Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome

Abstract

Bovine leukemia virus (BLV) causes Enzootic Bovine Leukosis (EBL), a persistent life-long disease resulting in immune dysfunction and shortened lifespan in infected cattle, severely impacting the profitability of the US dairy industry. Our group has found that 94% of dairy farms in the United States are infected with BLV with an average in-herd prevalence of 46%. This is partly due to the lack of clinical presentation during the early stages of primary infection and the elusive nature of BLV transmission. This study sought to validate a near-complete genomic sequencing approach for reliability and accuracy before determining its efficacy in characterizing the sequence identity of BLV proviral genomes collected from a pilot study made up of 14 animals from one commercial dairy herd. These BLV-infected animals were comprised of seven adult dam/daughter pairs that tested positive by ELISA and qPCR. The results demonstrate sequence identity or divergence of the BLV genome from the same samples tested in two independent laboratories, suggesting both vertical and horizontal transmission in this dairy herd. This study supports the use of Oxford Nanopore sequencing for the identification of viral SNPs that can be used for retrospective genetic contact tracing of BLV transmission.

Phaeoviruses found in recovering Nereocystis luetkeana kelp forest community

“…The reads obtained were reference assembled against the known MCP phaeoviral genes (McKeown et al, 2017) using Minimap 2.17 in Geneious Prime 2021.1.1 and manually curated to correct ambiguities where possible. Fidelity of the PCR plus ONT approach was previously confirmed using a bovine leukemia virus model (Pavliscak et al, 2021).…”

Identification of BoLA Alleles Associated with BLV Proviral Load in US Beef Cows

“…DNA quantity and quality was determined using the NanoDrop One/One c (ThermoFisher Scientific, Austin, TX, USA). The methods to determine PVL followed Pavliscak et al, 2020 [ 24 ], and PVL was reported as a ratio of BLV polymerase gene copies to Beta Actin gene copies.…”

Nanopore sequencing technology and its applications

“…Animal virus infections can spread rapidly and have severe socioeconomic consequences. Nanopore sequencing has been used to identify various animal viruses, including differentiating between three Capripox Viruses in outbreaks in the Middle East and Africa, 277 identifying African horse sickness in an outbreak in Thailand, 278 and tracking Enzootic Bovine Leukosis caused by Bovine leukemia virus transmission in US pastures 279 . It has also been used to reveal the complex transcriptional landscape of porcine reproductive and respiratory syndrome virus 280 …”