Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses

Abstract

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts

“…Briefly, three different protocols were used: (i) dsDNA was fragmented by ultrasound with Bioruptor (Diagenode), libraries were prepared using NEXTflex PCR-Free DNA-Seq kit (Bioo Scientific), and then sequenced using an 100 or 150 nucleotides single-end strategy on the HiSeq2500 platform or a 2 x 300 nucleotides paired-end strategy on the MiSeq platform, (ii) dsDNA was fragmented by NEBNext dsDNA fragmentase (New England Biolabs), libraries were prepared using NEBNext Ultra DNA Library Prep kit (New England Biolabs) and sequenced using an 100 nucleotides single-end strategy on the NextSeq500 platform, and (iii) dsDNA libraries were constructed using Nextera XT kit (Illumina) and sequenced using a 2 x 150 nucleotides paired-end strategy on the NextSeq500 platform. For nine remaining isolates (S1 Table), the viral RNAs were reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and then amplified using the whole-transcription amplification (WTA) protocol (QuantiTect Whole Transcriptome kit; Qiagen) as previously described [70]. dsDNA was fragmented by ultrasound, libraries were prepared using TruSeq protocol (Illumina) and sequenced using an 100 nucleotides single-end strategy on the HiSeq2000 platform.…”

Evaluation of High-Throughput Sequencing for Identifying Known and Unknown Viruses in Biological Samples

“…An Affymetrix PathogenID RMA, optimized for the detection and sequence determination of rhabdoviruses, was used as described previously (11).…”

“…Although DNA arrays can substantially increase the number of targets that can be explored simultaneously (24,26,36), currently only resequencing microarrays (RMA; Affymetrix) are able to identify divergent viral strains, by virtue of their large set of probes with mismatches to account for strain diversity. Both technologies, however, still suffer from a sensitivity lower than that of optimized (quantitative) (RT-)PCRs (3), even if this sensitivity does allow the detection of viruses present at high load in biological fluids and tissues (8,9,11). Some subtractive techniques, such as representational difference analysis (RDA), have allowed identification of previously unknown viruses, such as human herpesvirus type 8 (HHV8) (7), human GB virus (31), torque teno virus (TTV) (22), and bocavirus (2).…”

Lyssavirus Detection and Typing Using Pyrosequencing

“…Recently, microarray protocols for the detection of lyssaviruses have been able to demonstrate a high species-level concordance with standard reference assays (7,18). Although potentially suitable for a diagnostic purpose, the method is still not used on a routine basis.…”