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

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns

Application de la génomique virale pour comprendre l'épidémiologie et l'évolution des virus à ARN dans le contexte d'épidémies passées et en cours

From 2009 H1N1 influenza pandemic to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, emerging and re-emerging viral infectious diseases have constituted one of the greatest global challenges of the twenty-first century. For instance, in the last two years alone, Chikungunya virus (CHIKV) have caused major outbreaks in several countries across Southeast Asia, in addition to the growing burden of SARS-CoV-2, causing major consequences around the globe. While pandemics are not a new phenomenon, the growing popularity of next generation sequencing technologies, the increased pace of genome data generation - which are enlarging viral genome repositories - and advances in both phylodynamic methods and computer science made it possible to use viral genomes to answer crucial epidemiological questions, ultimately strengthening public health response to outbreaks. This work focused on investigating the origin, timing, and spread of viral infectious diseases of past and ongoing outbreaks. To address these questions we used genomic epidemiology, showing that it can be a very powerful tool to investigate infectious disease outbreaks at various steps. For instance, we succeeded in setting up a metagenomic deep sequencing protocol that allowed us to determine that the probable etiologic agent responsible for a series of meningitis cases in southern Spain during 2015-2018 was Toscana virus (TOSV), an arbovirus responsible for an increasing number of infections in countries enclosing the Mediterranean Sea. Next, using an in-house designed amplicon-based sequencing approach we succeeded to obtain the complete sequence of TOSV from samples of varying viral load and quality. We then went a step further on the analyses of CHIKV outbreaks in Cambodia. In light of the genomes that we generated, phylogenetic analysis allowed us to study the genetic diversity of CHIKV and by adding temporal data, to estimate the time at which it was introduced into the population. Subsequent phylogeographic analysis provided us with an additional level of detail, shedding light on the origins of the outbreak, connections to previous outbreaks in the same region and dispersal of the virus within the country. On a different scale, we were able to track the dynamics of the SARS-CoV-2 viral population during the long-term infection of an immunocompromised patient, highlighting the challenges of treating these vulnerable members of our society for whom there is still significant risk for severe illness. Together, this work provides a better understanding of the epidemiology and evolution of several RNA viruses representing important threats to human health and simultaneously highlights the importance and main contributions of genomic epidemiology for outbreak investigation.Les pandémies qui ont impacté le début du XXIe siècle (la grippe H1N1 en 2009 et la COVID-19 actuellement) soulignent l'importance du défi global que posent les maladies infectieuses virales émergentes et ré-émergentes à nos sociétés. Par exemple, au cours des deux dernières années seulement, le virus chikungunya (CHIKV) a provoqué des épidémies majeures dans plusieurs pays d'Asie du Sud-Est, auxquelles s'ajoute le fardeau croissant du syndrome respiratoire aigu sévère (SARS-CoV-2), avec des conséquences majeures dans le monde entier. Bien que les pandémies ne soient pas un phénomène nouveau, la popularité croissante des technologies de séquençage de nouvelle génération, l'accélération de la génération de données génomiques - qui élargissent les bases de données de génomes viraux - et les progrès des méthodes phylodynamiques ainsi que de l'informatique ont permis d'utiliser les génomes viraux pour répondre à des questions épidémiologiques cruciales, renforçant les réponses de la santé publique aux épidémies. Ce travail s'est concentré sur l'étude de l'origine, aussi bien spatiale que temporelle, et de la propagation des maladies infectieuses virales d'épidémies passées et en cours. Pour ce faire, nous avons utilisé l'épidémiologie génomique et montré qu'elle peut être un outil très puissant pour enquêter sur les épidémies de maladies infectieuses à différents niveaux. Par exemple, nous avons réussi à mettre en place un protocole de séquençage métagénomique profond qui nous a permis de découvrir que le probable agent étiologique responsable d'une série de cas de méningite dans le sud de l'Espagne, au cours de la période 2015-2018, était le virus Toscana (TOSV), un arbovirus responsable d'un nombre croissant d'infections dans les pays bordant la mer Méditerranée. Ensuite, nous avons développé une approche de séquençage basée sur des amplicons qui nous a permis d'obtenir la séquence complète du TOSV à partir d'échantillons de qualité et charge virale très variables. Nous sommes ensuite allés plus loin dans les analyses des épidémies de CHIKV au Cambodge. En effet, l'analyse phylogénétique des génomes que nous avons générés nous a permis d'étudier la diversité génétique du CHIKV et, en ajoutant des données temporelles, d'estimer le moment auquel il a été introduit dans la population. Dans un second temps, une analyse phylogéographique nous a fourni un niveau de détail supplémentaire en mettant en lumière les origines de l'épidémie, les liens avec les épidémies précédentes dans la même région et la dispersion du virus dans le pays. Enfin, à une autre échelle, nous avons pu suivre la dynamique de la population virale du SARS-CoV-2 lors de l'infection à long terme d'un patient immunodéprimé, ce qui a souligné les défis de la prise en charge de ces membres vulnérables de notre société pour lesquels il existe un risque accru de maladie grave. Pour conclure, ces travaux contribuent à une meilleure compréhension de l'épidémiologie et l'évolution de plusieurs virus à ARN qui représentent encore aujourd'hui des menaces importantes pour la santé humaine, et soulignent les apports de l'épidémiologie génomique contre les épidémies

