Assessment of Rapid MinION Nanopore DNA Virus Meta-Genomics Using Calves Experimentally Infected with Bovine Herpes Virus-1

Abstract

Bovine respiratory disease (BRD), which is the leading cause of morbidity and mortality in cattle, is caused by numerous known and unknown viruses and is responsible for the widespread use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines. Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer and sequence analysis with its associated user-friendly point-and-click Epi2ME cloud-based pathogen identification software has the potential for point-of-care/same-day/sample-to-result metagenomic sequence diagnostics of known and unknown BRD pathogens to inform a rapid response and vaccine design. We assessed this potential using in vitro viral cell cultures and nasal swabs taken from calves that were experimentally challenged with a single known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive optimisation of the standard Oxford Nanopore library preparation protocols, particularly a reduction in the PCR bias of library amplification, was required before BoHV-1 could be identified as the main virus in the in vitro cell cultures and nasal swab samples within approximately 7 h from sample to result. In addition, we observed incorrect assignment of the bovine sequence to bacterial and viral taxa due to the presence of poor-quality bacterial and viral genome assemblies in the RefSeq database used by the EpiME Fastq WIMP pathogen identification software.

Can the Revolution in mRNA-Based Vaccine Technologies Solve the Intractable Health Issues of Current Ruminant Production Systems?

“…Similarly, with appropriate monitoring systems, it may be feasible to formulate vaccines in an informed manner, similar to the use of autologous vaccines in some livestock industries. Underpinning the application of vaccines in this manner could be the emergence of next-generation sequencing technologies to rapidly inform the most appropriate pathogen repertoire for the feedlot of interest [ 166 , 167 , 168 ]. When combined with other types of genetic testing, pathogen detection via these emerging technologies has the potential to inform vaccine coverage in a highly cost-effective manner [ 169 ].…”

Genome Sequence of an Ungulate Tetraparvovirus 1 from a Nasal Swab of an Irish Beef-Suckler Calf

“…Prior to nucleic acid extraction, 1× phosphate-buffered saline (PBS) was added to the nasal swab and vortexed, 300 μL was removed, and then bead beating and RNase and DNase treatment steps were performed ( 6 ). Nucleic acids were extracted using the QIAamp MinElute virus spin kit (Qiagen, Manchester, UK) with carrier RNA substituted with 5 mg/mL linear acrylamide (ThermoFisher).…”

Whole blood transcriptome analysis in dairy calves experimentally challenged with bovine herpesvirus 1 (BoHV-1) and comparison to a bovine respiratory syncytial virus (BRSV) challenge

“…Foetal calf lung (FCL) primary cells were grown in a T75 tissue culture flask in 2% Glasgow minimal essential medium (G-MEM). A 1: 100 concentration solution of BoHV-1 strain 2011-415 (Esnault et al, 2022) was prepared in 2% G-MEM. The BoHV-1 dilution (5 mL) was added to the FCL cells in the T75 flask and incubated in a CO 2 incubator at 37 °C for 90 min.…”

“…Animals were restrained in a calf-restraining chute and the head was held to prevent movement during intranasal atomisation. In a previous study by our group (Esnault et al, 2022) we examined viral metagenomic sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer. We assessed in vitro viral cell cultures and nasal swabs taken from the same calves as employed in the current study that were experimentally challenged with BoHV-1.…”