Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories.

A breakthrough genetic testing method, which is as sensitive as the gold standard polymerase chain reaction assays, enables simultaneous testing for hundreds of different viruses and provides near complete sequence of their genomes promises to give clinicians a powerful new tool to detect and sequence viruses.

Scientists at Columbia University (New York, NY, USA) generated facsimiles of clinical specimens in a background of nucleic acid (NA) extracted from normal human lung tissue, EDTA-blood, or serum. The samples were spiked with viral NA and quantitated by virus-specific TaqMan real-time (reverse transcription) PCR (qPCR). They developed the Virome-Capture-Sequencing platform for Vertebrate viruses (VirCapSeq-VERT) which enables simultaneous testing for hundreds of different viruses and providing near complete sequence of their genomes.

Clinical samples included a human nasal swab sample known to be positive for Enterovirus D68 (EV-D68) and serum samples from hemophilia patients co-infected with Hepatitis C virus (HCV), GB virus C (GBV-C), human immunodeficiency virus (HIV), and torque teno virus (TTV). NA from cell culture or blood, serum, or tissue samples was extracted using the easyMAG system (bioMérieux; Marcy l’Etoile, France) or AllPrep DNA/RNA kits (Qiagen; Hilden, Germany). The PCR products were purified using Agencourt Ampure DNA purification beads (Beckman Coulter; Brea, CA, USA) and quantitated for Illumina sequencing.

The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero ribosomal ribonucleic acid (rRNA) subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics.

Thomas Briese, PhD, a professor and lead author of the study, said, “If you have patients you suspect has a viral disease, you can now for a very reasonable amount of money, definitively characterize all the viruses present in those individuals in order to figure out how they should be treated.” VirCapSeq-VERT costs approximately USD 40 when testing for 20 patients/samples, comparing favorably with other procedures like rRNA depletion which cost approximately USD 65 per sample. The study was published on September 22, 2015, in the journal mBIO.

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