Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing.
enterovirus
environmental surveillance
nanopore sequencing
poliovirus
stool
Journal
Journal of clinical microbiology
ISSN: 1098-660X
Titre abrégé: J Clin Microbiol
Pays: United States
ID NLM: 7505564
Informations de publication
Date de publication:
24 08 2020
24 08 2020
Historique:
received:
29
04
2020
accepted:
29
06
2020
pubmed:
3
7
2020
medline:
24
6
2021
entrez:
3
7
2020
Statut:
epublish
Résumé
Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.
Identifiants
pubmed: 32611795
pii: JCM.00920-20
doi: 10.1128/JCM.00920-20
pmc: PMC7448630
pii:
doi:
Substances chimiques
Poliovirus Vaccine, Oral
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : World Health Organization
ID : 001
Pays : International
Organisme : Medical Research Council
ID : MR/R015600/1
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 206298/Z/17/Z
Pays : United Kingdom
Informations de copyright
Copyright © 2020 Shaw et al.
Références
Nucleic Acids Res. 2012 Jan;40(Database issue):D593-8
pubmed: 22006842
Lancet. 2014 Oct 25;384(9953):1505-12
pubmed: 25018120
Mol Biol Evol. 2013 Apr;30(4):772-80
pubmed: 23329690
Biomed Environ Sci. 2017 Oct;30(10):718-726
pubmed: 29122092
Bioinformatics. 2012 Oct 1;28(19):2520-2
pubmed: 22908215
Bioinformatics. 2016 Jul 15;32(14):2103-10
pubmed: 27153593
Lancet. 2019 Jul 13;394(10193):148-158
pubmed: 31174831
J Virol Methods. 2011 Jun;174(1-2):128-30
pubmed: 21440569
J Virol Methods. 2018 Oct;260:21-25
pubmed: 29981296
Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10604-9
pubmed: 25002465
J Clin Microbiol. 2006 Aug;44(8):2698-704
pubmed: 16891480
J Infect Dis. 2018 Mar 28;217(8):1222-1230
pubmed: 29309594
J Mol Biol. 1990 Oct 5;215(3):403-10
pubmed: 2231712
J Clin Microbiol. 2015 Jan;53(1):73-8
pubmed: 25339406
J Clin Microbiol. 2013 Aug;51(8):2717-20
pubmed: 23698530
Clin Infect Dis. 2019 Oct 26;:
pubmed: 31665247
Mol Biol Evol. 2018 Jun 1;35(6):1547-1549
pubmed: 29722887
Cell. 2017 Mar 23;169(1):35-46.e19
pubmed: 28340348
J Virol. 2006 Sep;80(17):8653-63
pubmed: 16912313
Virus Evol. 2020 May 20;6(2):veaa040
pubmed: 32782825
Genome Res. 2017 May;27(5):737-746
pubmed: 28100585
J Virol Methods. 2013 Apr;189(1):7-14
pubmed: 23305817
Virus Res. 2018 Jul 2;252:8-12
pubmed: 29763627
Clin Infect Dis. 2016 Jan 15;62(2):190-198
pubmed: 26417032
J Virol Methods. 2005 Jun;126(1-2):127-34
pubmed: 15847928
PeerJ. 2016 Oct 18;4:e2584
pubmed: 27781170
Mol Biol Evol. 1993 May;10(3):512-26
pubmed: 8336541
Bioinformatics. 2011 Feb 15;27(4):592-3
pubmed: 21169378
J Clin Microbiol. 2009 Jun;47(6):1939-41
pubmed: 19386844