Dr.med.vet. thesis by Jakub Kubacki
Animal health in the wake of intensive globalization, climate change and globetrotting is challenged by an increasing number of viral diseases often caused by emerging, re-emerging or novel viral variants. Classical methods such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) are limited to circumscribed groups of known pathogens, require careful selection and a targeted approach. Therefore, in cases of disease outbreaks where standard routine diagnostics fail to identify the infective agent, next generation sequencing (NGS) technology enables to identify and discover viruses without the need to target a specific infectious agent because all viruses present in a sample are sequenced, the so-called virome. However, there is still a lack of applicable NGS protocols fitted to veterinary diagnostics purposes. Here, we present an animal sample preparation protocol for high-throughput metagenomic virus sequencing.
In order to establish a NGS protocol applicable in veterinary diagnostics, several points have to be attained. The protocol should be sensitive, fast, cost-efficient and adaptable to diverse samples materials and species. In a first step, we focused on the sample preparation. Parameters such as filter pore size, nuclease treatment, extraction method and amplification cycle numbers were tested and compared using samples spiked with RNA and DNA viruses. Effectiveness of each method for removal of host nucleic acid and preservation of viral genomes was measured by specific real-time (RT)-PCR and DNA concentration measurements on Qubit Fluorometer.
Analysis of the sequence data showed that enrichment for virus particles such as filtration, centrifugation and nuclease treatment as well as amplification have significant influence on the number of viral genomic sequences detected. In a second step, the enrichment protocol was tested in further sequencing runs, using clinical samples collected at different clinics of the Tierspital (University of Zurich) from several species with or without clinical symptoms. In all cases, NGS data reflected nicely the results of previously performed specific (RT)-PCR results. In addition, it provided full-length genome sequences of two hepatitis E virus genotype 3 (HEV-3) isolates, porcine circovirus type 2 (PCV-2) and all 7 segments of swine influenza virus (H1N1). In other cases, where previous tests were negative, new or unexpected viruses where detected such as Torque teno sus virus in the brain of a pig with neurological signs. In conclusion, the present study set the basis for the development of a diagnostically applicable metagenomic sequencing protocol. And indeed, after an extensive validation and testing phase it is now available from out diagnostic unit as ViroScreen (PDF, 376 KB)and represents the first publicly available NGS-based diagnostic tool for veterinary virology in Switzerland.