuroinvasive virus following challenge with a heterologous strain of ZIKV. These data provide a sound basis for the future development of this ZIKV vaccine candidate.

microbiology↗

bioRxiv2018

Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

Yellow fever virus (Flavivirus genus) is an arthropod-borne pathogen which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow Fever Ap7, by introducing a set of 4 synonymous and 6 non-synonymous mutations into a single subgenomic amplicon, derived from the sequence of the Asibi strain. The resulting strain, Yellow Fever Ap7M, induced a disease similar to that described for Ap7 in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from either Ap7M or Asibi viruses and investigated the role of each of Ap7M non-synonymous mutations in its in vivo phenotype. This allowed identifying key components of the virulence mechanism in hamsters. In Ap7M virus, the reversion of either E/Q27H or E/D155A mutations, led to an important reduction of both virulence and in vivo replicative fitness. In addition, the introduction of the single D155A Ap7M mutation within the E protein of the Asibi virus was sufficient to drastically modify its phenotype in hamsters towards both a greater replication efficiency and virulence. Finally, inspection of the Asibi strain E protein structure combined to in vivo testing revealed the importance of an exposed -helix in domain I, containing residues 154 and 155, for Ap7M virulence in hamsters.

microbiology↗

bioRxiv2018

Exploratory re-encoding of Yellow Fever Virus genome: analysis of in vitro and in vivo replicative phenotypes

Virus attenuation by genome re-encoding is a pioneering approach for generating effective live-attenuated vaccine candidates. Its core principle is to introduce a large number of synonymous substitutions into the viral genome to produce stable attenuation of the targeted virus. Introduction of large numbers of mutations has also been shown to maintain stability of the attenuated phenotype by lowering the risk of reversion and recombination of re-encoded genomes. Identifying mutations with low fitness cost is pivotal as this increases the number that can be introduced and generates more stable and attenuated viruses. Here, we sought to identify mutations with low deleterious impact on the in vivo replication and virulence of yellow fever virus (YFV). Following comparative bioinformatic analyses of flaviviral genomes, we categorized synonymous transition mutations according to their impact on CpG/UpA composition and secondary RNA structures. We then designed 17 re-encoded viruses with 100-400 synonymous mutations in the NS2A-to-NS4B coding region of YFV Asibi and Ap7M (hamster-adapted) genomes. Each virus contained a panel of synonymous mutations designed according to the above categorisation criteria. The replication and fitness characteristics of parent and re-encoded viruses were compared in vitro using cell culture competition experiments. In vivo laboratory hamster models were also used to compare relative virulence and immunogenicity characteristics. Most of the re-encoded strains showed no decrease in replicative fitness in vitro. However, they showed reduced virulence and, in some instances, decreased replicative fitness in vivo. Importantly, the most attenuated of the re-encoded strains induced robust, protective immunity in hamsters following challenge with Ap7M, a virulent virus. Overall, the introduction of transitions with no or a marginal increase in the number of CpG/UpA dinucleotides had the mildest impact on YFV replication and virulence in vivo. Thus, this strategy can be incorporated in procedures for the finely tuned creation of substantially re-encoded viral genomes.

microbiology↗