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.

Figure 1. Enhanced viral RNA amplification in VIDISCA using non-ribosomal hexamers during reverse transcription.

VIDISCA fragments are visualized on a 3% metaphor gel. A dilution series of echovirus 18 was used and the concentration per ml is indicated above each lane. NC = negative PBS control, M = 25 bp marker.(a) VIDISCA products were generated with primers Hinp-A/Mse-C. The viral fragments are 167 bp, 296 bp and 382 bp in size. (b) VIDISCA products amplified with primers Hinp-A/Mse-A. The product originating from rRNA (70 bp) is indicated by an arrow.

Figure 2. Enhanced amplification of viral fragments using one restriction enzyme in VIDISCA.

Visualization of VIDISCA fragments digested with HinP1-I+MseI or MseI alone. VIDISCA fragments are visualized on a 1% agarose gel, which were generated after a single first round PCR of 40 cycles. The dots indicate viral fragments which were only visible with MseI digestion.

Figure 3. rRNA-blocking oligo's decrease rRNA-cDNA synthesis in VIDISCA.

VIDISCA fragment of ribosomal RNA visualized on a 3% metaphor gel. A nasopharyngeal washing was used as input for VIDISCA with or without blocking oligo's. Lane 1 and 2 are without blocking oligo's whereas lane 3 and 4 are with blocking oligo's, M = 25 bp marker. The arrow indicates the rRNA fragment of which the amplification was decreased.

Figure 4. Schematic overview of VIDISCA-454.