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The potential of viral metagenomics in blood transfusion safety
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State of the art
The potential of viral metagenomics in blood transfusion safety La métagénomique virale : un nouvel outil de surveillance des agents viraux émergents au service de la sécurité transfusionnelle V. Sauvage ∗ , J. Gomez , L. Boizeau , S. Laperche Département d’études des agents transmissibles par le sang, Institut national de la transfusion sanguine (INTS), Centre national de référence risques infectieux transfusionnels, 75015 Paris, France
Abstract Thanks to the significant advent of high throughput sequencing in the last ten years, it is now possible via metagenomics to define the spectrum of the microbial sequences present in human blood samples. Therefore, metagenomics sequencing appears as a promising approach for the identification and global surveillance of new, emerging and/or unexpected viruses that could impair blood transfusion safety. However, despite considerable advantages compared to the traditional methods of pathogen identification, this non-targeted approach presents several drawbacks including a lack of sensitivity and sequence contaminant issues. With further improvements, especially to increase sensitivity, metagenomics sequencing should become in a near future an additional diagnostic tool in infectious disease field and especially in blood transfusion safety. © 2017 Elsevier Masson SAS. All rights reserved. Keywords: Viral metagenomics; High throughput sequencing; Viral discovery; Blood-borne viruses; Emerging viruses; Blood safety
Résumé Grâce aux avancées technologiques dans le domaine du séquenc¸age haut débit ces dix dernières années, il est maintenant possible via la métagénomique d’établir le microbiome d’un prélèvement clinique tel que le sang. Ainsi, la métagénomique par séquenc¸age direct apparaît comme une approche prometteuse pour l’identification et la surveillance d’agents viraux nouveaux, émergents et/ou inattendus qui pourraient impacter la sécurité transfusionnelle. Cependant, bien que cette approche sans a priori présente de considérables avantages comparés aux techniques traditionnelles d’identification des pathogènes, la métagénomique présente également plusieurs inconvénients et limitations techniques tels qu’un manque de sensibilité et la présence de nombreuses contaminants provenant notamment des réactifs. Malgré ces inconvénients qui demandent des améliorations majeures, nul doute que la métagénomique devienne dans un futur proche un outil diagnostique supplémentaire dans le domaine des maladies infectieuses, et en particulier en sécurité transfusionnelle. © 2017 Elsevier Masson SAS. Tous droits r´eserv´es. Mots clés : Métagénomique ; Séquenc¸age haut débit ; Découverte de virus ; Virus transmis par le sang ; Virus émergents ; Sécurité transfusionnelle
Thanks to the significant advent of high throughput sequencing (also referred to as deep sequencing) in the last ten years, it is now possible via metagenomics, which gives access to all nucleic acids present in a given sample, to define the spectrum of
∗
Corresponding author. E-mail address: [email protected] (V. Sauvage).
the microbial sequences present in environmental, human and animal samples. This untargeted approach (also called whole genome shotgun sequencing) has been first used to describe the microbial communities from various biological specimens (e.g. feces, urine and blood) and has rapidly become a very useful diagnostic tool for detecting unexpected or unknown infectious agents, and particularly viruses, in infectious diseases of unknown etiology.
http://dx.doi.org/10.1016/j.tracli.2017.06.018 1246-7820/© 2017 Elsevier Masson SAS. All rights reserved.
