Functional multi-organelle units control inflammatory lipid metabolism of macrophages.

Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multi-spectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (mitochondria-ER-LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (mitochondria-ER-peroxisome-LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid mediator synthesis. Together, we show that macrophages form functional multi-organellar units to support metabolic adaptation and provide an experimental strategy to identify organelle-metabolic signalling hubs.

Known and novel viruses in Belgian honey bees: yearly differences, spatial clustering, and associations with overwintering loss.

In recent years, managed honey bee colonies have been suffering from an increasing number of biotic and abiotic stressors, resulting in numerous losses of colonies worldwide. A pan-European study, EPILOBEE, estimated the colony loss in Belgium to be 32.4% in 2012 and 14.8% in 2013. In the current study, absolute viral loads of four known honey bee viruses (DWV-A, DWV-B, AmFV, and BMLV) and three novel putative honey bee viruses (Apis orthomyxovirus 1, apthili virus, and apparli virus) were determined in 300 Flemish honey bee samples, and associations with winter survival were determined. This revealed that, in addition to the known influence of DWV-A and DWV-B on colony health, one of the newly described viruses (apthili virus) shows a strong yearly difference and is also associated with winter survival. Furthermore, all scrutinized viruses revealed significant spatial clustering patterns, implying that despite the limited surface area of Flanders, local virus transmission is paramount. The vast majority of samples were positive for at least one of the seven investigated viruses, and up to 20% of samples were positive for at least one of the three novel viruses. One of those three, Apis orthomyxovirus 1, was shown to be a genuine honey bee-infecting virus, able to infect all developmental stages of the honey bee, as well as the Varroa destructor mite. These results shed light on the most prevalent viruses in Belgium and their roles in the winter survival of honey bee colonies. IMPORTANCE: The western honey bee (Apis mellifera) is a highly effective pollinator of flowering plants, including many crops, which gives honey bees an outstanding importance both ecologically and economically. Alarmingly high annual loss rates of managed honey bee colonies are a growing concern for beekeepers and scientists and have prompted a significant research effort toward bee health. Several detrimental factors have been identified, such as varroa mite infestation and disease from various bacterial and viral agents, but annual differences are often not elucidated. In this study, we utilize the viral metagenomic survey of the EPILOBEE project, a European research program for bee health, to elaborate on the most abundant bee viruses of Flanders. We complement the existing metagenomic data with absolute viral loads and their spatial and temporal distributions. Furthermore, we identify Apis orthomyxovirus 1 as a potentially emerging pathogen, as we find evidence for its active replication honey bees.

MSL2 ensures biallelic gene expression in mammals.

In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter-enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.

EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the blood system. Despite their multipotency, MPPs display poorly understood lineage bias. Here, we examine whether lineage-specifying transcription factors, such as the B-lineage determinant EBF1, regulate lineage preference in early progenitors. We detect low-level EBF1 expression in myeloid-biased MPP3 and lymphoid-biased MPP4 cells, coinciding with expression of the myeloid determinant C/EBPα. Hematopoietic deletion of Ebf1 results in enhanced myelopoiesis and reduced HSC repopulation capacity. Ebf1-deficient MPP3 and MPP4 cells exhibit an augmented myeloid differentiation potential and a transcriptome with an enriched C/EBPα signature. Correspondingly, EBF1 binds the Cebpa enhancer, and the deficiency and overexpression of Ebf1 in MPP3 and MPP4 cells lead to an up- and downregulation of Cebpa expression, respectively. In addition, EBF1 primes the chromatin of B-lymphoid enhancers specifically in MPP3 cells. Thus, our study implicates EBF1 in regulating myeloid/lymphoid fate bias in MPPs by constraining C/EBPα-driven myelopoiesis and priming the B-lymphoid fate.

The Virome of Healthy Honey Bee Colonies: Ubiquitous Occurrence of Known and New Viruses in Bee Populations.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales, of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales, which were strongly decreased in Czech bees compared to the Varroa- and DWV-naive Australian bees.

The virota and its transkingdom interactions in the healthy infant gut.

