key: cord-0007501-xyk6eu60
authors: Sauvage, V.; Eloit, M.
title: Viral metagenomics and blood safety
date: 2016-01-09
journal: Transfus Clin Biol
DOI: 10.1016/j.tracli.2015.12.002
sha: a4ff8569daa52602d8577186ac240223548e4413
doc_id: 7501
cord_uid: xyk6eu60

The characterization of the human blood-associated viral community (also called blood virome) is essential for epidemiological surveillance and to anticipate new potential threats for blood transfusion safety. Currently, the risk of blood-borne agent transmission of well-known viruses (HBV, HCV, HIV and HTLV) can be considered as under control in high-resource countries. However, other viruses unknown or unsuspected may be transmitted to recipients by blood-derived products. This is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. Several measures to prevent transfusion transmission of unknown viruses have been implemented including the exclusion of at-risk donors, leukocyte reduction of donor blood, and physicochemical treatment of the different blood components. However, up to now there is no universal method for pathogen inactivation, which would be applicable for all types of blood components and, equally effective for all viral families. In addition, among available inactivation procedures of viral genomes, some of them are recognized to be less effective on non-enveloped viruses, and inadequate to inactivate higher viral titers in plasma pools or derivatives. Given this, there is the need to implement new methodologies for the discovery of unknown viruses that may affect blood transfusion. Viral metagenomics combined with High Throughput Sequencing appears as a promising approach for the identification and global surveillance of new and/or unexpected viruses that could impair blood transfusion safety.

In developed countries, blood and organ donors are routinely screened for a range of blood-borne viruses (HIV, HBV, HCV and HTLV in some countries) with highly sensitive screening tests. This has dramatically improved the safety of blood supply. However, transmission by transfusion of unknown or unsuspected viruses, responsible for persistent and asymptomatic viremia, remains a continuing threat. This is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. Several measures to prevent transfusion transmission of unknown viruses have been implemented including the exclusion of at-risk donors, leukocyte reduction of donor blood, and physicochemical treatments of the different blood components aimed to inactivate infectious agents potentially present in blood. However, up to now there is no universal method for pathogen inactivation, which would be applicable for all types of blood components (currently there is no method applicable for red blood cell concentrates) and, equally effective for all viral families [1] . Several inactivation procedures are available but some of them are recognized to be less effective on non-enveloped viruses, such as human parvovirus B19 (B19 V), Hepatitis A (HAV) or Hepatitis E (HEV) viruses [2] . Moreover, recent publications have also confirmed that some viral reduction methods (solvent/detergent and heat treatment) are inadequate to inactivate high viremic titers of these viruses in plasma pools or derivates [3, 4] . Given this, the threat for blood safety due to emerging viruses is of crucial importance. Thus, it remains imperative to improve the blood product safety, not only by surveillance of well-characterized viral pathogens (HBV, HCV, HIV, B19 V) but also, by implementing new methodologies for discovery of unknown viruses that may affect blood transfusion safety.

The recorded emergence of infectious diseases has risen significantly since the last three decades. In a period of 64 years ) the emergence of 335 infectious diseases (all pathogens combined) was reported. Emerging infectious diseases (EIDs) are dominated by zoonoses, the majority of which (72%) has wildlife origin. The increasing and ongoing documentation of novel animal viruses identified worldwide predicts that additional animal pathogens may jump species barrier to potentially become novel human infectious diseases in the near future, some perhaps highly pathogenic. Viruses account for 25-44% of all EIDs [5] and are the pathogen class most likely to emerge [6] with an estimated number of undiscovered mammalian viruses of at least 320,000 [7] . This estimation suggests that zoonotic EIDs represent a serious threat to human health and emphasizes the importance to evaluate the factors that are involved in the contact between human and animals, responsible for the emergence of "zoonotic-human" infections [5] . [8] , as the increasing contact between humans and wildlife habitats mainly due to agricultural practices and the globalization of travel and trade. Other important factors include climate changes that promote vector expansion (mosquitoes, ticks) and mass human migrations (due to wars, natural disasters, poverty, and desertification) [9] . For these reasons, there is now a clear increased risk of arbovirus emergence in the future due to environmental and climate changes, extensive tropical urbanization and to the colonization of this expanding habitat by highly anthropophilic mosquitos (i.e. Chikungunya and dengue viruses both transmitted by Aedes aegypti). To date, dengue viruses are internationally recognized as an existing threat to blood safety due to a known transfusion-transmission and severe/fatal disease in recipients [10] . As all arboviruses are potentially transmissible by transfusion due to their capacity to induce an asymptomatic viremic phase, this represents new risks for blood safety.

In the last years, very broad and sensitive high throughput techniques based on metagenomics were developed to detect and characterize previously unknown or variant viruses associated with several human diseases. These methods based on the random amplification of genomes include pan-viral microarrays and High Throughput Sequencing (HTS) [11] . By overcoming conventional methods of viral identification, metagenomics, which gives access to all nucleic acids present in a given sample, allows the description of viral communities and their diversity in environmental [12, 13] , human [14] [15] [16] and animal samples [17] [18] [19] using HTS. Metagenomic sequencing has also demonstrated its usefulness in investigations when infectious diseases have unknown etiology, as reported in human Merkel cell carcinoma for which a polyomavirus was identified as responsible [20] , for the thrombocytopenia and leukopenia syndrome in China associated with a new bunyavirus [21] and for acute hemorrhagic fever outbreaks in Uganda [22] and Central Africa [23] due to new Ebola virus and rhabdovirus, respectively. This approach has also been used to identify novel viruses responsible of severe or fatal post-transplant associated diseases in grafted recipients [24, 25] . Besides the development of metagenomics as a diagnostic tool in clinical virology [26, 27] , this methodology was applied to viral safety for biotechnological and biological products [28, 29] . Using metagenomics, it is then possible to provide manufacturers the exhaustive viral bioburden of raw materials [30] [31] [32] [33] [34] and to detect adventitious viruses in live-attenuated vaccines [35] .

With more than 3 million blood components transfused annually in France alone, viral metagenomics appears as a promising approach for the identification and global surveillance of new and/or (re)-emerging viruses that could impair blood transfusion safety.

Metagenomic sequencing offers the considerable advantage compared to traditional methods of pathogen identification of being a molecular approach able to detect simultaneously multiple organisms in a complex sample without culturing and a priori knowledge of the microrganism's nucleic acid sequences. However, the application of viral metagenomics to clinical samples is made difficult by the fact that viral sequences represent a very low proportion compared to host DNA sequences and transcripts, bacterial and other contaminating genetic material (e.g. from environment and reagent). This leads to the requirement of high depth of sequencing and intensive bioinformatics analyses to increase the probability of virus detection. Despite the fact that amplification method, library construction and sequencing platform impact metagenome composition [36] [37] [38] [39] [40] , adequate sample preparation enhancing the ratio of viral to host nucleic acid loads remains a crucial step for biological samples. The pipeline presented below comprises key steps and recommendations for viral metagenomic analyses, especially as applied to plasma or serum sample (Fig. 1 ).

In order to increase the proportion of viruses, different purification and enrichment strategies can be applied on plasma samples prior sequencing, such as clearing centrifugation at low speed, membrane filtration (0.45 m and/or 0.2 m filter), ultracentrifugation, polyethylene glycol (PEG) virus precipitation, digestion of host cell-free DNA/RNA by nuclease(s) treatment and the removal of ribosomal RNA. These different techniques may be combined to maximize the amount of viral nucleic acids after extraction [39, [41] [42] [43] . All viral metagenomic studies agree on the fact that nuclease(s) pretreatment of biological sample is an essential step to importantly reduce the amount of background nucleic acids.

Different manual and automated methods to extract viral nucleic acids can be used ( Fig. 1 ) and require choosing between extracting DNA and RNA simultaneously or independently. Since (re)emerging viral threats are mainly caused by RNA viruses [5, 44] , viral nucleic acid isolation can be performed using TRIzol LS (Life Technologies) or reagents designed to obtain high quality RNA molecules in high yields. Following viral particles lysis and nucleic acid precipitation steps, viral genomic RNAs are purified using a spin column. Residual host and/or viral DNA are then removed by an additional DNAse digestion is performed directly on the silica membrane or the final eluate. In some protocol, unwanted ribosomal RNAs are also removed after RNA purification [43] , but removing ribosomal RNA at this stage by capture probes hybridization may cause viral sequences loss, especially those from unknown viruses. In addition, TRIzol extraction method offers the possibility to recover DNA virus genomes from the organic phase.

Once nucleic acids are extracted, RNAs are converted into single-stranded complementary DNA (ss cDNA) and randomly amplified together with DNA (if DNA and RNA were extracted simultaneously) to obtain the concentration required for library construction (1 g to 1 ng depending on library preparation kits and sequencing platform). Number of sequenceindependent amplification methods have been developed and used in combination with HTS for virus discovery. The most common are the sequence-independent single-primer amplification (SISPA) [45, 46] , Multiple Displacement Amplification (MDA) [36, 47] and cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) [48] . These three methodologies can amplify simultaneously cDNA and DNA, linear and circular, single-and double-stranded viral genomes in a same sample. The SISPA method is extensively used by a number of laboratories in the world and allowed the identification of many new viruses in animal [49] [50] [51] and human clinical samples in the last ten years [23, [52] [53] [54] . Compared to SISPA, MDA uses bacteriophage Phi29 polymerase. The MDA process presents the disadvantage to preferentially amplify circular ss DNA than linear viral genomes and thereby introduced a bias of amplification. However, this limitation does not prevent the detection of known RNA viruses [29, 36, 55] even at low concentrations (around 500-1000 copies/mL), and the discovery of new ones in biological samples [56] . Recently, MDA was successfully used in combination with HTS to discover a novel human astrovirus associated with a case of progressive encephalitis in a boy with X-linked agammaglobulinemia [57] . The cDNA-AFLP method was developed by Bachem et al. [58] and adapted for virus discovery (VIDISCA method) by van der Hoek et al. [59] for successful identification of a novel human coronavirus, HCoV-NL63, in a child suffering from bronchiolitis and conjunctivitis. Later, VIDISCA was used to discover novel parvoviruses [60, 61] and papillomavirus [62] in veterinary and clinical human samples, respectively.

Despite the efficiency of nuclease(s) pretreatment to decrease the host background, a significant proportion of reads after deep sequencing always correspond to host genome and transcripts. To increase the sensitivity of virus detection required for clinical applications, several methods were developed to selectively hybridize and amplify non-human sequences. Of them, the "virome capture sequencing platform for vertebrate viruses" (VirCapSeq-VERT) [63] , which comprises around 2 million probes covering all viral taxa known to infect vertebrates, the "Preferential Amplification of Pathogenic Sequences" (PATHseq) based on oligonucleotides that do not match the most abundant human transcripts [64] and the modified VIDISCA method using random hexamers that do not anneal to rRNAs [65, 66] . Today, VirCapSeq-VERT in combination with Illumina HiSeq appears to be the most sensitive method as it enabled detection of HHV-1 and West Nile Virus genomes, and enterovirus D68 at concentrations corresponding to 100 and 10 copies/mL, respectively, in human blood and serum samples [63] . Furthermore, according to the authors, the method can detect a virus whose the genome is 40% different to the probe sequence, making this method a suitable tool for viral discovery. However, this method is extremely time-consuming (hybridization step > 12 h) and, therefore, non-appropriate for clinical diagnostic applications.

Finally, particular precautions to avoid contamination of samples, nucleic acid extracts and amplification products by exogenous nucleic acids must be implemented throughout the process including ultra-violet light treatment of some kit reagents, substitution of commercial carrier RNA kits by neutral chemical carriers such as linear polyacrylamide (LPA) and good laboratory practices compliance.

Following reverse transcription and amplification steps, the resulting ds DNAs are sheared into defined DNA fragment size according to the sequencing platform by using enzymatic (e.g. Illumina Nextera ® technology) or mechanical techniques (e.g. ultrasonic fragmentation with the Bioruptor [Diagenode] or the Covaris E210 [Covaris] systems). The extremities of fragmented DNA are end-repaired (mechanical shearing only) and ligated to specific sequencing adapters, which can be barcoded to allow multiplexing of several samples in a same sequencing run. Adapter-ligated DNAs are then size purified (e.g. Agencourt AMPure XP beads from Beckman Coulter) to select the properly ligated DNA. A reconditioning step corresponding to few cycles of amplification (5-16 cycles according to the sequencing chemistry) can be performed. This adaptor-mediated amplification step is used to increase product yield and enrich for DNA fragments with the appropriate adapters combination for optimal sequencing (Fig. 1) . The library is then quantified by a fluorimetric-based method (e.g. Qubit ® , Life Technologies or quantitative real-time PCR) and quality controlled with 2100 Bioanalyzer ® (Agilent Technologies) to confirm the library size.

It should be noted that the choice of the library preparation method depends in part on the input DNA amount. For example, Illumina TruSeq ® DNA PCR-Free library preparation kit (based on mechanical fragmentation) requires 1-2 g of input DNA, while Illumina Nextera ® technology uses 1-50 ng of DNA. For Ion Torrent Technology, the library preparation kit based on enzymatic shearing (Ion Xpress ® from Life Technologies) requires a minimum of 50-100 ng. With the recent development of library preparation kits (e.g. Illumina Nextera XT ® DNA) from very low input DNA (≤ 1 ng), non-amplified ds DNA can be directly fragmented, associated to adapters and PCR amplified to obtain the required DNA concentration for sequencing. Removing the non-specific preamplification step significantly reduces duplicated sequences, and thus increases the genome coverage and the number of viruses detected [67] . Likewise, the linker-amplified (LA) protocol in combination with optimal sample preparation for viruses enabled detection of viral sequences from 1-10 pg of input DNA [37, 68] , even if is not recommended by the manufacturers.

The resulting libraries can be sequenced following the manufacturer's protocol for each of the sequencing platforms.

Novel sequencing technologies that are currently available comprise second-and third-single molecule generation of sequencers. Second-generation sequencers are currently the most widely used and include the Genome Analyzer (GA)/HiSeq/NextSeq/MiSeq from Illumina, GS FLX System/GS Junior from Roche (whose marketing will be stopped mid-2016) and the Ion Personal Genome Machine (PGM)/Ion Proton from Ion Torrent (Life Technologies) ( Table 1 ). Note that Ion Torrent recently launched (September 2015) a newest sequencer, the Ion S5 instrument (Table 1) , which offers in a unique device, chips with output (number of reads) and read lengths comparable to those of the Ion PGM and the Ion Proton (e.g. the 520 and 540 chips on S5 system are similar to the 318 chip on the PGM and the PI chip on the Proton, respectively). While the second-generation platforms present distinct sequencing chemistries and detection technologies [69] , the sequencing workflow is based for all on libraries amplification to produce clonal clusters and sequencing using massive parallel synthesis (Fig. 1) . On the other hand, given the number of second-generation sequencers, several factors must be considered in the choice of the technology, including cost performance, run time, output, flexibility, convenience and type of application. For example, the desktop sequencers, such as the Illumina MiSeq and the Ion Torrent PGM (both launched in 2011), are positioned for rapid targeted resequencing [70, 71] , and whole viral [72, 73] and bacterial genome sequencing [74, 75] . While they are both also used for metagenomic analysis [38, [76] [77] [78] , they offer a lower depth of sequencing (25 million reads per run for the MiSeq and 5-6 million reads for the Ion PGM) compared to the Illumina HiSeq (3 billion pair-end reads) and the Ion Proton (60-80 million reads with Ion PI chip), which therefore are more appropriate for whole metagenomics. Nowadays, the number of scientific publications using HiSeq instrument is considerably higher than those using Ion Proton, which is mainly attributable to its recent availability (September 2012) and lower capacity in terms of samples multiplexing (Ion PII chip announced for 2016 (but needs confirmation), will increase Ion Proton throughput to 300 million reads per run). However, the Ion Proton is a much more affordable, faster and compact system, theoretically capable of generating long reads (400b announced in 2016 but needs confirmation) compared to the HiSeq instrument. Recently, this platform was successfully used in viral metagenomics to identify two novel papillomaviruses in the vaginal microbiome of HIV positive women [79] and to detect different known viruses in plasma samples spiked at low viral concentrations [29] .

To date, the use of the third generation of sequencers in viral metagenomics remains anecdotal [80, 81] . Third-generation sequencers include the Single-Molecule Real-Time technology (SMRT) from Pacific Biosciences with the PacBio RS II Table 1 Features of the most commonly used next-generation sequencing platforms in research and clinical diagnostic laboratories. system and the Nanopore sequencing technology from Oxford Nanopore Technologies with the MinION and PromethION devices (currently in prerelease testing through the MinION and PromethION Access Programmes). Compared to the previous generation, the third generation is characterized by the absence of template amplification prior sequencing (and for this reason it's also called single-molecule sequencing), a signal registered in real-time and the ability to read long DNA fragments (several kilobases). Although obtaining long reads is revolutionary, these platforms still suffer from lower accuracy and the need of high input amounts of DNA (at least for Min-ION device according to our experience). However, the newest SMRT sequencer, the Sequel System (announced in Septembre 2015 by Pacifc Biosciences), may provide higher accuracy and throughput compared to PacBioRS II. The continuous performance improvements made by these two companies should secure an important place for these technologies in the field of viral metagenomics in the near future.