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1. Principle Metagenome analysis by high throughput sequencing comprises the following five distinct steps: extraction and purification of nucleic acids (DNA and/or RNA) from the sample, reverse transcription of RNAs into complementary DNA (cDNA) and random amplification of DNA molecules (still required for all available deep sequencing technologies when a microbiome analysis is performed), fragmentation of double-strand DNA (ds DNA) by using enzymatic or mechanical techniques prior library construction, deep sequencing and bioinformatics analysis of raw sequencing data (see review [1]). Another method can also be applied to sequence all RNA molecules present in a sample, the RNA-Seq approach, also called RNA shotgun sequencing, where RNA molecules are first fragmented, ligated to specific adapters, reverse transcribed into complementary DNA (cDNA), which are then amplified prior to sequencing. Similarly, this approach can be applied to sequence only DNA molecules (DNA-Seq) in a given sample. 2. Advantages Metagenomics sequencing offers considerable advantages compared to other methods of pathogen identification such as polymerase chain reaction (PCR) amplification or DNA microarrays. Firstly, compared to PCR amplification, this molecular approach is able to detect simultaneously multiple organisms without a priori knowledge of the pathogen’s nucleic acid sequences. Consequently, there is no need to design a set of sequence-specific primers able to target multiple pathogens (and their different variants), and with the capacity to function without any interference or competition to produce nonspecific amplifications. Furthermore, nucleic acid detection by PCR amplifications can be laborious, time consuming and expensive in the case where no pathogen is clearly suspected. Compared to DNA microarrays, which contain representative sequences from all known viruses (e.g. Virochip [2]) and/or bacteria [3], deep sequencing detects the full spectrum of pathogens with a higher sensitivity and offers the possibility to get full genomes. Metagenomics sequencing also allows analyzing simultaneously and in parallel several samples in a same sequencing run, decreasing significantly the cost of this approach. One of the most attractive advantages is the capacity to identify sequences of new microorganisms including the description of novel species [4] and even novel genera [5]. The power of this approach to discover new pathogens rests in large part on a robust in depth bioinformatics analysis that requires a skilled analyst, and thus represents a major limitation for numerous laboratories with limited resources. To date, the bioinformatics analysis still remains a critical step in metagenomics. 3. Limits Like all molecular techniques, metagenomics sequencing presents several drawbacks. In virology, the application of metagenomics to clinical samples is made difficult by the fact that viral sequences represent a very low proportion compared
to the host DNA, ribosomal RNA and bacterial sequences. To improve the detection of viruses, different steps can be included to enrich viral sequences during sample preparation such as filtering out cellular material, removal of ribosomal RNA and/or host cell-free DNA/RNA sequences or viral genomes capture [6]. Once viral nucleic acids are extracted, a random amplification is required both to optimize the detection of small amount of viruses and to obtain the amount of DNA required by the sequencing library preparation kits (1 g to 1 ng depending on library preparation kits and sequencing platform). This step also amplifies remaining host nucleic acids leading to a significant proportion of host reads after sequencing. To get around this major disadvantage, at least in part, a high depth of sequencing is required for good sample coverage and to detect viruses present at low level. The question is then, how many reads are needed to obtain the analytical sensitivity desired? As the sensitivity of the approach depends of the sample matrix and is the result of all successive steps of the workflow, from the nucleic acid extraction method to the bioinformatics analysis, a rigorous pilot study on spiked clinical samples must be imperatively carried out before implementation of a metagenomics pipeline in a laboratory [7]. Very recently, the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, UK) produced a reagent containing 25 target viruses (Virus Multiplex Reagent 11/242001) intended for use as a reference materials in adventitious virus detection assays employing deep sequencing technology. This first common material reference constitutes a very useful control to ensure satisfactory assay performance and sensitivity to enable comparisons between laboratories [8]. Reagent and laboratory contaminants also represent a real problem in metagenomics that could lead to misinterpretations of data. The most representative example is the discovery of a novel parvoviruslike hybrid genome, which was described as a possible new causative agent of seronegative hepatitis [9] and was in fact a contaminant found in Qiagen nucleic acid extraction columns [10,11]. Other studies have reported viral contaminants from extraction columns including circoviruses/densoviruses and iridoviruses [12] or from reagents used for random amplifications including plant viruses [13,14]. To address this issue, it is recommended to sequence in parallel of samples, a negative control, from which the abundance and the nature of reads is subtracted by the bioinformatics pipeline. This measure is only available if samples and negative controls (e.g. water samples) are prepared with reagents from a same batch number. On the other hand, if this measure, expensive given the cost of sequencing, is fully justified for microbiome analyses, this one can be discussed for viral metagenomics. Indeed, viral contaminant sequences from reagents and environment being much less abundant than bacterial contaminants, it is less expensive and equally effective, to investigate the presence of the detected viral sequence(s) using a specific PCR assay in negative extraction and random amplification controls. Contaminations occurring after ligation of adapters onto the insert fragment do not constitute an issue since they cannot be sequenced and then, detected. The technique is also particularly propitious to cross-contaminations between samples and especially with samples containing high viral-titers. Using different barcoded adapters for each sample can help to
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trace such contaminations. However, any cross-contaminations that would have occurred before the ligation step of adapters would not be detected. Contamination issues may depend of sequencing platforms and library workflows. All together, these observations clearly illustrate the importance to implement a rigorous control strategy in any metagenomics study. 4. Viral metagenomics applied to blood transfusion safety The characterization of the human blood-associated viral community (also called human blood virome) is essential for epidemiological surveillance and to detect new potential emerging threats to blood transfusion safety. Viruses known to be commonly detected in blood are Anelloviruses (from the three human genera: alpha, beta and gamma) and Human Pegivirus1 (formerly known as GB virus type C [GBV-C] or Human Pegivirus A) [15,16] which are both nonpathogenic and considered as commensal viruses. Other viral sequences are reported in the blood of healthy individuals including sequences from herpesviruses, papillomaviruses, polyomaviruses, poxviruses and picornaviruses [17,18]. Metagenomics studies on plasma samples have also led to the identification of new viruses such as the identification of two novel rhabdoviruses, Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), in healthy individuals from West Africa [19]. These few examples clearly demonstrate the potential of viral metagenomics sequencing for the identification and the surveillance of unknown or unexpected viruses that could be transmitted to recipients by blood products. In this context, recent reports have used metagenomics to characterize the nature and composition of viruses in blood donations from healthy and ineligible donors for transfusion, in blood from multi-transfused recipients [16,20–22], in final blood products eligible for transfusion [23] as well as in plasma pools used for manufacture of plasma derivated-products [24]. Hence, a novel human Flavivirus, named Human Hepegivirus 1 (HHPgV-1) or Human Pegivirus 2 (HPgV-2) was identified in plasma samples from blood donors and multi-transfused recipients from the United States (US) and the United Kingdom, suggesting that this virus could be transmitted by transfusion. [20,21]. HHPgV-1/HPgV-2 viremia and seroprevalence were demonstrated strongly, but not exclusively, associated with active or recovered HCV infection [20,21,25,26]. Very recently, HHPgV-1 infection was also detected at a high prevalence (10.9%) in a cohort of injection drug users [27]. On the other hand, no related sequences were detected in additional plasma pools originating from the US and Europe as reported in two recent metagenomics analysis [23,24]. However, these results could be explained by a lack of sensitivity of the whole process due to the dilution factor introduced according to the pool size. Despite a low frequency of HHPgV-1/HPgV-2 viremia in HCVnon-infected individuals, these preliminary data justify further investigations using large cohorts of eligible blood donors to estimate the risk of exposition to this new virus through transfusion. During a study investigating the virome in final blood products eligible for transfusion, Lau et al., [23] detected
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sequences from common viruses such as Anellovirus and HPgV1, but also sequences derived from Merkel cell Polyomavirus (MCPyV) and Human Papillomavirus 27, and more unexpectedly, sequences from Human astrovirus MLB2 in a fresh-frozen plasma unit. The presence of this virus was confirmed by RTPCR in the donor’s blood and corresponded to an asymptomatic viremia. Human astroviruses, which are ssRNA belonging to the family Astroviridae, are known as important agents of viral gastroenteritis, especially in young children, elderly and immunocompromised individuals [28]. The Human astrovirus MLB2 was initially discovered in diarrheal stools of children from India and the United States [29], and it has since been detected in the plasma of a febrile child [30], 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 [31] and immunocompromised individuals [32,33]. Very recently, Astrovirus MLB2 was clearly associated with meningitis infection in an immunocompetent adult patient [34]. Reports of astrovirus-associated encephalitis were also described in allogeneic hematopoietic stem cells [35] and bone marrow transplant recipients [36]. The study of Lau et al. clearly demonstrates that viral metagenomics sequencing is a suitable tool for the diagnosis of unexpected viral infections in blood products. Similarly, Zhang et al. [24] investigated the viral landscape in plasma fractionation pools assembled from blood donors from US and the United Kingdom. Beside commensal viruses, they detected few sequences of human papillomavirus and two distinct gemycircularvirus genomes (DB1 and DB2). Gemycircularvirus are ssDNA virus belonging to the recently proposed genus Gemycircularvirus within the Genomoviridae family. There is to date only one representative isolate of the family, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), which is a virus infecting fungi. However, several other gemycircularvirus genomes have been identified in environmental samples (e.g. sewage), plant and animal tissues [37]. Recently, a divergent Gemycirculavirus (GemyC1c) was described from an HIV1-positive blood donation [38]. In the framework of a viral discovery research program using metagenomics, we have also detected several sequences related to gemycircularvirus in blood donations collected in sub-Saharan Africa (data not published). The presence of gemycircularvirus genomes in plasma samples raises the question of their origin. Are they human infectious agents or contaminants from different sources? Similarly, the detection of a new Giant virus belonging to the Marseilleviridae family, the Giant Blood Marseillevirus (GMB), in healthy donors and post-transfusion sera from thalassemia patients [39,40] may potentially reflect contaminations of plasma samples from kit reagents or environment [24,41]. This emphasizes the rigorous approach needed to establish the validity of new viral genomes identified by metagenomics sequencing before concluding to the discovery of new agents. Finally, viral metagenomics has also been used in completion of routine diagnostics that failed to identify an etiologic agent in unexplained infectious disease after transplantation [42–45].
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Thereby, Lipowski et al., [45] provide the first description of transmission of Tick-Borne Encephalitis Virus (TBEV) through transplantation of solid organs. This virus may represent an emerging viral infection in transplant recipients. In conclusion, the increasing and ongoing documentation of novel animal viruses identified worldwide predicts that additional animal pathogens may cross species barrier to potentially become novel human infectious diseases in the near future, some perhaps highly pathogenic. Viruses are the pathogen class most likely to emerge with an estimated number of undiscovered mammalian viruses of at least 320,000 [46]. This estimation suggests that zoonotic emerging viral infections (EIDs) represent a serious threat to human health and justify implementations of pro-active surveillance of EIDs in blood donor populations from different parts of the world especially from geographic areas with the highest rates of EIDs. Viral metagenomics can help us to prevent such a threat. Thanks to the new sequencing technologies (e.g. nanopore sequencing) allowing the progressive increase of read length and the continual development of bioinformatics tools for pathogen identification, this approach should become an additional diagnostic tool [47,48]. However, this will become possible only after additional improvements, especially to increase sensitivity. Moreover, as mentioned during the first International Conference on Clinical Metagenomics, there is a necessary evolution towards a universal clinical metagenomics pipeline [49]. Disclosure of interest The authors have not supplied their declaration of competing interest. References [1] Sauvage V, Eloit M. Viral metagenomics and blood safety. Transfus Clin Biol 2016;23:28–38. [2] Chen EC, Miller SA, DeRisi JL, Chiu CY. Using a pan-viral microarray assay (Virochip) to screen clinical samples for viral pathogens. J Vis Exp 2011;50. [3] Berthet N, Dickinson P, Filliol I, Reinhardt AK, Batejat C, Vallaeys T, et al. Massively parallel pathogen identification using high-density microarrays. Microb Biotechnol 2008;1:79–86. [4] Parras-Moltó M, Suárez-Rodríguez P, Eguia A, Aguirre-Urizar JM, LópezBueno A. Genome sequence of two novel species of torque teno minivirus from the human oral cavity. Genome Announc 2014:2. [5] Sauvage V, Ar Gouilh M, Cheval J, Muth E, Pariente K, Burguiere A, et al. A member of a new Picornaviridae genus is shed in pig feces. J Virol 2012;86:10036–46. [6] Briese T, Kapoor A, Mishra N, Jain K, Kumar A, Jabado OJ, et al. Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis. MBio 2015;6 [pii: e01491-15]. [7] Cheval J, Sauvage V, Frangeul L, Dacheux L, Guigon G, Dumey N, et al. Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples. J Clin Microbiol 2011;49:3268–75. [8] Mee ET, Preston MD, Minor PD, Schepelmann S. CS533 study participants. Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing. Vaccine 2016;34:2035–43. [9] Xu B, Zhi N, Hu G, Wan Z, Zheng X, Liu X, et al. Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing. Proc Natl Acad Sci USA 2013;110:10264–9.
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State of the art
The potential of viral metagenomics in blood transfusion safety La métagénomique virale : un nouvel outil de surveillance des agents viraux émergents au service de la sécurité transfusionnelle V. Sauvage ∗ , J. Gomez , L. Boizeau , S. Laperche Département d’études des agents transmissibles par le sang, Institut national de la transfusion sanguine (INTS), Centre national de référence risques infectieux transfusionnels, 75015 Paris, France
Abstract Thanks to the significant advent of high throughput sequencing in the last ten years, it is now possible via metagenomics to define the spectrum of the microbial sequences present in human blood samples. Therefore, metagenomics sequencing appears as a promising approach for the identification and global surveillance of new, emerging and/or unexpected viruses that could impair blood transfusion safety. However, despite considerable advantages compared to the traditional methods of pathogen identification, this non-targeted approach presents several drawbacks including a lack of sensitivity and sequence contaminant issues. With further improvements, especially to increase sensitivity, metagenomics sequencing should become in a near future an additional diagnostic tool in infectious disease field and especially in blood transfusion safety. © 2017 Elsevier Masson SAS. All rights reserved. Keywords: Viral metagenomics; High throughput sequencing; Viral discovery; Blood-borne viruses; Emerging viruses; Blood safety
Résumé Grâce aux avancées technologiques dans le domaine du séquenc¸age haut débit ces dix dernières années, il est maintenant possible via la métagénomique d’établir le microbiome d’un prélèvement clinique tel que le sang. Ainsi, la métagénomique par séquenc¸age direct apparaît comme une approche prometteuse pour l’identification et la surveillance d’agents viraux nouveaux, émergents et/ou inattendus qui pourraient impacter la sécurité transfusionnelle. Cependant, bien que cette approche sans a priori présente de considérables avantages comparés aux techniques traditionnelles d’identification des pathogènes, la métagénomique présente également plusieurs inconvénients et limitations techniques tels qu’un manque de sensibilité et la présence de nombreuses contaminants provenant notamment des réactifs. Malgré ces inconvénients qui demandent des améliorations majeures, nul doute que la métagénomique devienne dans un futur proche un outil diagnostique supplémentaire dans le domaine des maladies infectieuses, et en particulier en sécurité transfusionnelle. © 2017 Elsevier Masson SAS. Tous droits r´eserv´es. Mots clés : Métagénomique ; Séquenc¸age haut débit ; Découverte de virus ; Virus transmis par le sang ; Virus émergents ; Sécurité transfusionnelle
Thanks to the significant advent of high throughput sequencing (also referred to as deep sequencing) in the last ten years, it is now possible via metagenomics, which gives access to all nucleic acids present in a given sample, to define the spectrum of
∗
Corresponding author. E-mail address: [email protected] (V. Sauvage).
the microbial sequences present in environmental, human and animal samples. This untargeted approach (also called whole genome shotgun sequencing) has been first used to describe the microbial communities from various biological specimens (e.g. feces, urine and blood) and has rapidly become a very useful diagnostic tool for detecting unexpected or unknown infectious agents, and particularly viruses, in infectious diseases of unknown etiology.
