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1. J Virol Methods. 2015 Dec 15;226:7-14. doi: 10.1016/j.jviromet.2015.09.004. Epub

2015 Sep 14.

 

Rapid next-generation sequencing of dengue, EV-A71 and RSV-A viruses.

 

Baronti C(1), Piorkowski G(2), Leparc-Goffart I(3), de Lamballerie X(2),

Dubot-Pérès A(4).

 

Author information:

(1)Aix Marseille Université, IRD French Institute of Research for Development,

EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies

Virales", 13005 Marseille, France; IHU Méditerranée Infection, APHM Public

Hospitals of Marseille, 13005 Marseille, France. Electronic address:

Cecile.baronti@univ-amu.fr. (2)Aix Marseille Université, IRD French Institute of

Research for Development, EHESP French School of Public Health, EPV UMR_D 190

"Emergence des Pathologies Virales", 13005 Marseille, France; IHU Méditerranée

Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France. (3)Aix

Marseille Université, IRD French Institute of Research for Development, EHESP

French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies

Virales", 13005 Marseille, France; French National Reference Centre for

Arboviruses, IRBA, 13013 Marseille, France. (4)Aix Marseille Université, IRD

French Institute of Research for Development, EHESP French School of Public

Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille,

France; Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU),

Microbiology Laboratory, Mahosot Hospital, PDR Vientiane, Lao Democratic People's

Republic; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine,

University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK.

 

Accurate characterisation of viral strains constitutes a crucial objective for

the management of modern virus collections. Next-generation sequencing (NGS)

provides technical solution for fast and cost-effective full genome sequencing.

Here, we report protocols for rapid full-genome characterisation of RNA viruses

of medical importance: dengue virus, enterovirus A71 and respiratory syncytial

virus A, based on a specific amplification step followed by NGS-sequencing. A

subset of full-length genome sequences representing the genetic diversity of each

virus type was selected in GenBank and used to design primer sets allowing the

amplification of the complete genome in 3-8 overlapping PCR fragments. The

technique was used for characterising 53 strains (33 DENV, 8 EV-A71, 12 RSV-A)

from various genotypes and origins. In a single assay, and in just 4 days, it

provided for all strains an excellent genomic coverage (∼ 99% including complete

ORF for all strains) and accurate sequences with high number of reads per

position (250-3500 on average). The elaboration of specific PCR-based full-genome

sequencing protocols for diverse virus groups is likely to revolutionise the

characterisation of viral isolates in modern collection, but also to contribute

in the next future to the study of RNA viruses directly from biological samples.

 

Copyright © 2015 Elsevier B.V. All rights reserved.

 

DOI: 10.1016/j.jviromet.2015.09.004

PMID: 26376168  [PubMed - indexed for MEDLINE]

 

 

2. Adv Exp Med Biol. 2016;885:11-23. doi: 10.1007/5584_2015_186.

 

Next-Generation Sequencing of 5' Untranslated Region of Hepatitis C Virus in

Search of Minor Viral Variant in a Patient Who Revealed New Genotype While on

Antiviral Treatment.

 

Caraballo Cortes K(1), Bukowska-Ośko I(2), Pawełczyk A(1), Perlejewski K(1),

Płoski R(3), Lechowicz U(4), Stawiński P(4), Demkow U(5), Laskus T(1), Radkowski

M(1).

 

Author information:

(1)Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw

Medical University, 3c Pawińskiego St., 02-106, Warsaw, Poland. (2)Department of

Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University,

3c Pawińskiego St., 02-106, Warsaw, Poland. ibukowska@wum.edu.pl. (3)Department

of Medical Genetics, Warsaw Medical University, 3c Pawińskiego St., 02-106,

Warsaw, Poland. (4)Department of Genetics, Institute of Physiology and Pathology

of Hearing, 10 Mochnackiego St., 02-042, Warsaw, Poland. (5)Department of

Laboratory Medicine and Clinical Immunology of Developmental Age, Warsaw Medical

University, 24 Marszałkowska St., 00-576, Warsaw, Poland.

 

The role of mixed infections with different hepatitis C virus (HCV) genotypes in

viral persistence, treatment effects, and tissue tropism is unclear.