Application de la génomique virale pour comprendre l'épidémiologie et l'évolution des virus à ARN dans le contexte d'épidémies passées et en cours

From 2009 H1N1 influenza pandemic to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, emerging and re-emerging viral infectious diseases have constituted one of the greatest global challenges of the twenty-first century. For instance, in the last two years alone, Chikungunya virus (CHIKV) have caused major outbreaks in several countries across Southeast Asia, in addition to the growing burden of SARS-CoV-2, causing major consequences around the globe. While pandemics are not a new phenomenon, the growing popularity of next generation sequencing technologies, the increased pace of genome data generation - which are enlarging viral genome repositories - and advances in both phylodynamic methods and computer science made it possible to use viral genomes to answer crucial epidemiological questions, ultimately strengthening public health response to outbreaks. This work focused on investigating the origin, timing, and spread of viral infectious diseases of past and ongoing outbreaks. To address these questions we used genomic epidemiology, showing that it can be a very powerful tool to investigate infectious disease outbreaks at various steps. For instance, we succeeded in setting up a metagenomic deep sequencing protocol that allowed us to determine that the probable etiologic agent responsible for a series of meningitis cases in southern Spain during 2015-2018 was Toscana virus (TOSV), an arbovirus responsible for an increasing number of infections in countries enclosing the Mediterranean Sea. Next, using an in-house designed amplicon-based sequencing approach we succeeded to obtain the complete sequence of TOSV from samples of varying viral load and quality. We then went a step further on the analyses of CHIKV outbreaks in Cambodia. In light of the genomes that we generated, phylogenetic analysis allowed us to study the genetic diversity of CHIKV and by adding temporal data, to estimate the time at which it was introduced into the population. Subsequent phylogeographic analysis provided us with an additional level of detail, shedding light on the origins of the outbreak, connections to previous outbreaks in the same region and dispersal of the virus within the country. On a different scale, we were able to track the dynamics of the SARS-CoV-2 viral population during the long-term infection of an immunocompromised patient, highlighting the challenges of treating these vulnerable members of our society for whom there is still significant risk for severe illness. Together, this work provides a better understanding of the epidemiology and evolution of several RNA viruses representing important threats to human health and simultaneously highlights the importance and main contributions of genomic epidemiology for outbreak investigation.Les pandémies qui ont impacté le début du XXIe siècle (la grippe H1N1 en 2009 et la COVID-19 actuellement) soulignent l'importance du défi global que posent les maladies infectieuses virales émergentes et ré-émergentes à nos sociétés. Par exemple, au cours des deux dernières années seulement, le virus chikungunya (CHIKV) a provoqué des épidémies majeures dans plusieurs pays d'Asie du Sud-Est, auxquelles s'ajoute le fardeau croissant du syndrome respiratoire aigu sévère (SARS-CoV-2), avec des conséquences majeures dans le monde entier. Bien que les pandémies ne soient pas un phénomène nouveau, la popularité croissante des technologies de séquençage de nouvelle génération, l'accélération de la génération de données génomiques - qui élargissent les bases de données de génomes viraux - et les progrès des méthodes phylodynamiques ainsi que de l'informatique ont permis d'utiliser les génomes viraux pour répondre à des questions épidémiologiques cruciales, renforçant les réponses de la santé publique aux épidémies. Ce travail s'est concentré sur l'étude de l'origine, aussi bien spatiale que temporelle, et de la propagation des maladies infectieuses virales d'épidémies passées et en cours. Pour ce faire, nous avons utilisé l'épidémiologie génomique et montré qu'elle peut être un outil très puissant pour enquêter sur les épidémies de maladies infectieuses à différents niveaux. Par exemple, nous avons réussi à mettre en place un protocole de séquençage métagénomique profond qui nous a permis de découvrir que le probable agent étiologique responsable d'une série de cas de méningite dans le sud de l'Espagne, au cours de la période 2015-2018, était le virus Toscana (TOSV), un arbovirus responsable d'un nombre croissant d'infections dans les pays bordant la mer Méditerranée. Ensuite, nous avons développé une approche de séquençage basée sur des amplicons qui nous a permis d'obtenir la séquence complète du TOSV à partir d'échantillons de qualité et charge virale très variables. Nous sommes ensuite allés plus loin dans les analyses des épidémies de CHIKV au Cambodge. En effet, l'analyse phylogénétique des génomes que nous avons générés nous a permis d'étudier la diversité génétique du CHIKV et, en ajoutant des données temporelles, d'estimer le moment auquel il a été introduit dans la population. Dans un second temps, une analyse phylogéographique nous a fourni un niveau de détail supplémentaire en mettant en lumière les origines de l'épidémie, les liens avec les épidémies précédentes dans la même région et la dispersion du virus dans le pays. Enfin, à une autre échelle, nous avons pu suivre la dynamique de la population virale du SARS-CoV-2 lors de l'infection à long terme d'un patient immunodéprimé, ce qui a souligné les défis de la prise en charge de ces membres vulnérables de notre société pour lesquels il existe un risque accru de maladie grave. Pour conclure, ces travaux contribuent à une meilleure compréhension de l'épidémiologie et l'évolution de plusieurs virus à ARN qui représentent encore aujourd'hui des menaces importantes pour la santé humaine, et soulignent les apports de l'épidémiologie génomique contre les épidémies

