key: cord-0870283-qw7gcrqa authors: Xu, Lili; Zhu, Yun; Ren, Lili; Xu, Baoping; Liu, Chunyan; Xie, Zhengde; Shen, Kunling title: Characterization of the nasopharyngeal viral microbiome from children with community‐acquired pneumonia but negative for Luminex xTAG respiratory viral panel assay detection date: 2017-07-25 journal: J Med Virol DOI: 10.1002/jmv.24895 sha: 3fc6971f4f4e0eabb1262fc6b1203e6d6cff9d04 doc_id: 870283 cord_uid: qw7gcrqa In the present study, 50 nasopharyngeal swabs from children with community‐acquired pneumonia (CAP) but negative for 18 common respiratory viruses, as measured by the Luminex xTAG Respiratory Viral Panel Assay, were subjected to multiplex metagenomic analyses using a next‐generation sequencing platform. Taxonomic analysis showed that all sequence reads could be assigned to a specific species. An average of 95.13% were assigned to the Bacteria kingdom, whereas, only 0.72% were potentially virus derived. This snapshot of the respiratory tract virome revealed most viral reads to be respiratory tract related, classified into four known virus families: Paramyxoviridae, Herpesviridae, Anelloviridae, and Polyomaviridae. Importantly, we detected a novel human parainfluenza virus 3 (HPIV 3) strain with a 32‐bp insertion in the haemagglutinin‐neuraminidase (HN) gene that produced a negative result in the Luminex assay, highlighting the strength of virome metagenomic analysis to identify not only novel viruses but also viruses likely to be missed by ordinary clinical tests. Thus, virome metagenomic analysis could become a viable clinical diagnostic method. In the present study, 50 nasopharyngeal swabs from children with communityacquired pneumonia (CAP) but negative for 18 common respiratory viruses, as measured by the Luminex xTAG Respiratory Viral Panel Assay, were subjected to multiplex metagenomic analyses using a next-generation sequencing platform. Taxonomic analysis showed that all sequence reads could be assigned to a specific species. An average of 95.13% were assigned to the Bacteria kingdom, whereas, only 0.72% were potentially virus derived. This snapshot of the respiratory tract virome revealed most viral reads to be respiratory tract related, classified into four known virus families: Paramyxoviridae, Herpesviridae, Anelloviridae, and Polyomaviridae. Importantly, we detected a novel human parainfluenza virus 3 (HPIV 3) strain with a 32-bp insertion in the haemagglutinin-neuraminidase (HN) gene that produced a negative result in the Luminex assay, highlighting the strength of virome metagenomic analysis to identify not only novel viruses but also viruses likely to be missed by ordinary clinical tests. Thus, virome metagenomic analysis could become a viable clinical diagnostic method. Virome results for the pediatric respiratory tract have been reported by several groups. [4] [5] [6] [7] We also previously compared respiratory tract viromes between children with severe acute respiratory infection (SARI) and those without SARI and showed that members of the Paramyxoviridae, Coronaviridae, Parvoviridae, Orthomyxoviridae, Picornaviridae, Anelloviridae, and Adenoviridae families represented the most abundant families identified in the respiratory tracts of children with SARI. The viral population in the respiratory tracts of children without SARI was less diverse and mainly dominated by the Anelloviridae family, with only a small proportion of common epidemic respiratory viruses. 8 Although the agent most commonly detected in CAP is Streptococcus pneumoniae, 9 new, highly sensitive techniques have allowed us to detect many respiratory viruses associated with CAP, 9, 10 mostly influenza virus, respiratory syncytial virus, parainfluenza virus, and human metapneumovirus. [10] [11] [12] Rhinovirus and human coronavirus have also been detected. 10, 11, 13 Whether other still unidentified novel pathogens could cause CAP remains to be investigated. Comprehensive and detailed metagenomic analyses of the respiratory tracts of children with CAP but not carrying common respiratory viruses have not yet been reported. Therefore, in the present study, clinical specimens from children with CAP but negative with common respiratory pathogens were screened using the FDA-cleared Luminex xTAG Respiratory Viral Panel Assay. Fifty specimens that were negative for 18 common respiratory viruses were then subjected to multiplex metagenomic analyses using a next-generation sequencing platform. We provide a picture of the viral content and diversity in the respiratory tracts of children with CAP but uninfected with common respiratory viruses. Table S1 ). Most sequence reads could be assigned to a specific species, and taxonomic analysis showed that an average of 95.13% (range from 44.65% to 