Next-generation sequencing (NGS), which is suitable for analysis of large,

genetically diverse populations offers unparalleled advantages for the study of

mixed infections. The aim of the study was to determine, using two different deep

sequencing strategies (pyrosequencing - 454 Life Sciences/Roche and reversible

terminator sequencing-by-synthesis by Illumina), the origin of a novel HCV

genotype transiently detectable during antiviral therapy (pre-existing minor

population vs. de novo superinfection). Secondly, we compared 5' untranslated

region (5'-UTR) variants obtained by the two NGS approaches. 5' UTR amplification

products from 9 samples collected from genotype 1b infected patient before,

during, and after treatment (4 serum and 5 peripheral blood mononuclear cell -

PBMC - samples) were subjected to the next-generation sequencing. The sequencing

revealed the presence of two (454/Roche) and one (Illumina) genotype 4 variants

in PBMC at Week 16. None of these variants were present either in the preceding

or following samples as revealed by both platforms. 454/Roche sequencing detected

24 different 5'-UTR variants: 8 were present in serum and PBMC, 4 only in serum

and 12 only in PBMC. Illumina sequencing detected 11 different 5'-UTR variants: 5

in serum and PBMC, 4 only in serum and 2 only in PBMC. Six variants were

identical for both sequencing platforms. The difference in variants number was

primarily due to variability in two 5'-UTR homopolymeric regions. In conclusion,

longitudinal analysis of HCV variants, employing two independent deep sequencing

methods, suggests that the transient presence of a different genotype strain in

PBMC was a result of superinfection and not a selection of pre-existing minor

variant.

 

DOI: 10.1007/5584_2015_186

PMID: 26747069  [PubMed - indexed for MEDLINE]

 

 

3. 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.

 

Perlejewski K(1), Popiel M(2), Laskus T(3), Nakamura S(4), Motooka D(5), Stokowy

T(6), Lipowski D(7), Pollak A(8), Lechowicz U(9), Caraballo Cortés K(10), Stępień

A(11), Radkowski M(12), Bukowska-Ośko I(13).

 

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.

 

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.

 

Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

 

DOI: 10.1016/j.jviromet.2015.09.010

PMID: 26424618  [PubMed - indexed for MEDLINE]

 

 

4. J Clin Microbiol. 2014 Oct;52(10):3722-30. doi: 10.1128/JCM.01641-14. Epub 2014

Aug 6.

 

Exploring the potential of next-generation sequencing in detection of respiratory

viruses.

 

Prachayangprecha S(1), Schapendonk CM(2), Koopmans MP(3), Osterhaus AD(4),

Schürch AC(2), Pas SD(2), van der Eijk AA(2), Poovorawan Y(1), Haagmans BL(2),

Smits SL(5).

 

Author information:

(1)Center of Excellence in Clinical Virology, Department of Pediatrics,

Chulalongkorn University and Hospital, Bangkok, Thailand. (2)Department of

Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands. (3)Department of

Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands Virology

Division, Centre for Infectious Diseases Research, Diagnostics and Screening,

National Institute for Public Health and the Environment, Bilthoven, the

Netherlands. (4)Department of Viroscience, Erasmus Medical Center, Rotterdam, the

Netherlands Viroclinics Biosciences, Rotterdam, the Netherlands. (5)Department of

Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands Viroclinics

Biosciences, Rotterdam, the Netherlands s.smits@erasmusmc.nl.

 

Efficient detection of human respiratory viral pathogens is crucial in the

management of patients with acute respiratory tract infection.

Sequence-independent amplification of nucleic acids combined with next-generation

sequencing technology and bioinformatics analyses is a promising strategy for

identifying pathogens in clinical and public health settings. It allows the

characterization of hundreds of different known pathogens simultaneously and of

novel pathogens that elude conventional testing. However, major hurdles for its

routine use exist, including cost, turnaround time, and especially sensitivity of

the assay, as the detection limit is dependent on viral load, host genetic

material, and sequencing depth. To obtain insights into these aspects, we

analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with

respiratory disease for the presence of respiratory viruses using a

sequence-independent next-generation sequencing approach and routinely used

diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With

respect to the detection of rhinovirus and human metapneumovirus, the

next-generation sequencing approach was at least as sensitive as diagnostic

real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus,

next-generation sequencing was less sensitive than real-time RT-PCR. The

advantage of the sequencing approach over real-time RT-PCR was the immediate

availability of virus-typing information. Considering the development of

platforms capable of generating more output data at declining costs,

next-generation sequencing remains of interest for future virus diagnosis in

clinical and public health settings and certainly as an additional tool when

screening results from real-time RT-PCR are negative.

 

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

 

DOI: 10.1128/JCM.01641-14

PMCID: PMC4187785

PMID: 25100822  [PubMed - indexed for MEDLINE]

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