Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019

Background: Various pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels. Aim: In this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail. Methods: Cerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed. Results: Among 23 idiopathic cases, TOSV was identified in eight (all male; median age:  39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event. Conclusions: Our study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.ESL laboratory received funding from Institut Pasteur, from the INCEPTION programme (Investissements d’Avenir grant ANR-16-CONV-0005), from the NIH PICREID program (Award Number U01AI151758) and from the Labex IBEID (ANR-10-LABX-62-IBEID). MDFG acknowledges funding from the Junta de Andalucia (PI-0216-2019) and Instituto de Salud Carlos III (Acción Estratégica en Salud Intramural PI20CIII/00005).S

Enhanced Zika virus susceptibility of globally invasive Aedes aegypti populations

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito Aedes aegypti is a major arbovirus vector native to Africa that invaded most of the world’s tropical belt over the past four centuries, after the evolution of a “domestic” form that specialized in biting humans and breeding in water storage containers. Here, we show that human specialization and subsequent spread of A. aegypti out of Africa were accompanied by an increase in its intrinsic ability to acquire and transmit the emerging human pathogen Zika virus. Thus, the recent evolution and global expansion of A. aegypti promoted arbovirus emergence not solely through increased vector–host contact but also as a result of enhanced vector susceptibility

Interactions entre le flavivirus spécifique d'insecte CFAV, son hôte moustique aedes aegypti et les arbovirus co-infectants

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause major public health problems worldwide. These arthropod-borne viruses (arboviruses) are RNA viruses of the Flavivirus genus that are primarily transmitted among humans by the mosquito Aedes aegypti. In addition, Ae. aegypti mosquitoes are naturally infected with cell-fusing agent virus (CFAV), the first-described insect-specific flavivirus (ISF). Little is known about CFAV evolution, interactions with arboviruses in coinfected mosquitoes, and mosquito immune responses to CFAV. This PhD work addressed such understudied aspects of CFAV biology. First, novel full-genome sequences of CFAV allowed a global phylogenetic analysis. A lack of phylogenetic congruence between CFAV and Ae. aegypti indicates that other factors than host population structure shape CFAV genetic diversity. Second, in coinfection experiments, CFAV inhibited DENV and ZIKV replication in vitro and their dissemination in vivo. These results support the hypothesis that ISFs may reduce arbovirus transmission in nature. Third, a CFAV-derived endogenous viral element (EVE) in the Ae. aegypti genome was found to regulate CFAV replication in the ovaries through the piRNA pathway. This finding suggests that EVEs may help to minimize reproductive costs associated with infection by cognate viruses. Overall, this PhD thesis shed light on the complex interactions between CFAV, Ae. aegypti, and arboviruses.Les virus de la dengue (DENV) et du Zika (ZIKV) sont des virus transmis par les moustiques qui causent de graves problèmes de santé publique à travers le monde. Ces virus transmis par des arthropodes (arbovirus) sont des virus à ARN du genre Flavivirus principalement transmis à l'homme par le moustique Aedes aegypti. De plus, les moustiques Ae. aegypti sont naturellement infectés par le cell-fusing agent virus (CFAV), le premier flavivirus spécifique d'insecte (FSI) ayant été décrit. L'évolution du CFAV, ses interactions avec les arbovirus chez les moustiques co-infectés et les réponses immunitaires des moustiques vis-à-vis du CFAV, sont mal connus. Ce travail doctoral a abordé plusieurs de ces aspects peu étudiés de la biologie du CFAV. Premièrement, de nouvelles séquences du génome complet du CFAV ont permis une analyse phylogénétique globale. Un manque de congruence phylogénétique entre le CFAV et Ae. aegypti indique que d’autres facteurs que la structure de la population hôte façonnent la diversité génétique du CFAV. Deuxièmement, dans des expériences de co-infection, le CFAV a inhibé la réplication du DENV et du ZIKV in vitro et leur dissémination in vivo. Ces résultats étayent l'hypothèse que les FSIs pourraient réduire la transmission des arbovirus dans la nature. Troisièmement, il a été montré qu’un élément viral endogène (EVE) dérivé du CFAV dans le génome d’Ae. aegypti régule la réplication du CFAV dans les ovaires via la voie des piARNs. Ce résultat suggère que les EVEs pourraient minimiser des coûts de reproduction associés à l'infection par des virus apparentés. Cette thèse a éclairé les interactions complexes entre le CFAV, Ae. aegypti et les arbovirus

Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo.

International audienceAedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly , A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and den-gue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. ae-gypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission

Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti

International audienceArthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations

Non-retroviral Endogenous Viral Element Limits Cognate Virus Replication in Aedes aegypti Ovaries

International audienceEndogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction

Novel genome sequences of cell-fusing agent virus allow comparison of virus phylogeny with the genetic structure of Aedes aegypti populations

International audienceFlaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity

Introductions and early spread of SARS-CoV-2 in France, 24 January to 23 March 2020

International audienceFollowing SARS-CoV-2 emergence in China, a specific surveillance was implemented in France. Phylogenetic analysis of sequences retrieved through this surveillance suggests that detected initial introductions, involving non-clade G viruses, did not seed local transmission. Nevertheless, identification of clade G variants subsequently circulating in the country, with the earliest from a patient who neither travelled to risk areas nor had contact with travellers, suggests that SARS-CoV-2 might have been present before the first recorded local cases

Novel genome sequences of cell-fusing agent virus allow comparison of virus phylogeny with the genetic structure of Aedes aegypti populations

Flaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity.status: publishe

A novel SARS-CoV-2 related coronavirus in bats from Cambodia

International audienceKnowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses