The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease

Flavivirus study : molecular epidemiology in Bolivia and interaction analysis of TLR3-dependent interferon response

Le genre Flavivirus regroupe plus de 70 espèces dont plusieurs sont des pathogènes humains de première importance responsables dans les formes les plus graves de manifestations hémorragiques ou d’encéphalites parfois mortelles. L’absence de traitements antiviraux spécifiques et l’augmentation croissante des flaviviroses, surtout dans les régions tropicales, justifient un effort de recherche et développement pour lutter contre ces maladies.Ce travail a abordé deux aspects de l’infection à flavivirus : un aspect épidémiologique et un aspect plus fondamental sur l’immunité innée et les contremesures flavivirales. L’étude épidémiologique a été menée en collaboration avec le CENETROP (Centro national de enfermedades tropicales) de Bolivie grâce à la contribution de l’IRD. De par l’analyse des différentes souches circulantes dans ce pays, elle a permis une meilleure compréhension de l’épidémiologie de la dengue et de la fièvre jaune et nous a fait prendre conscience de la variabilité génétique de ces virus. Devant le peu de données répertoriées en Bolivie, nos travaux serviront de référence pour comprendre les épidémies futures, peut-être améliorer les techniques de diagnostic et permettre le développement de stratégies de prévention adaptées et l’amélioration des politiques de lutte contre la fièvre jaune en Amérique du Sud. La cohabitation entre le virus et l’hôte immunocompétent est le résultat d’un équilibre subtil entre le taux de réplication virale et la clairance du système immunitaire pour garantir la survie des deux espèces. Chacun a évolué en développant des mécanismes de défenses contre l’autre. Notre second travail visait à analyser l’influence de la protéine flavivirale non structurale NS1 sur la réponse interféron de l’hôte. L’identification de stratégies virales d’évasion face à l’immunité de l’hôte et l’analyse de leurs fonctions dans l’infection virale permettrait de mieux comprendre le système immunitaire ainsi que l’interaction virus–hôte. Ceci aiderait au développement de nouvelles stratégies antivirales afin de traiter les pathologies associées à ces arbovirus.The Flavivirus genus consists of sevevral human pathogens responsible for hemorragic syndrome or encephalitis. The absence of specific antiviral treatment and an increase in Flavivirus incidence has led to a greater research effort in fighting these diseases. The study takes an epidemiological and a fundamental approach in its analysis of the innate immune response to flavivirus infection as well as flaviviral adaptation to evade this response. The analysis of circulating strains in Bolivia has led to a better understanding of dengue and yellow fever and also an awareness of their genetic variability. Given the limited information available in Bolivia, our studies could be used as a reference to understand future epidemics, improve diagnostic methods and allow the development of prevention strategies to fight against yellow fever in south Africa. The relationship between virus and host results from a subtle balance between viral replication and immunity clearance allowing the survival of both species. Each one as developed defence mechanisms against the other. We also examined the role of the non structural protein NS1 in the interferon respons to Flaviviral infection. Knowledge on viral escape strategies from host immunity could help to develop antiviral treatment for these arbovirus disease

Mycoplasma removal : simple curative methods for viral supernatants

As a partner of the European Virus Archive (EVA) FP7 infrastructure, our research group is maintaining and developing a large virus collection. To meet the standards of the quality management system adopted by all European Virus Archive partners, the detection and eradication of mycoplasma in cell culture supernatants (stored at -80 degrees C or freeze-dried) has to be improved. Although the methods for mycoplasma elimination from infected cell lines were largely described, the decontamination procedures of precious cell culture supernatants was poorly documented. In this study, a large panel of mycoplasma-contaminated virus stocks (enveloped and non enveloped, RNA and DNA viruses) was tested successfully for mycoplasma removal using two simple optimized methods. These easy-to-perform protocols, using respectively Plasmocin (TM) (InvivoGen, Cayla, France) and chloroform, were shown to remove mycoplasma completely from cell supernatant without incidence in viral infectivity

Comparative production analysis of three phlebovirus nucleoproteins under denaturing or non-denaturing conditions for crystallographic studies.

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle

Rapid next-generation sequencing of dengue, EV-A71 and RSV-A viruses

Accurate characterisation of viral strains constitutes a crucial objective for the management of modern virus collections. Next-generation sequencing (NGS) provides technical solution for fast and cost-effective full genome sequencing. Here, we report protocols for rapid full-genome characterisation of RNA viruses of medical importance: dengue virus, enterovirus A71 and respiratory syncytial virus A, based on a specific amplification step followed by NGS-sequencing. A subset of full-length genome sequences representing the genetic diversity of each virus type was selected in GenBank and used to design primer sets allowing the amplification of the complete genome in 3-8 overlapping PCR fragments. The technique was used for characterising 53 strains (33 DENV, 8 EV-A71,12 RSV-A) from various genotypes and origins. In a single assay, and in just 4 days, it provided for all strains an excellent genomic coverage (similar to 99% including complete ORF for all strains) and accurate sequences with high number of reads per position (250-3500 on average). The elaboration of specific PCR-based full-genome sequencing protocols for diverse virus groups is likely to revolutionise the characterisation of viral isolates in modern collection, but also to contribute in the next future to the study of RNA viruses directly from biological samples

In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant

Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against influenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human fibroblasts (MRC-5 lung cells) (IC50 < 10 mu g/ml). A CHIKV resistant mutant was then selected and adapted to growth in the presence of 30 mu g/ml ARB in MRC5 cells; its complete sequence analysis revealed a single amino acid substitution (G407R) localized in the E2 envelope protein. To confirm the G407R role in the molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand the molecular basis of ARB activity

Toscana, West Nile, Usutu and tick-borne encephalitis viruses : external quality assessment for molecular detection of emerging neurotropic viruses in Europe, 2017

Background: Neurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response. Aim: To improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV). Methods: Sixty-nine laboratories were invited. The EQA panel included two WNV RNA- positive samples (lineages 1 and 2), two TOSV RNA- positive samples (lineages A and B), one TBEV RNA- positive sample (Western subtype), one USUV RNA- positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed. Results: The final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%). Conclusion: We observed wide variety in extraction methods and RT- PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories

Effect of chemical stabilizers on the thermostability and infectivity of a representative panel of freeze dried viruses

As a partner of the European Virus Archive (EVA) FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20 degrees C, +4 degrees C and +20 degrees C) for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID50) assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection