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J Virol Methods. 2015 Dec 15;226:1-6. doi: 10.1016/j.jviromet.2015.09.010. Epub 2015 Sep 28.

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

Author information

1
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.
2
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.
3
Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: tlaskus@yahoo.com.
4
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.
5
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.
6
Department of Clinical Science, University of Bergen, 5021 Bergen, Norway. Electronic address: tomasz.stokowy@k2.uib.no.
7
Municipal Hospital for Infectious Diseases, 37 Wolska Street, 01-201 Warsaw, Poland. Electronic address: dariusz.lipowski@wum.edu.pl.
8
Department of Genetics, Institute of Physiology and Pathology of Hearing, Mochnackiego 10, 02-042 Warsaw, Poland. Electronic address: a.pollak@ifps.org.pl.
9
Department of Genetics, Institute of Physiology and Pathology of Hearing, Mochnackiego 10, 02-042 Warsaw, Poland. Electronic address: u.lechowicz@ifps.org.pl.
10
Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: kcaraballo@wum.edu.pl.
11
Department of Neurology, Military Institute of Medicine, 128 Szaserów Street, 04-141 Warsaw, Poland. Electronic address: astepien@wim.mil.pl.
12
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.
13
Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawinskiego Street, 02-106 Warsaw, Poland. Electronic address: ibukowska@wum.edu.pl.

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.

CONCLUSION:

In summary, it seems that NGS preceded by nucleic acid amplification could supplement currently used diagnostic methods in encephalitis.

KEYWORDS:

Encephalitis; Next-generation sequencing; Unknown etiology

PMID:
26424618
DOI:
10.1016/j.jviromet.2015.09.010
[Indexed for MEDLINE]
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