SignificanceMicrobes colonizing the infant gut during the first year(s) of life play an important role in immune system development. We show that after birth the (nearly) sterile gut is rapidly colonized by bacteria and their viruses (phages), which often show a strong cooccurrence. Most viruses infecting the infant do not cause clinical signs and their numbers strongly increase after day-care entrance. The infant diet is clearly reflected by identification of plant-infecting viruses, whereas fungi and parasites are not part of a stable gut microbiota. These temporal high-resolution baseline data about the gut colonization process will be valuable for further investigations of pathogenic viruses, dynamics between phages and their bacterial host, as well as studies investigating infants with a disturbed microbiota.

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970's Egypt.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Successional Stages in Infant Gut Microbiota Maturation.

Disturbances in the primary colonization of the infant gut can result in lifelong consequences and have been associated with a range of host conditions. Although early-life factors have been shown to affect infant gut microbiota development, our current understanding of human gut colonization in early life remains limited. To gain more insights into the unique dynamics of this rapidly evolving ecosystem, we investigated the microbiota over the first year of life in eight densely sampled infants (n = 303 total samples). To evaluate the gut microbiota maturation transition toward an adult configuration, we compared the microbiome composition of the infants to that of the Flemish Gut Flora Project (FGFP) population (n = 1,106). We observed the infant gut microbiota to mature through three distinct, conserved stages of ecosystem development. Across these successional gut microbiota maturation stages, the genus predominance was observed to shift from Escherichia over Bifidobacterium to Bacteroides. Both disease and antibiotic treatment were observed to be associated occasionally with gut microbiota maturation stage regression, a transient setback in microbiota maturation dynamics. Although the studied microbiota trajectories evolved to more adult-like constellations, microbiome community typing against the background of the FGFP cohort clustered all infant samples within the (in adults) potentially dysbiotic Bacteroides 2 (Bact2) enterotype. We confirmed the similarities between infant gut microbial colonization and adult dysbiosis. Profound knowledge about the primary gut colonization process in infants might provide crucial insights into how the secondary colonization of a dysbiotic adult gut can be redirected. IMPORTANCE After birth, microbial colonization of the infant intestinal tract is important for health later in life. However, this initial process is highly dynamic and influenced by many factors. Studying this process in detail requires a dense longitudinal sampling effort. In the current study, the bacterial microbiota of >300 stool samples was analyzed from 8 healthy infants, suggesting that the infant gut microbial population matures along a path involving distinct microbial constellations and that the timing of these transitions is infant specific and can temporarily retrace upon external events. We also showed that the infant microbial populations show similarities to suboptimal bacterial populations in the guts of adults. These insights are crucial for a better understanding of the dynamics and characteristics of a "healthy gut microbial population" in both infants and adults and might allow the identification of intervention targets in cases of microbial disturbances or disease.

A Previously Undescribed Highly Prevalent Phage Identified in a Danish Enteric Virome Catalog.

Gut viruses are important, yet often neglected, players in the complex human gut microbial ecosystem. Recently, the number of human gut virome studies has been increasing; however, we are still only scratching the surface of the immense viral diversity. In this study, 254 virus-enriched fecal metagenomes from 204 Danish subjects were used to generate the Danish Enteric Virome Catalog (DEVoC) containing 12,986 nonredundant viral scaffolds, of which the majority was previously undescribed, encoding 190,029 viral genes. The DEVoC was used to compare 91 healthy DEVoC gut viromes from children, adolescents, and adults that were used to create the DEVoC. Gut viromes of healthy Danish subjects were dominated by phages. While most phage genomes (PGs) only occurred in a single subject, indicating large virome individuality, 39 PGs were present in more than 10 healthy subjects. Among these 39 PGs, the prevalences of three PGs were associated with age. To further study the prevalence of these 39 prevalent PGs, 1,880 gut virome data sets of 27 studies from across the world were screened, revealing several age-, geography-, and disease-related prevalence patterns. Two PGs also showed a remarkably high prevalence worldwide-a crAss-like phage (20.6% prevalence), belonging to the tentative AlphacrAssvirinae subfamily, and a previously undescribed circular temperate phage infecting Bacteroides dorei (14.4% prevalence), called LoVEphage because it encodes lots of viral elements. Due to the LoVEphage's high prevalence and novelty, public data sets in which the LoVEphage was detected were de novo assembled, resulting in an additional 18 circular LoVEphage-like genomes (67.9 to 72.4 kb). IMPORTANCE Through generation of the DEVoC, we added numerous previously uncharacterized viral genomes and genes to the ever-increasing worldwide pool of human gut viromes. The DEVoC, the largest human gut virome catalog generated from consistently processed fecal samples, facilitated the analysis of the 91 healthy Danish gut viromes. Characterizing the biggest cohort of healthy gut viromes from children, adolescents, and adults to date confirmed the previously established high interindividual variation in human gut viromes and demonstrated that the effect of age on the gut virome composition was limited to the prevalence of specific phage (groups). The identification of a previously undescribed prevalent phage illustrates the usefulness of developing virome catalogs, and we foresee that the DEVoC will benefit future analysis of the roles of gut viruses in human health and disease.

