Éric Bergeron

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread

BackgroundSevere acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available.ResultsWe report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations.ConclusionChloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.

Structure, Function, and Evolution of the Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.

Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012.

In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

Efficient Reverse Genetics Generation of Infectious Junin Viruses Differing in Glycoprotein Processing

ABSTRACT The New World arenaviruses, Junin, Machupo, Guanarito, Sabia, and Chapare, are associated with rapidly progressing severe hemorrhagic fever with a high rate of case fatality in various regions of South America. The threat of natural or deliberate outbreaks associated with these viruses makes the development of preventive or therapeutic measures important. Here we describe a Junin virus functional minigenome system and a reverse genetics system for production of infectious Junin virus. This robust, highly efficient system involves transfection of cells with only two plasmids which transcribe the virus S and L antigenomic RNAs. The utility of the system is demonstrated by generating Junin viruses which encode a glycoprotein precursor (GPC) containing the following: (i) the wild-type (SKI-1/S1P peptidase) cleavage site, (ii) no cleavage site, or (iii) a cleavage site where the SKI-1/S1P motif (RSLK) is replaced by a furin cleavage site (RRKR). In contrast to the wild-type virus, Junin virus lacking a GPC cleavage site replicated within successfully transfected cells but failed to yield infectious virus particles. This confirms observations with other arenaviruses suggesting that GPC cleavage is essential for arenavirus infectivity. In contrast, infectious Junin virus which encoded GPC cleaved by furin-like proteases was easily generated. The two-plasmid, high efficiency aspects of this Junin virus reverse genetics system show great promise for addressing important questions regarding arenavirus hemorrhagic fever disease and for development of precisely attenuated live arenavirus vaccines.

The Major Determinant of Attenuation in Mice of the Candid1 Vaccine for Argentine Hemorrhagic Fever Is Located in the G2 Glycoprotein Transmembrane Domain

ABSTRACT Candid1, a live-attenuated Junin virus vaccine strain, was developed during the early 1980s to control Argentine hemorrhagic fever, a severe and frequently fatal human disease. Six amino acid substitutions were found to be unique to this vaccine strain, and their role in virulence attenuation in mice was analyzed using a series of recombinant viruses. Our results indicate that Candid1 is attenuated in mice through a single amino acid substitution in the transmembrane domain of the G2 glycoprotein. This work provides insight into the molecular mechanisms of attenuation of the only arenavirus vaccine currently available.

Prognostic Indicators for Ebola Patient Survival

Odds of survival were greatest when first Ebola virus–positive blood sample collected had low viral load.

Reverse Genetics Generation of Chimeric Infectious Junin/Lassa Virus Is Dependent on Interaction of Homologous Glycoprotein Stable Signal Peptide and G2 Cytoplasmic Domains

ABSTRACT The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses ☆

Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention.

Seroepidemiological Studies of Crimean-Congo Hemorrhagic Fever Virus in Domestic and Wild Animals.

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, tick-borne viral disease. Humans are the only species known to develop illness after CCHF virus (CCHFV) infection, characterized by a nonspecific febrile illness that can progress to severe, often fatal, hemorrhagic disease. A variety of animals may serve as asymptomatic reservoirs of CCHFV in an endemic cycle of transmission. Seroepidemiological studies have been instrumental in elucidating CCHFV reservoirs and in determining endemic foci of viral transmission. Herein, we review over 50 years of CCHFV seroepidemiological studies in domestic and wild animals. This review highlights the role of livestock in the maintenance and transmission of CCHFV, and provides a detailed summary of seroepidemiological studies of wild animal species, reflecting their relative roles in CCHFV ecology.

A Virus-Like Particle System Identifies the Endonuclease Domain of Crimean-Congo Hemorrhagic Fever Virus

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus) is an extremely pathogenic member of the Bunyaviridae family. Since handling of the virus requires a biosafety level 4 (BSL-4) facility, little is known about pathomechanisms and host interactions. Here, we describe the establishment of a transcriptionally competent virus-like particle (tc-VLP) system for CCHFV. Recombinant polymerase (L), nucleocapsid protein (N) and a reporter minigenome expressed in human HuH-7 cells resulted in formation of transcriptionally active nucleocapsids that could be packaged by coexpressed CCHFV glycoproteins into tc-VLPs. The tc-VLPs resembled authentic virus particles in their protein composition and neutralization sensitivity to anti-CCHFV antibodies and could recapitulate all steps of the viral replication cycle. Particle attachment, entry, and primary transcription were modeled by infection of naive cells. The subsequent steps of genome replication, secondary transcription, and particle assembly and release can be obtained upon passaging the tc-VLPs on cells expressing CCHFV structural proteins. The utility of the VLP system was demonstrated by showing that the endonuclease domain of L is located around amino acid D693, as was predicted in silico by B. Morin et al. (PLoS Pathog 6:e1001038, 2010, http://dx.doi.org/10.1371/journal.ppat.1001038). The tc-VLP system will greatly facilitate studies and diagnostics of CCHFV under non-BSL-4 conditions. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is an extremely virulent pathogen of humans. Since the virus can be handled only at the highest biosafety level, research is restricted to a few specialized laboratories. We developed a plasmid-based system to produce virus-like particles with the ability to infect cells and transcribe a reporter genome. Due to the absence of viral genes, the virus-like particles are unable to spread or cause disease, thus allowing study of aspects of CCHFV biology under relaxed biosafety conditions.