In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the ...genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.
•The genomic sequence of 2019-nCoV indicates that the virus clusters with betacoronaviruses of lineage b.•2019-nCoV S-protein sequence has a specific furin-like cleavage site absent in lineage b CoV including SARS-CoV sequences.•The furin-like cleavage site in the S-protein of 2019-nCoV may have implications for the viral life cycle and pathogenicity.•Campaigns to develop anti-2019-nCoV therapeutics should include the evaluation of furin inhibitors.
In the context of the COVID-19 pandemic, virus collections such as EVA-GLOBAL play a key role in the supply of viruses and related products for research. Freeze-drying techniques for viruses ...represent a method of choice for the preservation of strains and their distribution without the need for a demanding cold chain. Here, we describe an optimised lyophilisation protocol usable for SARS-CoV-2 strains that improves preservation and thermostability. We show that sucrose used as an adjuvant represents a simple and efficient stabilizer providing increased protection for long-term preservation and shipment of the virus under different climatic conditions.
The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a ...novel class of small molecules (1,2,3triazolo4,5-dpyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
Cardamones variant Wēnzhōu virus (CVWV) is a rodent-borne virus and part of Arenaviridae family. It causes occasionally fatal hemorrhagic in human, but is generally neglected among others zoonotic ...disease. In Cambodia, the presence of CVWV was detected in 2009 in two Rattus species. It shares more than 85% of identity in S and L segments compared to Wenzhou Mammarenavirus from China, and was shown to be pathogenic in human. To assess the molecular epidemiology of CVWV amount rodents, and the seroprevalence in human and rodents populations in Cambodia
The study was conducted using rodent's serum and pool of organs, and human serum samples, collected in Phnom Penh (the Capital city of Cambodia) and two provinces (Preah Sihanouk and Kampong Cham) representing rural setting, in 2020 and 2022. The presence of CVWV RNA was tested on rodent's pool organs using RT-PCR of GPC and L genes of Arenavirus and Sanger sequencing. The detection of anti-CVWV IgG antibody was performed on rodent and human serum samples using in-house ELISA.
Between 2020 and 2022, 750 pools of organs from rodents and 788 human serum samples were collected. In rodents, the overall prevalence of CVWV was 5.2% (39/750) with the highest prevalence observed in Preah Sihanouk site 3.6% (27/750). Only Rattus exulans 15.4% (32/280) and Rattus norvegicus 4.9% (7/144) species were affected by CVWV. Seroprevalence of anti-CVWV IgG was significantly highest in Rattus norvegicus (58.1%, 72/124), followed by Rattus exulans (13.3%, 24/181) (p < 0.001). In human, seroprevalence of anti-CVWV IgG was between 12.7% (100/788) to 20.3% (112/555) among two visits of 788 participants. Twenty-eight participant remained sero-positive within 2 years period.
Our findings provide updates on prevalence and genetic characterization diversity of Mammarenavirus in Cambodia. Further investigations are needed to determine the risk behavior and impact of Arenavirus infection at human-rodent interface.
Antibody kinetic curves obtained during a viral infection are often fitted using aggregated patient data, hiding the heterogeneity of individual humoral immune responses. Individual antibody ...responses can be modeled using the Wood equation and grouped according to their profile. Such modeling takes into account several important kinetic parameters, such as the day when antibody detection becomes positive daypos, the day of the maximal response daymax, the maximum antibody level levelmax, and the day when antibody detection becomes negative dayneg. Potential associations between these profiles and studied factors can then be tested.
•The day of Antibodies’ appearance, maximal concentration, and Ab response disappearance characterized the humoral response•Aggregating Ab responses from patients to obtain an average kinetic curve hides the heterogeneity of individuals’.•The Wood equation modeled individual Ab response and complete missing data.•The Wood equation model allows identification of clusters as a function of the severity of disease or immunological scar.
The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that ...could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.
•The EVAg was created as an international organization aiming to provide a gold standard resource to the scientific community.•The EVAg operates as a non-profit consortium of 26 partners and 20 associated partners from EU and non-EU countries.•Members and associated members retain ownership of the viruses that they disseminate via the EVAg infrastructure.•The EVAg approach to quality management is directed by the project's own quality standard, based upon OECD guidelines.•The ultimate objective is to make the EVAg a permanent archive that can provide access to viruses and reagents globally.
Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying ...constructs that are likely to be amenable to structural analysis. A variety of expression‐screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6‐tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small‐scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi‐parameter problems faced by crystallographers, such as crystallization.
•Production of two 74-mer capped RNA substrates, GpppA2′OMe-RNA74 and 7MeGpppA-RNA74, for DENV N7- and 2′-O-MTase.•Development of a robust, specific and high-throughput N7-MTase inhibition ...assay.•Selective screening of known MTase inhibitors against DENV 2′-O- and N7-MTase.
Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, 7MeGpppA2′OMe-RNA, at the 5′-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, 7MeGpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA2′OMe-RNA74, by ligation of two RNA fragments. Then, we use GpppA2′OMe-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate 7MeGpppA-RNA74 in order to characterize the DENV 2′-O-MTase activity specifically on long capped RNA.
We next compared the N7- and 2′-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.
Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an ...EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.
The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, ...insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.
PDF