Whole genome analysis of Aichivirus A, isolated from a child, suffering from gastroenteritis, in Pakistan.

Viruses are the primary cause of acute gastroenteritis in children all over the world. Understanding the emergence and genetic variation of these viruses may help to prevent infections. Aichivirus (AiV) is a member of the Kobuvirus genus, which currently contains six officially recognized species: Aichivirus A-F. The species AiV A contains six types including Aichivirus 1 (AiV 1) and eventually, three genotypes have been identified in the human AiV 1 (named A to C). The present study describes the identification and sequencing of the polyprotein gene of a human AiV 1 strain PAK419 via NGS in Pakistani children with acute gastroenteritis. Our study strain PAK419 was classified as AiV 1 genotype A, most commonly found in Japan and Europe, and closely related to non-Japanese and European strains on the phylogenetic tree. PAK419 showed 95-98 % nucleotide sequence identity with strains isolated from Ethiopia (ETH/2016/P4), Australia (FSS693) and China (Chshc7). On phylogenetic observation PAK419 formed a distinct cluster in the AiV 1 genotype A with the above mentioned and other human AiV strains detected around the world (Germany, Brazil, Japan, Thailand, Korea and Vietnam). The data clearly showed that Pakistani AiV strains and human strains identified from all over the world are distinct from Aichivirus strains found in bovine, swine, canine, feline, caprine, ferret, bat, and environmental samples. The distinguishing characteristics of the AiV genome showed a lower probability of inter-genotypic recombination events, which may support the lack of AiV serotypes. PAK419 also had a high content of C nucleotide (37.4 %), as found in previous studies, which could also restrict the possible genetic variation of AiV. This study demonstrate the power of NGS in uncovering unknown gastroenteric etiological agents circulating in the population.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

NCBI's Virus Discovery Codeathon: Building "FIVE" -The Federated Index of Viral Experiments API Index.

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.

Metagenomic Approach with the NetoVIR Enrichment Protocol Reveals Virus Diversity within Ethiopian Honey Bees (Apis mellifera simensis).

Metagenomics studies have accelerated the discovery of novel or divergent viruses of the honey bee. However, most of these studies predominantly focused on RNA viruses, and many suffer from the relatively low abundance of viral nucleic acids in the samples (i.e., compared to that of the host). Here, we explored the virome of the Ethiopian honey bee, Apis mellifera simensis, using an unbiased metagenomic approach in which the next-generation sequencing step was preceded by an enrichment protocol for viral particles. Our study revealed five well-known bee viruses and 25 atypical virus species, most of which have never been found in A. mellifera before. The viruses belong to Iflaviridae, Dicistroviridae, Secoviridae, Partitiviridae, Parvoviridae, Potyviridae, and taxonomically unclassified families. Fifteen of these atypical viruses were most likely plant-specific, and the remaining ten were presumed to be insect-specific. Apis mellifera filamentous virus (AmFV) was found in one sampling site out of 10. Two samples contained high read counts of a virus similar to Diatraea saccharales densovirus (DsDNV), which is a virus that causes high mortality in the sugarcane borer. AmFV and the DsDNV-like virus were the only DNA viruses found. Three viruses that primarily infect Drosophila spp. were also discovered: La Jolla virus (LJV), Kilifi virus (KiV), and Thika virus. Our study suggests that phoretic varroa mites are involved in the transmission of LJV and KiV and that both viruses replicate in mites and adult bees. We also found an overwhelming dominance of the deformed wing virus type B variant, which fits well with the apparently harmless infestation by Varroa destructor. It was suggested that Ethiopian bees have developed tolerance against virus infections as the result of natural selection.

