Equine Encephalosis Virus in India, 2008

A virus isolated from a sick horse from India in 2008 was confirmed by next-generation sequencing analysis to be equine encephalosis virus (EEV). EEV in India is concerning because several species of Culicoides midge, which play a major role in EEV natural maintenance and transmission, are present in this country

Etudes des mécanismes cellulaires et moléculaires de Plasmodium falciparum impliqués dans les résistances aux combinaisons à bases de dérivés de l’artémisinine

Artemisinin-based combination therapies (ACTs) are one of the pillars of the current strategies implemented for fighting malaria. Over the last decade, ACTs have played a major role in decreasing malaria burden. However, this progress is being jeopardized by the emergence of artemisinin-resistant Plasmodium falciparum parasites. Artemisinin (ART) resistance was first detected in western Cambodia in 2008 and has since been observed in neighboring countries in Southeast Asia. The problem of antimalarial drug resistance has recently worsened in Cambodia, with reports of parasites resistant to piperaquine (PPQ), the latest generation of partner drug used in combination with dihydroartemisinin, leading to worrying rates of clinical treatment failure. The monitoring and the comprehension of both types of resistance are crucial to prevent the spread of multi-drug resistant parasites outside Southeast Asia, and particularly to Africa, where the public health consequences would be catastrophic. To this end, new tools are required for studies of the biological and molecular mechanisms underlying resistance to antimalarial drugs and for monitoring the geographic distribution of the resistant parasites.In the first section of this thesis, centered on artemisinin resistance, we first present the discovery of mutations within the K13 gene as a molecular marker for ART resistance. Then we confirm their role as a major determinant of such a resistance. Finally, we analyze the extent of ART resistance through a global mapping of K13 polymorphisms. In the second section, we present our work on the emergence of piperaquine resistance in Cambodia, by initially confirming that multiresistant parasites now circulate in the country. Then we detail the development of a new phenotypical test for the detection of PPQ-resistant parasites, the Piperaquine Survival Assay or PSA. Lastly, we report the discovery of the PfPM2 gene amplification as a candidate molecular marker for PPQ-resistance.Les combinaisons thérapeutiques à base d’artémisinine (ou CTAs) sont une des clés de voûte des stratégies actuelles de lutte contre le paludisme : ces thérapies ont en effet joué un rôle important dans la réduction de l’impact du paludisme au cours de la dernière décennie. Cependant, ces progrès sont aujourd’hui compromis par l’émergence de parasites Plasmodium falciparum résistants aux dérivés de l’artémisinine (ART). Les premiers parasites résistants ont été détectés pour la première fois en 2008 dans l’ouest du Cambodge, puis dans plusieurs pays avoisinants d’Asie du Sud-Est. Le problème de la multirésistance aux antipaludiques s’est récemment aggravé au Cambodge : plusieurs études ont rapporté l’émergence de parasites résistants à la pipéraquine (PPQ), la dernière génération de molécule partenaire utilisée en combinaison avec la dihydroartémisinine, entraînant des taux alarmants d’échecs cliniques. Pour préserver l’efficacité de ces combinaisons, la surveillance et la compréhension de ces deux types de résistance sont cruciales afin d’éviter la dissémination de parasites multirésistants en dehors d’Asie du Sud-Est, et particulièrement en Afrique où les conséquences sanitaires seraient désastreuses. À cette fin, le développement de nouveaux outils est nécessaire pour étudier les mécanismes biologiques et moléculaires impliqués dans la résistance aux CTAs et pour surveiller la distribution géographique des parasites multirésistants. Dans la première partie de cette thèse, axée sur la résistance à l’artémisinine, nous présentons tout d’abord la découverte de mutations au sein du gène K13, marqueur moléculaire pour la résistance à l'ART. Puis nous confirmons leur rôle comme déterminant majeur de cette résistance. Enfin, nous analysons l’étendue de cette résistance au travers d'une cartographie mondiale des polymorphismes de K13. Dans la seconde partie de cette thèse, nous présentons nos travaux portant sur l’émergence de la résistance à la pipéraquine au Cambodge, en confirmant dans un premier temps que des parasites multirésistants à l’ART et à la PPQ circulent désormais dans le pays. Puis en détaillant la mise en point d’un nouveau test phénotypique pour la détection des parasites PPQ-résistants, le Piperaquine Survival Assay ou PSA. Pour finir, nous exposons la découverte de l’amplification du gène PfPM2 comme marqueur moléculaire pour la résistance à la PPQ

Phylogenetic analysis of available full-length RESTV genomes.

