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.

Keywords: Oxford Nanopore Sequencing; bovine leukemia virus; phylogenetics; proviral load; retroviral evolution; targeted sequencing.

Figure 1

DNA dot plot matrix showing 100% similarity between sequences derived from Oxford Nanopore Technology (ONT) and Scheme 16s rDNA (A) or Thioredoxin gene (B) from two different mastitis-causing pathogens. (A): Corynebacterium bovis and (B): Mycoplasma bovis.

Figure 2

Experimental workflow for inter-laboratory technical replication of long-range BLV genome PCR amplification and Oxford Nanopore Sequencing.

Figure 3

Electrophoretograms showing the amplification, size, and integrity of BLV amplicons used for Oxford Nanopore Sequencing developed by the UMN and CentralStar methods. The coverage and number of trimmed reads used for mapping to the BLV genome is provided below each lane corresponding to the sample. Samples from CentralStar and UMN laboratories are appended with the letters “CS” and “UMN” respectively following the dam or daughter pair number. NTC: Non-Template Control.

Figure 4

Phylogenetic tree of BLV genomes derived from amplicons generated in the CentralStar and University of Minnesota laboratories displaying vertical and horizontal transmission between the dam-daughter pairs. Trees were inferred using maximum likelihood methods and supported with bootstrap value above 70. Different pairs are shown in different colors. Samples from CentralStar and UMN laboratories are appended with the letters “CS” and “UMN” respectively following the dam or daughter pair number.

Figure 5

Phylogenetic tree of env gene derived from sequences generated from the CentralStar laboratory. Different pairs are shown in different colors. Trees were inferred using maximum likelihood methods and supported with bootstrap value above 70.