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

BackgroundVarious 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.AimIn this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail.MethodsCerebrospinal 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.ResultsAmong 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.ConclusionsOur 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.

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.

Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner.

Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.

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

We 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.

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

Following 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.

Evidence of increasing diversification of Zika virus strains isolated in the American continent.

Zika virus (ZIKV) is a member of the family Flaviviridae. ZIKV emerged in Brazil in 2015, causing an unprecedented epidemic and since then the virus has rapidly spread throughout the Americas. These facts highlight the need of detailed phylogenetic studies to understand the emergence, spread, and evolution of ZIKV populations. For these reasons, a Bayesian coalescent Markov Chain Monte Carlo analysis of complete genome sequences of ZIKV strains recently isolated in the American continent was performed. The results of these studies revealed an increasing diversification of ZIKV strains in different genetic lineages and co-circulation of distinct genetic lineages in several countries in the region. The time of the most recent common ancestor (tMRCA) was established to be around February 20, 2014 for ZIKV strains circulating in the American region. A mean rate of evolution of 1.55 × 10-3 substitutions/site/year was obtained for ZIKV strains included in this study. A Bayesian skyline plot indicate a sharp increase in population size from February 2014 to July 2015 and a decline during 2016. These results are discussed in terms of the emergence and evolution of ZIKV populations in the American continent.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space.

Emergence and dissemination of SARS-CoV-2 XBB.1.5 in New York.

The recombinant SARS-CoV-2 Omicron XBB.1.5 variant was first detected in New York City (NYC) and rapidly became the predominant variant in the area by early 2023. The increased occurrence of circulating variants within the SARS-CoV-2 XBB-sublineage prompted the modification of COVID-19 mRNA vaccines by Moderna and Pfizer-BioNTech. This update, implemented in mid-September 2023, involved the incorporation of a monovalent XBB.1.5 component. Considering that NYC probably played a central role in the emergence of the XBB.1.5 variant, we conducted phylogeographic analysis to investigate the emergence and spread of this variant in the metropolitan area. Our analysis confirms that XBB.1.5 emerged within or near the NYC area and indicates that XBB.1.5 had a diffusion velocity similar to that of the variant Alpha in the same study area. Additionally, the analysis of 2,392 genomes collected in the context of the genomic surveillance program at NYU Langone Health system showed that there was no increased proportion of XBB.1.5, relative to all cocirculating variants, in the boosted compared to unvaccinated individuals. This study provides a comprehensive description of the emergence and dissemination of XBB.1.5.

Mixed Infections Unravel Novel HCV Inter-Genotypic Recombinant Forms within the Conserved IRES Region.

Hepatitis C virus (HCV) remains a significant global health challenge, affecting millions of people worldwide, with chronic infection a persistent threat. Despite the advent of direct-acting antivirals (DAAs), challenges in diagnosis and treatment remain, compounded by the lack of an effective vaccine. The HCV genome, characterized by high genetic variability, consists of eight distinct genotypes and over ninety subtypes, underscoring the complex dynamics of the virus within infected individuals. This study delves into the intriguing realm of HCV genetic diversity, specifically exploring the phenomenon of mixed infections and the subsequent detection of recombinant forms within the conserved internal ribosome entry site (IRES) region. Previous studies have identified recombination as a rare event in HCV. However, our findings challenge this notion by providing the first evidence of 1a/3a (and vice versa) inter-genotypic recombination within the conserved IRES region. Utilizing advanced sequencing methods, such as deep sequencing and molecular cloning, our study reveals mixed infections involving genotypes 1a and 3a. This comprehensive approach not only confirmed the presence of mixed infections, but also identified the existence of recombinant forms not previously seen in the IRES region. The recombinant sequences, although present as low-frequency variants, open new avenues for understanding HCV evolution and adaptation.

spread.gl: visualising pathogen dispersal in a high-performance browser application.

Phylogeographic analyses are able to exploit the location data associated with sampled molecular sequences to reconstruct the spatio-temporal dispersal history of a pathogen. Visualisation software is commonly used to facilitate the interpretation of the accompanying estimation results, as these are not always easily interpretable. spread.gl is a powerful, open-source and feature-rich browser application that enables smooth, intuitive and user-friendly visualisation of both discrete and continuous phylogeographic inference results, enabling the animation of pathogen geographic dispersal through time. spread.gl can render and combine the visualisation of several data layers, including a geographic layer (e.g., a world map), multiple layers that contain information extracted from the input phylogeny, and different types of layers that represent environmental data. As such, users can explore which environmental data may have shaped pathogen dispersal patterns, that can subsequently be formally tested through more principled statistical analyses. We showcase the visualisation features of spread.gl on several representative pathogen dispersal examples, including the smooth animation of a phylogeny encompassing over 17,000 genomic sequences resulting from a large-scale SARS-CoV-2 analysis.

RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities.

Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed that nonvesicular exRNA profiles are highly biased toward those RNAs with intrinsic resistance to extracellular ribonucleases. These highly resistant exRNAs are interesting from a biomarker point of view, but are not representative of the actual bulk of RNAs released to the extracellular space. In order to understand exRNA dynamics and capture both stable and unstable RNAs, we developed a method based on size-exclusion chromatography (SEC) fractionation of RNase inhibitor (RI)-treated cell-conditioned medium (RI-SEC-seq). This method has allowed us to identify and study extracellular ribosomes and tRNAs, and offers a dynamical view of the extracellular RNAome which can impact biomarker discovery in the near future. Graphical abstract: Overview of the RI-SEC-seq protocol: sequencing of size-exclusion chromatography fractions from nonvesicular extracellular samples treated or not with RNase inhibitors (+/- RI).

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

New 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.

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

Knowledge 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.

Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries.

Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130-150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients' management and outcome prediction in developing countries.