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1. Principle Metagenome analysis by high throughput sequencing comprises the following five distinct steps: extraction and purification of nucleic acids (DNA and/or RNA) from the sample, reverse transcription of RNAs into complementary DNA (cDNA) and random amplification of DNA molecules (still required for all available deep sequencing technologies when a microbiome analysis is performed), fragmentation of double-strand DNA (ds DNA) by using enzymatic or mechanical techniques prior library construction, deep sequencing and bioinformatics analysis of raw sequencing data (see review [1]). Another method can also be applied to sequence all RNA molecules present in a sample, the RNA-Seq approach, also called RNA shotgun sequencing, where RNA molecules are first fragmented, ligated to specific adapters, reverse transcribed into complementary DNA (cDNA), which are then amplified prior to sequencing. Similarly, this approach can be applied to sequence only DNA molecules (DNA-Seq) in a given sample. 2. Advantages Metagenomics sequencing offers considerable advantages compared to other methods of pathogen identification such as polymerase chain reaction (PCR) amplification or DNA microarrays. Firstly, compared to PCR amplification, this molecular approach is able to detect simultaneously multiple organisms without a priori knowledge of the pathogen’s nucleic acid sequences. Consequently, there is no need to design a set of sequence-specific primers able to target multiple pathogens (and their different variants), and with the capacity to function without any interference or competition to produce nonspecific amplifications. Furthermore, nucleic acid detection by PCR amplifications can be laborious, time consuming and expensive in the case where no pathogen is clearly suspected. Compared to DNA microarrays, which contain representative sequences from all known viruses (e.g. Virochip [2]) and/or bacteria [3], deep sequencing detects the full spectrum of pathogens with a higher sensitivity and offers the possibility to get full genomes. Metagenomics sequencing also allows analyzing simultaneously and in parallel several samples in a same sequencing run, decreasing significantly the cost of this approach. One of the most attractive advantages is the capacity to identify sequences of new microorganisms including the description of novel species [4] and even novel genera [5]. The power of this approach to discover new pathogens rests in large part on a robust in depth bioinformatics analysis that requires a skilled analyst, and thus represents a major limitation for numerous laboratories with limited resources. To date, the bioinformatics analysis still remains a critical step in metagenomics. 3. Limits Like all molecular techniques, metagenomics sequencing presents several drawbacks. In virology, the application of metagenomics to clinical samples is made difficult by the fact that viral sequences represent a very low proportion compared
to the host DNA, ribosomal RNA and bacterial sequences. To improve the detection of viruses, different steps can be included to enrich viral sequences during sample preparation such as filtering out cellular material, removal of ribosomal RNA and/or host cell-free DNA/RNA sequences or viral genomes capture [6]. Once viral nucleic acids are extracted, a random amplification is required both to optimize the detection of small amount of viruses and to obtain the amount of DNA required by the sequencing library preparation kits (1 g to 1 ng depending on library preparation kits and sequencing platform). This step also amplifies remaining host nucleic acids leading to a significant proportion of host reads after sequencing. To get around this major disadvantage, at least in part, a high depth of sequencing is required for good sample coverage and to detect viruses present at low level. The question is then, how many reads are needed to obtain the analytical sensitivity desired? As the sensitivity of the approach depends of the sample matrix and is the result of all successive steps of the workflow, from the nucleic acid extraction method to the bioinformatics analysis, a rigorous pilot study on spiked clinical samples must be imperatively carried out before implementation of a metagenomics pipeline in a laboratory [7]. Very recently, the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, UK) produced a reagent containing 25 target viruses (Virus Multiplex Reagent 11/242001) intended for use as a reference materials in adventitious virus detection assays employing deep sequencing technology. This first common material reference constitutes a very useful control to ensure satisfactory assay performance and sensitivity to enable comparisons between laboratories [8]. Reagent and laboratory contaminants also represent a real problem in metagenomics that could lead to misinterpretations of data. The most representative example is the discovery of a novel parvoviruslike hybrid genome, which was described as a possible new causative agent of seronegative hepatitis [9] and was in fact a contaminant found in Qiagen nucleic acid extraction columns [10,11]. Other studies have reported viral contaminants from extraction columns including circoviruses/densoviruses and iridoviruses [12] or from reagents used for random amplifications including plant viruses [13,14]. To address this issue, it is recommended to sequence in parallel of samples, a negative control, from which the abundance and the nature of reads is subtracted by the bioinformatics pipeline. This measure is only available if samples and negative controls (e.g. water samples) are prepared with reagents from a same batch number. On the other hand, if this measure, expensive given the cost of sequencing, is fully justified for microbiome analyses, this one can be discussed for viral metagenomics. Indeed, viral contaminant sequences from reagents and environment being much less abundant than bacterial contaminants, it is less expensive and equally effective, to investigate the presence of the detected viral sequence(s) using a specific PCR assay in negative extraction and random amplification controls. Contaminations occurring after ligation of adapters onto the insert fragment do not constitute an issue since they cannot be sequenced and then, detected. The technique is also particularly propitious to cross-contaminations between samples and especially with samples containing high viral-titers. Using different barcoded adapters for each sample can help to