<p>Phylogenetic analysis was done with a Bayesian algorithm using Geneious 6.1.5. Clades I to V are indicated with brackets, Asterisks indicate the 9 sequences determined in this study. Posterior probabilities are listed next to each nodes. Scale bar indicates nucleotide substitutions per site.</p

Sequence comparison of full-length viral genomes of representative RESTV isolates.

<p>Sequence identity (%) is indicated in the lower diagonal half, while the number of differing residues is indicated in the upper diagonal half. Asterisks indicate the 9 sequences determined in this study.</p

Sequences analyzed in this study.

<p>Sequences analyzed in this study.</p

Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds

<div><p>Sosuga virus (SOSV) is a recently discovered zoonotic paramyxovirus isolated from a single human case in 2012; it has been ecologically and epidemiologically associated with transmission by the Egyptian rousette bat (<i>Rousettus aegyptiacus</i>). Bats have long been recognized as sources of novel zoonotic pathogens, including highly lethal paramyxoviruses like Nipah virus (NiV) and Hendra virus (HeV). The ability of SOSV to cause severe human disease supports the need for studies on SOSV pathogenesis to better understand the potential impact of this virus and to identify effective treatments. Here we describe a reverse genetics system for SOSV comprising a minigenome-based assay and a replication-competent infectious recombinant reporter SOSV that expresses the fluorescent protein ZsGreen1 in infected cells. First, we used the minigenome assay to rapidly screen for compounds inhibiting SOSV replication at biosafety level 2 (BSL-2). The antiviral activity of candidate compounds was then tested against authentic viral replication using the reporter SOSV at BSL-3. We identified several compounds with anti-SOSV activity, several of which also inhibit NiV and HeV. Alongside its utility in screening for potential SOSV therapeutics, the reverse genetics system described here is a powerful tool for analyzing mechanisms of SOSV pathogenesis, which will facilitate our understanding of how to combat the potential public health threats posed by emerging bat-borne paramyxoviruses.</p></div

Selected compounds’ 50% effective concentrations (EC₅₀), 50% cytotoxic concentrations (CC₅₀), and selectivity indices (SI) determined by minigenome and reporter virus screening assays.

<p>Selected compounds’ 50% effective concentrations (EC₅₀), 50% cytotoxic concentrations (CC₅₀), and selectivity indices (SI) determined by minigenome and reporter virus screening assays.</p

Using SOSV/ZsG as a tool to screen antiviral compounds.

<p><b>a.</b> Representative concentration-response curves of Huh7 cells treated with 2-fold serial dilutions of compound 1 h prior to infection with rSOSV/ZsG at MOI 0.2. ZsG fluorescence (green) was measured at 48 hpi and normalized to mock-treated cells. Cell viability was assessed concurrently by determining ATP content (blue), with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments. <b>b.</b> Confirmatory counter-screening of each compound with wild-type rSOSV. Huh7 cells were treated with serial dilutions of compound 1 h prior to infection with rSOSV at MOI 0.2. At 2 days post infection cells were fixed, and SOSV proteins stained and quantified using immunofluorescence microscopy. Relative fluorescence (red; total fluorescence normalized to mock-treated cells) in each well was determined, with cell viability assessed concurrently by determining ATP content (blue). Each point represents the mean of quadruplicate wells, with error bars showing standard deviation. Values stated are the 50% effective concentration (EC₅₀), 50% cytotoxic concentration (CC₅₀), and selectivity index (SI).</p

Design and optimization of the Sosuga virus minigenome screening assay.

<p><b>a.</b> Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. <b>b.</b> The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). <b>c.</b> Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.</p