Abstract
Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state.
Highlights
Deep nucleotide sequencing, a process by which a genomic locus is sequenced multiple times, is currently changing the field of clinical virology by providing a platform for the unbiased, broad-spectrum detection of viruses without requiring an a priori hypothesis for the source of infection [1, 2, 3]
Viral genomes are orders of magnitude smaller compared to the human genome and through deep whole genome sequencing of clinical samples only a small fraction of the sequencing information is related to viral genomes
The ViroFind probes are used in a hybridization reaction to enrich viral sequences out of all the nucleotide sequences present in a clinical sample and enhance the detection of viruses via deep sequencing
Summary
A process by which a genomic locus is sequenced multiple times, is currently changing the field of clinical virology by providing a platform for the unbiased, broad-spectrum detection of viruses without requiring an a priori hypothesis for the source of infection [1, 2, 3]. The use of deep sequencing for virus detection and discovery in clinical samples is challenged by low cost-effectiveness. To make deep sequencing of viral genomes more cost-effective, we designed a target-enrichment platform, which we named ViroFind. The ViroFind probes are used in a hybridization reaction to enrich viral sequences out of all the nucleotide sequences present in a clinical sample and enhance the detection of viruses via deep sequencing
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