There are a lot of different pipelines that can be used for viral metagenomics. Basically, reads are first trimmed for quality and often, at least for the Illumina technology, duplicates (i.e. identical reads) are suppressed. In a second step, reads are filtered against the host human genome with a high stringency, meaning that in principle only human sequences are removed. There are then two possibilities.

There is often a step of assembly of single reads into contigs. Use of contigs allows for identifying hits with distant identities that would not have been distinguished from background on short single reads. As reads from new generation of sequencers become longer and longer, the need for assembly should decrease. Assembly of metagenomic reads into contigs poses specific difficulties due to the uneven abundance and thus coverage of viral species; moreover, random amplification also leads to uneven coverage of each genomes, the combination of both heterogeneity increasing the difficulty for assembly (see discussions on the tools in [82, 83] ). The following step is to conduct a blast analysis against a comprehensive database like the NCBI nr database and to identify sequences with a virus as best hit (a less ressource demanding protocol is to blast first a viral database like NCBI vrl and then confirm viral best hits by checking them against the NCBI nr). Blast analysis can be done with singletons and also with reads assembled into contigs. For contigs non assigned at the nucleotide level, a protein Blast (Blastx) can be used. Results need to be carefully screened by a virologist with some practical experience in metagenomic analysis, as there are frequent false positive results when the blast is conducted with a high e-value. Coverage of the putative viral genome is a good criterion to assess the pertinence of identification of new viral sequences.

A second possibility is to map the reads against a database of viruses using stringent rules and thus identify known viruses (already present in databases) and close relatives. This type of pipeline is quicker as less resource-demanding and is particularly well suited for diagnostic or as a first comprehensive filter before more demanding Blast analysis (see, for example, the computational pipeline SURPI [84] ).

Viral metagenomics associated with HTS has successfully demonstrated its capacity to detect and discover viruses in complex biological samples such as serum and plasma, and therefore appears as a promising approach for the identification and surveillance of unknown or unexpected viruses that may be transmitted to recipients by blood products.

To further assess the performance and the limits of this methodology, we recently conducted a metagenomic analysis on plasma-pooled samples collected from at risk subjects, including recipients of multiple blood transfusions and HBV-, HCV-and HIV-infected blood donors. A large amount of various viral sequences has been found in all pools, among which, anelloviruses and human pegivirus (HPgV), formerly known as GB virus C, were the most frequently identified agents. The presence of HBV DNA and HCV RNA was confirmed in plasma pools from blood donors previously found HBV or HCV infected. In these plasma pools, HBV DNA and HCV RNA accounted for 3.8 × 10 3 IU/mL and 1.7 × 10 5 IU/mL, respectively. In contrast, HIV RNA accounting for 150 copies/mL in the pool, was not detected (data not published). We also detected numerous short nucleotide sequences distantly related to viral sequences reported in the current databases, and for which it was not possible to know if they represent bona fide viruses or they derive from other organisms. Despite the inclusion of at risk individuals and the efficacy of the pipeline to discover new viruses [36, 56, 85, 86] , no sequence suggestive of a new viral species was identified in the plasma samples analyzed in the study. Hence, these findings confirmed that viral metagenomics combined to HTS is able to identify the viral diversity present in blood samples, but also highlighted the difficulty to detect low levels of virus (lower than 500 equivalent genome/mL at the time of the study) and underlined some other issues including uncertainty of taxonomic assignment of short sequences and reagent contaminants. For instance, we retrieved sequences from the Parvovirus-like hybrid genome (PHV/NIH-CQV) in all pools, which although initially described as a possible new causative agent of seronegative hepatitis [87] , was actually found as a background contaminant in water eluted from some nucleic acid extraction silica-binding columns [88, 89] . Other viral contaminants from spin columns including circoviruses/densoviruses and iridoviruses were also previously reported [15] . All together, these findings demonstrate that HTS results must be interpreted with caution before discovery of a new virus is claimed.