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trace such contaminations. However, any cross-contaminations that would have occurred before the ligation step of adapters would not be detected. Contamination issues may depend of sequencing platforms and library workflows. All together, these observations clearly illustrate the importance to implement a rigorous control strategy in any metagenomics study. 4. Viral metagenomics applied to blood transfusion safety The characterization of the human blood-associated viral community (also called human blood virome) is essential for epidemiological surveillance and to detect new potential emerging threats to blood transfusion safety. Viruses known to be commonly detected in blood are Anelloviruses (from the three human genera: alpha, beta and gamma) and Human Pegivirus1 (formerly known as GB virus type C [GBV-C] or Human Pegivirus A) [15,16] which are both nonpathogenic and considered as commensal viruses. Other viral sequences are reported in the blood of healthy individuals including sequences from herpesviruses, papillomaviruses, polyomaviruses, poxviruses and picornaviruses [17,18]. Metagenomics studies on plasma samples have also led to the identification of new viruses such as the identification of two novel rhabdoviruses, Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), in healthy individuals from West Africa [19]. These few examples clearly demonstrate the potential of viral metagenomics sequencing for the identification and the surveillance of unknown or unexpected viruses that could be transmitted to recipients by blood products. In this context, recent reports have used metagenomics to characterize the nature and composition of viruses in blood donations from healthy and ineligible donors for transfusion, in blood from multi-transfused recipients [16,20–22], in final blood products eligible for transfusion [23] as well as in plasma pools used for manufacture of plasma derivated-products [24]. Hence, a novel human Flavivirus, named Human Hepegivirus 1 (HHPgV-1) or Human Pegivirus 2 (HPgV-2) was identified in plasma samples from blood donors and multi-transfused recipients from the United States (US) and the United Kingdom, suggesting that this virus could be transmitted by transfusion. [20,21]. HHPgV-1/HPgV-2 viremia and seroprevalence were demonstrated strongly, but not exclusively, associated with active or recovered HCV infection [20,21,25,26]. Very recently, HHPgV-1 infection was also detected at a high prevalence (10.9%) in a cohort of injection drug users [27]. On the other hand, no related sequences were detected in additional plasma pools originating from the US and Europe as reported in two recent metagenomics analysis [23,24]. However, these results could be explained by a lack of sensitivity of the whole process due to the dilution factor introduced according to the pool size. Despite a low frequency of HHPgV-1/HPgV-2 viremia in HCVnon-infected individuals, these preliminary data justify further investigations using large cohorts of eligible blood donors to estimate the risk of exposition to this new virus through transfusion. During a study investigating the virome in final blood products eligible for transfusion, Lau et al., [23] detected
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sequences from common viruses such as Anellovirus and HPgV1, but also sequences derived from Merkel cell Polyomavirus (MCPyV) and Human Papillomavirus 27, and more unexpectedly, sequences from Human astrovirus MLB2 in a fresh-frozen plasma unit. The presence of this virus was confirmed by RTPCR in the donor’s blood and corresponded to an asymptomatic viremia. Human astroviruses, which are ssRNA belonging to the family Astroviridae, are known as important agents of viral gastroenteritis, especially in young children, elderly and immunocompromised individuals [28]. The Human astrovirus MLB2 was initially discovered in diarrheal stools of children from India and the United States [29], and it has since been detected in the plasma of a febrile child [30], 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 [31] and immunocompromised individuals [32,33]. Very recently, Astrovirus MLB2 was clearly associated with meningitis infection in an immunocompetent adult patient [34]. Reports of astrovirus-associated encephalitis were also described in allogeneic hematopoietic stem cells [35] and bone marrow transplant recipients [36]. The study of Lau et al. clearly demonstrates that viral metagenomics sequencing is a suitable tool for the diagnosis of unexpected viral infections in blood products. Similarly, Zhang et al. [24] investigated the viral landscape in plasma fractionation pools assembled from blood donors from US and the United Kingdom. Beside commensal viruses, they detected few sequences of human papillomavirus and two distinct gemycircularvirus genomes (DB1 and DB2). Gemycircularvirus are ssDNA virus belonging to the recently proposed genus Gemycircularvirus within the Genomoviridae family. There is to date only one representative isolate of the family, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), which is a virus infecting fungi. However, several other gemycircularvirus genomes have been identified in environmental samples (e.g. sewage), plant and animal tissues [37]. Recently, a divergent Gemycirculavirus (GemyC1c) was described from an HIV1-positive blood donation [38]. In the framework of a viral discovery research program using metagenomics, we have also detected several sequences related to gemycircularvirus in blood donations collected in sub-Saharan Africa (data not published). The presence of gemycircularvirus genomes in plasma samples raises the question of their origin. Are they human infectious agents or contaminants from different sources? Similarly, the detection of a new Giant virus belonging to the Marseilleviridae family, the Giant Blood Marseillevirus (GMB), in healthy donors and post-transfusion sera from thalassemia patients [39,40] may potentially reflect contaminations of plasma samples from kit reagents or environment [24,41]. This emphasizes the rigorous approach needed to establish the validity of new viral genomes identified by metagenomics sequencing before concluding to the discovery of new agents. Finally, viral metagenomics has also been used in completion of routine diagnostics that failed to identify an etiologic agent in unexplained infectious disease after transplantation [42–45].
