Optimisation of rapid untargeted nanopore DNA virus metagenomics using cell cultures and calves experimentally infected with bovine herpes virus-1

Abstract Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle in Ireland, and internationally. This disease is caused by many well-known, and an ever-increasing number of newly associated viruses and bacteria. Consequently, diagnosis of BRD pathogens by targeted real-time polymerase chain reaction (qPCR) diagnostics is too expensive and slow to enable a same-day response that is targeted at the causative pathogen(s). To address this, we developed a same-day, sample to result, untargeted metagenomic MinION sequencing protocol for the identification of DNA viruses associated with BRD from nasal swabs. The procedure comprises non-viral nucleic acid depletion, nucleic acid extraction, rapid transposase-based tagmentation with barcoded adapters, non-biased PCR amplification of tagmented nucleic acid, sequencing on a MinION device, then rapid analysis of resulting sequences on cloud-based software EPI2ME WIMP. The protocol was developed using BoHV1-infected foetal lung cell cultures where we achieved 96% enrichment of the BoHV-1 sequence. Subsequently, the protocol was successfully applied to untargeted detection of BoHV-1 in nasal swabs from calves experimentally challenged with BoHV-1.