99.67) were assigned to the Bacteria kingdom and only 0.72% were potentially virus derived ( Figure 1A and Supplementary Table S1 ). The latter data were valuable for further analyzing the CAPassociated children respiratory virome. The remaining eukaryote-like reads included unscreened human-derived sequences and some animal or plant sequences, as food remains in the pharyngeal passage. Meanwhile, there were still some undefined sequences that lacked a known homolog, or for which almost equally close homologs were found in more than one category. These eukaryote-like and undefined reads were excluded from further analysis. In our data set, only 0.72% of the valid metagenomic sequences were useful for virus detection due to the large amount of host-derived sequences (Supplementary Table S1 ). Human-derived sequence contamination of greater than 90% has been reported by other researchers when using nasal specimens for metagenomic studies. 16, 17 Hence, a high percentage of human-derived sequences is likely an inherent problem of directly extracting RNA/DNA from respiratory samples. Although current computational subtraction methods can efficiently remove host contamination, additional sample-filtering steps before sequencing to eliminate host cells and enrich for microbial genomes are undoubtedly required to detect microbial pathogens in a more efficient and cost-effective manner. The bacterial contigs were split further into genera, as defined by their closest homology ( Figure 1B) . Table 2 . Only one virus in the Paramyxoviridae family, HPIV 3, was detected by metagenomic analysis in these CAP specimens. Almost all children encounter HPIVs within the first few years after birth, but immunity is incomplete, and re-infections occur throughout life. 23 This insertion leads to an advanced stop codon at residue 58 in the haemagglutinin-neuraminidase glycoprotein and has not been reported previously ( Figure 2B ). In The outcome of reactivation strongly depends on the host immunological status. Mostly, hCMV causes severe respiratory disease, whereas the role of EBV in pneumonia is debated. 26 In this study, 5/50 patients were EBV positive by metagenomic analysis, with two patients (BCHNP6 and BCHNP14) having more than 200 reads. At least three members of the Anelloviridae family were found in respiratory specimens of children with CAP, including the torque teno diseases, with a prevalence ranging from 1.1% to 7%. [34] [35] [36] [37] [38] In this study, we reported that children with CAP could also be carrying WUPyV. However, WUPyV was found at similar frequencies in control groups without respiratory diseases. 39 Thus, the link between WUPyV and acute respiratory diseases and CAP remains speculative. However, in all above mentioned specimens which were positive In addition to these respiratory-related viruses, sequences matching other eukaryotic viruses were also detected within the respiratory The fact that micro-mass sequencing detected these canonical viruses in some of the specimens, despite conventional diagnostic testing by EIA and PCR, underscores the sensitivity limits of conventional diagnostics. In this study, a large proportion of the sequences from viruses and bacteria have been confirmed to be a part of the human microflora and many were in fact from known pathogens of the nasopharyngeal tract. However, the nasopharyngeal aspirates collected are likely to contain virus which mainly replicate in the upper respiratory tract. The mucosa may also contain temporary microorganisms that are not part of the normal microflora. They may come from the environment, for example, from dust and from food and water. It is likely that a proportion of the sequences came from such microorganisms. Viral metagenomics clearly provides a crucial tool for virus discovery. With this approach, not priori information is needed, in contrast to directed PCR or Luminex assays, and viruses that are difficult to propagate in cell culture can be discovered. In this study, we detected a novel HPIV3 strain with a 32-bp insertion in the HN gene, highlighting the strength of the method to identify not only novel viruses but also viruses likely to be missed by ordinary clinical tests. Lysholm et al 5 also identified one novel type of Rhinovirus C and a number of previously undescribed viral genetic fragments of unknown origin using a metagenomic sequencing strategy. Using the high-throughput capacity of ultra-deep sequencing, Yang et al 7 detected a co-infected case of HEV with HRV that was missed by regular PCR testing. As sequencing continuously becomes more available and inexpensive, viral metagenomic analysis could also become a viable clinical diagnostic method in the future. 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