title: Next-generation sequencing (NGS) in the identification of encephalitis-causing viruses: Unexpected detection of human herpesvirus 1 while searching for RNA pathogens.
abstract: BACKGROUND
  Encephalitis is a severe neurological syndrome usually caused by viruses. Despite significant progress in diagnostic techniques, the causative agent remains unidentified in the majority of cases. The aim of the present study was to test an alternative approach for the detection of putative pathogens in encephalitis using next-generation sequencing (NGS).
  
  
  METHODS
  RNA was extracted from cerebrospinal fluid (CSF) from a 60-year-old male patient with encephalitis and subjected to isothermal linear nucleic acid amplification (Ribo-SPIA, NuGen) followed by next-generation sequencing using MiSeq (Illumina) system and metagenomics data analysis.
  
  
  RESULTS
  The sequencing run yielded 1,578,856 reads overall and 2579 reads matched human herpesvirus I (HHV-1) genome; the presence of this pathogen in CSF was confirmed by specific PCR. In subsequent experiments we found that the DNAse I treatment, while lowering the background of host-derived sequences, lowered the number of detectable HHV-1 sequences by a factor of 4. Furthermore, we found that the routine extraction of total RNA by the Chomczynski method could be used for identification of both DNA and RNA pathogens in typical clinical settings, as it results in retention of a significant amount of DNA.
  
  
  CONCLUSIONS
  In summary, it seems that NGS preceded by nucleic acid amplification could supplement currently used diagnostic methods in encephalitis.
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: karol.perlejewski@wum.edu.pl.
authors.forename: Karol
authors.initials: K
authors.lastname: Perlejewski
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland; Postgraduate School of Molecular Medicine, 61 Żwirki i Wigury Street, 02-091 Warsaw, Poland. Electronic address: mszabl@wp.pl.
authors.forename: Marta
authors.initials: M
authors.lastname: Popiel
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: tlaskus@yahoo.com.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Laskus
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka, Suita-City, Osaka, Japan. Electronic address: nshota@gen-info.osaka-u.ac.jp.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka, Suita-City, Osaka, Japan. Electronic address: daisukem@gen-info.osaka-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Motooka
authors.affiliation: Department of Clinical Science, University of Bergen, 5021 Bergen, Norway. Electronic address: tomasz.stokowy@k2.uib.no.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Stokowy
authors.affiliation: Municipal Hospital for Infectious Diseases, 37 Wolska Street, 01-201 Warsaw, Poland. Electronic address: dariusz.lipowski@wum.edu.pl.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Lipowski
authors.affiliation: Department of Genetics, Institute of Physiology and Pathology of Hearing, Mochnackiego 10, 02-042 Warsaw, Poland. Electronic address: a.pollak@ifps.org.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pollak
authors.affiliation: Department of Genetics, Institute of Physiology and Pathology of Hearing, Mochnackiego 10, 02-042 Warsaw, Poland. Electronic address: u.lechowicz@ifps.org.pl.
authors.forename: Urszula
authors.initials: U
authors.lastname: Lechowicz
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: kcaraballo@wum.edu.pl.
authors.forename: Kamila
authors.initials: K
authors.lastname: Caraballo Cortés
authors.affiliation: Department of Neurology, Military Institute of Medicine, 128 Szaserów Street, 04-141 Warsaw, Poland. Electronic address: astepien@wim.mil.pl.
authors.forename: Adam
authors.initials: A
authors.lastname: Stępień
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: marek.radkowski@wum.edu.pl.
authors.forename: Marek
authors.initials: M
authors.lastname: Radkowski
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: ibukowska@wum.edu.pl.
authors.forename: Iwona
authors.initials: I
authors.lastname: Bukowska-Ośko
mesh_terms: D018792:Encephalitis, Viral / Q000175:diagnosis* / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000134:cerebrospinal fluid*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: Encephalitis; Next-generation sequencing; Unknown etiology