Dimerization confers increased stability to nucleases in 5′ halves from glycine and glutamic acid tRNAs

International audienceWe have previously shown that 5′ halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5′ halves can form homodimers or heterodimers with tRNAGlu 5′ halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5′ halves inside cells. Considering recent reports have shown that 5′ tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments

Genomic surveillance of enterovirus associated with aseptic meningitis cases in southern Spain, 2015–2018

International audienceNew circulating Enterovirus (EV) strains often emerge through recombination. Upsurges of recombinant non-polio enteroviruses (NPEVs) associated with neurologic manifestations such as EVA71 or Echovirus 30 (E30) are a growing public health concern in Europe. Only a few complete genomes of EVs circulating in Spain are available in public databases, making it difficult to address the emergence of recombinant EVs, understand their evolutionary relatedness and the possible implication in human disease. We have used metagenomic (untargeted) NGS to generate full-length EV genomes from CSF samples of EV-positive aseptic meningitis cases in Southern Spain between 2015 and 2018. Our analyses reveal the co-circulation of multiple Enterovirus B (EV-B) types (E6, E11, E13 and E30), including a novel E13 recombinant form. We observed a genetic turnover where emergent lineages (C1 for E6 and I [tentatively proposed in this study] for E30) replaced previous lineages circulating in Spain, some concomitant with outbreaks in other parts of Europe. Metagenomic sequencing provides an effective approach for the analysis of EV genomes directly from PCR-positive CSF samples. The detection of a novel, disease-associated, recombinant form emphasizes the importance of genomic surveillance to monitor spread and evolution of EVs

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

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

Recent African strains of Zika virus display higher transmissibility and fetal pathogenicity than Asian strains

The global emergence of Zika virus (ZIKV) revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compare seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We find that recent African ZIKV strains display higher transmissibility in mosquitoes and higher lethality in both adult and fetal mice than their Asian counterparts. We emphasize the high epidemic potential of African ZIKV strains and suggest that they could more easily go unnoticed by public health surveillance systems than Asian strains due to their propensity to cause fetal loss rather than birth defects