Interestingly, Laupouicheung et al., [90] reported during the XXVIIth National Congress of the French Society of Blood Transfusion, the detection of Human astrovirus-MLB2 sequences in transfusion plasma. Human astroviruses are known as important agents of viral gastroenteritis, especially in young children, elderly and immunocompromised individuals [91] . The Human astrovirus-MLB2 was initially discovered in diarrheal stools of children from India and the United States [92] , and it has since been detected in the plasma of a febrile child [93] , suggesting that astrovirus-MLB2 may spread from gastrointestinal tract to other tissues via the bloodstream. Several recent studies have reported the potential association of a new clade of astroviruses with neurological diseases and especially with encephalitis cases in both animals [94] and immunocompromised individuals [95, 96] . In humans, the latest reported case concerned a boy with X-linked agammaglobulinemia associated with progressive global motor decline, cerebellar syndrome, and epilepsy, who was found to be infected with a new astrovirus strain from the VA1/HMO clade [57] . Reports of astrovirus-associated encephalitis were also described in allogeneic hematopoietic stem cells [97] and bone marrow transplant recipients [98] . These examples clearly demonstrate the usefulness of viral metagenomic sequencing for the diagnosis of unexpected viral infections.

Other examples of viruses recently described in human blood samples by metagenomics and HTS can be cited such as the human hepegivirus 1 (HHpgV-1), discovered in post-transfusion serum samples from blood recipients [99] ; a divergent Gemycirculavirus (GemyC1c), found in an HIV1-positive blood donation [100] ; and Giant Blood Marseillevirus (GMB), discovered initially in a pool of samples collected from asymptomatic blood donors from the South of France [101] and subsequently found by PCR in 4% of 174 studied healthy donors and in 9.1% of post-transfusion sera from thalassemia patients [102] . However, in two recent studies reported (339 [103] and 187 [104] subjects, respectively), GMB viral DNA was not detected by PCR in any of the human plasma samples analyzed. Further investigations are needed to confirm the reality of these novel viral infections in humans, their potential pathogenicity and transfusion transmissibility.

Despite the undeniable analytical strength of this methodology, further investigations of the blood virome in a research context are needed to enforce the potential use of viral metagenomics and HTS for the discovery and surveillance of viruses of concern in blood safety.

As it is unlikely that the first occurrence of an emerging infection will be seen in transfused recipients, it is of paramount importance to implement a proactive surveillance of emerging viral infections in blood donor populations from different parts of the world especially from geographic areas with the highest rates of EIDs. After the identification of a new agent, if any, a process for evaluating its transmission routes by blood transfusion and its potential pathogenicity according to the immune status of the recipients will be implemented. Moreover, the risk assessment will help to define the need for implementation of new preventive strategies to reduce the transfusion transmission risk associated to this emerging agent. Such project will generate several recommendations to help health authorities to issue guidelines. This approach will become part of blood safety vigilance and will be extended to address more general public health issues regarding emerging infectious agents.

VS declares that she has no competing interest. ME is CSO of a spin out of Institut Pasteur, PathoQuest, dedicated to the diagnostic of infectious diseases by Next Generation Sequencing.

Viral inactivation in hemotherapy: systematic review on inactivators with action on nucleic acids

Hepatitis E transmission by transfusion of Intercept blood system-treated plasma

Emerging infectious disease agents and their potential threat to transfusion safety

Parvovirus B19 V DNA contamination in Chinese plasma and plasma derivatives

Global trends in emerging infectious diseases

Overviews of pathogen emergence: which pathogens emerge, when and why?

A strategy to estimate unknown viral diversity in mammals

Origins of major human infectious diseases

The changing face of pathogen discovery and surveillance

Current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections

Metagenomics for the discovery of novel human viruses

High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage

Previously unknown and highly divergent ssDNA viruses populate the oceans

The human gut virome: inter-individual variation and dynamic response to diet

Characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing

Human skin microbiota: high diversity of DNA viruses identified on the human skin by high throughput sequencing

The fecal viral flora of wild rodents

The fecal virome of pigs on a high-density farm

A preliminary study of viral metagenomics of French bat species in contact with humans: identification of new mammalian viruses

Clonal integration of a polyomavirus in human Merkel cell carcinoma

Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new bunyavirus

Newly discovered Ebola virus associated with hemorrhagic fever outbreak in Uganda

A novel rhabdovirus associated with acute hemorrhagic fever in central Africa

Identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy

A new arenavirus in a cluster of fatal transplant-associated diseases

Nextgeneration sequencing technologies in diagnostic virology

Deep sequencing: becoming a critical tool in clinical virology

Overview of emerging technologies to detect adventitious agents

Whole genome: next-generation sequencing as a virus safety test for biotechnological products

