title: A sensitive assay for virus discovery in respiratory clinical samples.
abstract: In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Michel
authors.initials: M
authors.lastname: de Vries
authors.forename: Martin
authors.initials: M
authors.lastname: Deijs
authors.forename: Marta
authors.initials: M
authors.lastname: Canuti
authors.forename: Barbera D C
authors.initials: BD
authors.lastname: van Schaik
authors.forename: Nuno R
authors.initials: NR
authors.lastname: Faria
authors.forename: Martijn D B
authors.initials: MD
authors.lastname: van de Garde
authors.forename: Loes C M
authors.initials: LC
authors.lastname: Jachimowski
authors.forename: Maarten F
authors.initials: MF
authors.lastname: Jebbink
authors.forename: Marja
authors.initials: M
authors.lastname: Jakobs
authors.forename: Angela C M
authors.initials: AC
authors.lastname: Luyf
authors.forename: Frank E J
authors.initials: FE
authors.lastname: Coenjaerts
authors.forename: Eric C J
authors.initials: EC
authors.lastname: Claas
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
authors.forename: Sylvie M
authors.initials: SM
authors.lastname: Koekkoek
authors.forename: Christine
authors.initials: C
authors.lastname: Lammens
authors.forename: Frank
authors.initials: F
authors.lastname: Leus
authors.forename: Herman
authors.initials: H
authors.lastname: Goossens
authors.forename: Margareta
authors.initials: M
authors.lastname: Ieven
authors.forename: Frank
authors.initials: F
authors.lastname: Baas
authors.forename: Lia
authors.initials: L
authors.lastname: van der Hoek
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D003948:Diagnostic Techniques, Respiratory System / Q000592:standards*; D059014:High-Throughput Nucleotide Sequencing; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D012335:RNA, Ribosomal