Clinical relevance of plasma virome dynamics in liver transplant recipients.

BACKGROUND: The role of the microbiome in liver transplantation (LT) outcome has received a growing interest in the past decades. In contrast to bacteria, the role of endogenous viral communities, known as the virome, is poorly described. Here, we applied a viral metagenomic approach to study the dynamic evolution of circulating viruses in the plasma of LT recipients and its effect on the clinical course of patients. METHODS: Patients chronically infected with hepatitis B virus (HBV) that received a LT due to endstage liver disease were included in this study. Longitudinal plasma samples were collected pre- and post-LT. Intact viral particles were isolated and sequenced on an Illumina HiSeq 2500 platform. Short read libraries were analysed with an in-house bioinformatics pipeline. Key endpoints were the dynamics of viral families and post-LT complications. FINDINGS: The initiation of immunosuppression induced a bloom of the Anelloviridae that dominated the post-LT plasma virome. A variety of post-LT complication were observed. Nephrotoxicity was reported in 38% of the patients and was associated with a high abundance of anelloviruses. Besides nephrotoxicity, 16 (67%) patients experienced flares of viral or bacterial infections in post-transplant follow-up. These flares were recognized by an increased burden of anelloviruses (p < 0.05). Interestingly, no mortality was observed in patients infected with human pegivirus. INTERPRETATION: These findings suggest a diagnostic potential for the Anelloviridae family in post-LT complications. Furthermore, the impact of human pegivirus infection on post-transplant survival should be further investigated. FUNDING: This trial was supported by Gilead Sciences grant number BE-2017-000133.

Honey-bee-associated prokaryotic viral communities reveal wide viral diversity and a profound metabolic coding potential.

Honey bees (Apis mellifera) produce an enormous economic value through their pollination activities and play a central role in the biodiversity of entire ecosystems. Recent efforts have revealed the substantial influence that the gut microbiota exert on bee development, food digestion, and homeostasis in general. In this study, deep sequencing was used to characterize prokaryotic viral communities associated with honey bees, which was a blind spot in research up until now. The vast majority of the prokaryotic viral populations are novel at the genus level, and most of the encoded proteins comprise unknown functions. Nevertheless, genomes of bacteriophages were predicted to infect nearly every major bee-gut bacterium, and functional annotation and auxiliary metabolic gene discovery imply the potential to influence microbial metabolism. Furthermore, undiscovered genes involved in the synthesis of secondary metabolic biosynthetic gene clusters reflect a wealth of previously untapped enzymatic resources hidden in the bee bacteriophage community.

NCBI's Virus Discovery Hackathon: Engaging Research Communities to Identify Cloud Infrastructure Requirements.

A wealth of viral data sits untapped in publicly available metagenomic data sets when it might be extracted to create a usable index for the virological research community. We hypothesized that work of this complexity and scale could be done in a hackathon setting. Ten teams comprised of over 40 participants from six countries, assembled to create a crowd-sourced set of analysis and processing pipelines for a complex biological data set in a three-day event on the San Diego State University campus starting 9 January 2019. Prior to the hackathon, 141,676 metagenomic data sets from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) were pre-assembled into contiguous assemblies (contigs) by NCBI staff. During the hackathon, a subset consisting of 2953 SRA data sets (approximately 55 million contigs) was selected, which were further filtered for a minimal length of 1 kb. This resulted in 4.2 million (Mio) contigs, which were aligned using BLAST against all known virus genomes, phylogenetically clustered and assigned metadata. Out of the 4.2 Mio contigs, 360,000 contigs were labeled with domains and an additional subset containing 4400 contigs was screened for virus or virus-like genes. The work yielded valuable insights into both SRA data and the cloud infrastructure required to support such efforts, revealing analysis bottlenecks and possible workarounds thereof. Mainly: (i) Conservative assemblies of SRA data improves initial analysis steps; (ii) existing bioinformatic software with weak multithreading/multicore support can be elevated by wrapper scripts to use all cores within a computing node; (iii) redesigning existing bioinformatic algorithms for a cloud infrastructure to facilitate its use for a wider audience; and (iv) a cloud infrastructure allows a diverse group of researchers to collaborate effectively. The scientific findings will be extended during a follow-up event. Here, we present the applied workflows, initial results, and lessons learned from the hackathon.