Ensuring the safety of vaccine cell substrates by massively parallel sequencing of the transcriptome

Massively parallel sequencing: a new tool in virus discovery and vaccine safety

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

Analysis by high throughput sequencing of Specific Pathogen Free eggs

Viral diversity in swine intestinal mucus used for the manufacture of heparin as analyzed by high-throughput sequencing

Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus

Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples

Sequencing platform and library preparation choices impact viral metagenomes

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

Protocol for metagenomic virus detection in clinical specimens

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

Laboratory procedures to generate viral metagenomes

Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

Detecting the emergence of novel, zoonotic viruses pathogenic to humans

Sequence-independent, single-primer amplification (SISPA) of complex DNA populations

A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species

Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR

Detection of new viruses by VIDISCA. Virus discovery based on cDNA-amplified fragment length polymorphism

Identification and complete genome characterization of a novel picornavirus in turkey (Meleagris gallopavo)

Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes

DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe

Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis

Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa

Using next generation sequencing to identify yellow fever virus in Uganda

Live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency

A member of a new Picornaviridae genus is shed in pig feces

Next-generation sequencing for diagnosis and tailored therapy: a case report of astrovirus-associated progressive encephalitis

Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development

Identification of a new human coronavirus

Two novel parvoviruses in frugivorous New and Old World bats

Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma

Metagenomic analysis of a sample from a patient with respiratory tract infection reveals the presence of a ␥-papillomavirus

Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis

Preferential amplification of pathogenic sequences

A sensitive assay for virus discovery in respiratory clinical samples

Performance of VIDISCA-454 in feces-suspensions and serum

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method

Next generation sequencing technology: advances and applications

A deep-sequencing method detects drug-resistant mutations in the hepatitis B virus in Indonesians

Deep sequencing analysis of Human T cell Lymphotropic Virus type 1 (HTLV-1) long terminal repeat (LTR) 5' region from patients with Tropical Spatic Paraparesis (TSP)/HTLV-1 associated myelopathy (HAM) and asymptomatic carriers

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Wholegenome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season

Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples

Utility of whole-genome sequencing of Escherichia coli O157 for outbreak detection and epidemiological surveillance

Next-generation sequencing (NGS) in the identification of encephalitis-causing viruses: unexpected detection of human herpesvirus 1 while searching for RNA pathogens

The human urine virome in association with urinary tract infections

Metagenomic profile of the viral communities in Rhipicephalus spp. ticks from Yunnan, China

Comprehensive profiling of the vaginal microbiome in HIV positive women using massive parallel semiconductor sequencing

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing

Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

Recovering full-length viral genomes from metagenomes

Comparison of different assembly and annotation tools on analysis of simulated viral metagenomic communities in the gut

A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples

Identification of the first human gyrovirus, a virus related to chicken anemia viru

Human polyomavirus related to African green monkey lymphotropic polyomavirus

Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing

The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns

Novel hybrid parvovirus-like virus, NIH-CQV/PHV, contaminants in silica column-based nucleic acid extraction kits

Étude du virome d'une banque de produits sanguins labiles par séquençage haut débit. In: SFTS, editor. Transfusion clinique et biologique. XXVII ème congrès de la Société française de transfusion sanguine

Human astroviruses

Human stool contains a previously unrecognized diversity of novel astroviruses

Astrovirus MLB2 viremia in febrile child

Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe

Astrovirus encephalitis in boy with X-linked agammaglobulinemia

Acute encephalitis in the immunocompromised individual

Astrovirus infection in hospitalized infants with severe combined immunodeficiency after allogeneic hematopoietic stem cell transplantation

Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing

Virome analysis of transfusion recipients reveals a novel human virus that shares genomic features with hepaciviruses and pegiviruses

Divergent gemycircularvirus in HIV-positive blood

Marseillevirus-like virus recovered from blood donated by asymptomatic humans

Marseillevirus prevalence in multitransfused patients suggests blood transmission

No evidence of Marseillevirus-like virus presence in blood donors and recipients of multiple blood transfusions

Absence of giant blood Marseille-like virus DNA detection by polymerase chain reaction in plasma from healthy US blood donors and serum from multiply transfused patients from Cameroon

Our thoughts are with Jean-Jacques Lefrère. We thank Syria Laperche for her confidence.