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Thereby, Lipowski et al., [45] provide the first description of transmission of Tick-Borne Encephalitis Virus (TBEV) through transplantation of solid organs. This virus may represent an emerging viral infection in transplant recipients. In conclusion, the increasing and ongoing documentation of novel animal viruses identified worldwide predicts that additional animal pathogens may cross species barrier to potentially become novel human infectious diseases in the near future, some perhaps highly pathogenic. Viruses are the pathogen class most likely to emerge with an estimated number of undiscovered mammalian viruses of at least 320,000 [46]. This estimation suggests that zoonotic emerging viral infections (EIDs) represent a serious threat to human health and justify implementations of pro-active surveillance of EIDs in blood donor populations from different parts of the world especially from geographic areas with the highest rates of EIDs. Viral metagenomics can help us to prevent such a threat. Thanks to the new sequencing technologies (e.g. nanopore sequencing) allowing the progressive increase of read length and the continual development of bioinformatics tools for pathogen identification, this approach should become an additional diagnostic tool [47,48]. However, this will become possible only after additional improvements, especially to increase sensitivity. Moreover, as mentioned during the first International Conference on Clinical Metagenomics, there is a necessary evolution towards a universal clinical metagenomics pipeline [49]. Disclosure of interest The authors have not supplied their declaration of competing interest. References [1] Sauvage V, Eloit M. Viral metagenomics and blood safety. Transfus Clin Biol 2016;23:28–38. [2] Chen EC, Miller SA, DeRisi JL, Chiu CY. Using a pan-viral microarray assay (Virochip) to screen clinical samples for viral pathogens. J Vis Exp 2011;50. [3] Berthet N, Dickinson P, Filliol I, Reinhardt AK, Batejat C, Vallaeys T, et al. Massively parallel pathogen identification using high-density microarrays. Microb Biotechnol 2008;1:79–86. [4] Parras-Moltó M, Suárez-Rodríguez P, Eguia A, Aguirre-Urizar JM, LópezBueno A. Genome sequence of two novel species of torque teno minivirus from the human oral cavity. Genome Announc 2014:2. [5] Sauvage V, Ar Gouilh M, Cheval J, Muth E, Pariente K, Burguiere A, et al. A member of a new Picornaviridae genus is shed in pig feces. J Virol 2012;86:10036–46. [6] Briese T, Kapoor A, Mishra N, Jain K, Kumar A, Jabado OJ, et al. Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis. MBio 2015;6 [pii: e01491-15]. [7] Cheval J, Sauvage V, Frangeul L, Dacheux L, Guigon G, Dumey N, et al. Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples. J Clin Microbiol 2011;49:3268–75. [8] Mee ET, Preston MD, Minor PD, Schepelmann S. CS533 study participants. Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing. Vaccine 2016;34:2035–43. [9] Xu B, Zhi N, Hu G, Wan Z, Zheng X, Liu X, et al. Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing. Proc Natl Acad Sci USA 2013;110:10264–9.
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