Stable distinct core eukaryotic viromes in different mosquito species from Guadeloupe, using single mosquito viral metagenomics.

BACKGROUND: Mosquitoes are the most important invertebrate viral vectors in humans and harbor a high diversity of understudied viruses, which has been shown in many mosquito virome studies in recent years. These studies generally performed metagenomics sequencing on pools of mosquitoes, without assessment of the viral diversity in individual mosquitoes. To address this issue, we applied our optimized viral metagenomics protocol (NetoVIR) to compare the virome of single and pooled Aedes aegypti and Culex quinquefasciatus mosquitoes collected from different locations in Guadeloupe, in 2016 and 2017. RESULTS: The total read number and viral reads proportion of samples containing a single mosquito have no significant difference compared with those of pools containing five mosquitoes, which proved the feasibility of using single mosquito for viral metagenomics. A comparative analysis of the virome revealed a higher abundance and more diverse eukaryotic virome in Aedes aegypti, whereas Culex quinquefasciatus harbors a richer and more diverse phageome. The majority of the identified eukaryotic viruses were mosquito-species specific. We further characterized the genomes of 11 novel eukaryotic viruses. Furthermore, qRT-PCR analyses of the six most abundant eukaryotic viruses indicated that the majority of individual mosquitoes were infected by several of the selected viruses with viral genome copies per mosquito ranging from 267 to 1.01 × 108 (median 7.5 × 106) for Ae. aegypti and 192 to 8.69 × 106 (median 4.87 × 104) for Cx. quinquefasciatus. Additionally, in Cx. quinquefasciatus, a number of phage contigs co-occurred with several marker genes of Wolbachia sp. strain wPip. CONCLUSIONS: We firstly demonstrate the feasibility to use single mosquito for viral metagenomics, which can provide much more precise virome profiles of mosquito populations. Interspecific comparisons show striking differences in abundance and diversity between the viromes of Ae. aegypti and Cx. quinquefasciatus. Those two mosquito species seem to have their own relatively stable "core eukaryotic virome", which might have important implications for the competence to transmit important medically relevant arboviruses. The presence of Wolbachia in Cx. quinquefasciatus might explain (1) the lower overall viral load compared to Ae. aegypti, (2) the identification of multiple unknown phage contigs, and (3) the difference in competence for important human pathogens. How these viruses, phages, and bacteria influence the physiology and vector competence of mosquito hosts warrants further research.

Signature of natural resistance in NS3 protease revealed by deep sequencing of HCV strains circulating in Iran.

A tremendous upscale of screening and treatment strategies is required to achieve elimination of the hepatitis C virus (HCV) in Iran by 2030. Among treated patients, at least 5-10% is expected to experience treatment failure. To efficiently retreat cases with prior exposure to NS5A and NS5B drugs, knowledge on the natural prevalence of NS3 resistance is key. The NS3 region of 32 samples from sixteen Iranian HCV patients, among which 6 injecting drug users, was amplified and subjected to deep sequencing. Amplification and sequencing were successful in 29 samples. The reads were assembled to consensus sequences and showed that 6 patients were infected with HCV1a (37.5%), 7 with HCV1b (43.8%) and 3 with HCV3a (18.7%). Nucleotide identities were shared for >97% between intra-host sequences. Two patients were infected with natural resistant viruses, of which one solely comprising low frequency variants. Inferred phylogenies showed that Iranian sequences clustered together for HCV1a and HCV1b, while for HCV3a a potential recombination event was detected. We firstly report the use of deep sequencing for HCV in Iran, demonstrate the use of NS3 inhibitors as salvage therapy in case of retreatment and stress the importance for Iran to prioritize drug users for screening and treatment.

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.