Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome
Abstract
:1. Introduction
2. Results
2.1. ONT Sequencing of Long-Range PCR Amplicons from TOPO ® Vectors Containing Bacterial Inserts
2.2. ONT Sequencing of BLV Whole Genome Sequences
2.3. Phylogenetic Analysis of BLV Whole Genome Amplicons
2.4. Amino Acid Substitution among BLV Positive Animals
3. Discussion
3.1. Fidelity of Approach
3.2. Reflection of Findings and Implications for the BLV Field
3.3. Application
3.4. Limitations to This Pilot Study
4. Materials and Methods
4.1. Sample Collection
4.2. BLV Antibodies
4.3. Proviral Load Diagnosis of BLV-Infected Animals
4.4. High Molecular Weight DNA Isolation
4.5. BLV Whole Genome PCR Amplification
4.5.1. CentralStar BLV Whole Genome PCR
4.5.2. University of Minnesota Veterinary Diagnostic Laboratory (MVDL) BLV Whole Genome PCR
4.6. Use of Vectors Containing Bacterial Inserts as Positive Sequencing Controls
4.7. Bead Clean Up and Library Preparation for Oxford Nanopores Sequencing
4.8. Bioinformatic Analysis of Oxford Nanopore Sequencing and Consensus Sequence Generation
4.9. Phylogenetic and Amino Acid Analysis of BLV Whole-Genome Consensus Sequences
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Direction | Sequence | Length | Tm | %GC | Accession |
---|---|---|---|---|---|---|
BLV_CS | Forward | 5′-AACCTTCTGCAAAGCGCGCAAA-3′ | 22 | 68 | 50 | AF033818 |
Reverse | 5′-AAGGCGGGAGAGCCATTCATTTTC-3 | 24 | 67 | 50 | ||
BLV_UMN | Forward | 5′-ATTGATCACCCCGGAACCCTAAC-3′ | 23 | 66 | 52 | AP019598.1 |
Reverse | 5′-CTCAAAAAAGGCGGGAGAGCCATTC-3′ | 25 | 68 | 52 | ||
Topo-BLV | Forward | 5′-AACCTTCTGCAAAGCGCGCAAA GCGGCCAACTTACTTCTGACAAC-3′ | 45 | 70.1 | 51.1 | PCR ®2.1-TOPO ® |
Reverse | 5′-AAGGCGGGAGAGCCATTCATTT AATAGTGTATGCGGCGACCGAGT-3′ | 45 | 69.7 | 51.1 | ||
M. bovis | Forward | 5′-GGTTAATTCTATGCCCAGCATT-3′ | 22 | 60 | 41 | CP058514 |
Reverse | 5′- TTCAGCTTCAATTGCATCCAC-3′ | 21 | 60 | 43 | ||
C. bovis | Forward | 5′-GTGCTTTAGTGTGTGCGGTGG-3′ | 21 | 62 | 57 | NZ_AENJ01000027 |
Reverse | 5′-CGTGTCTCAGTCCCAATGTGG-3′ | 21 | 60 | 57 |
Pair | Relationship | Animal ID | Date of Birth | Age (MO) | Lactaion | PVl | PVl Category | ELISA OD |
---|---|---|---|---|---|---|---|---|
1 | Daughter | 1437 | 25 June 2017 | 39 | 1 | 0.91 | High | 2.799 |
Dam | 1182 | 11 June 2015 | 63 | 3 | 1.02 | High | 2.626 | |
2 | Daughter | 1479 | 11 November 2017 | 34 | 1 | 0.37 | Low | 2.5 |
Dam | 1118 | 29 November 2014 | 70 | 4 | 0.71 | Low | 1.287 | |
3 | Daughter | 1503 | 21 December 2017 | 33 | 1 | 0.39 | Low | 1.704 |
Dam | 980 | 6 October 2013 | 84 | 5 | 0.41 | Low | 2.266 | |
4 | Daughter | 1283 | 10 February 2016 | 55 | 2 | 1.18 | High | 1.56 |
Dam | 909 | 27 February 2013 | 91 | 5 | 0.58 | Low | 2.559 | |
5 | Daughter | 1523 | 2 February 2018 | 32 | 1 | 0.86 | High | 1.48 |
Dam | 1280 | 2 February 2016 | 56 | 3 | 1.18 | High | 0.705 | |
6 | Daughter | 1560 | 22 May 2018 | 28 | 0 | 0.41 | Low | 0.893 |
Dam | 1303 | 27 March 2016 | 54 | 2 | 0.01 | Low | 0.5 | |
7 | Daughter | 1513 | 3 January 2018 | 33 | 1 | 0.04 | Low | 0.768 |
Dam | 1209 | 15 August 2015 | 61 | 3 | 0.87 | High | 2.335 | |
8 | Daughter | 1606 | 4 April 2018 | 25 | 0 | 0.32 | Low | 2.793 |
Dam | 1176 | 23 May 2015 | 64 | 3 | N/A | Negative | 2.476 |
Protein and Amino Acid Locations | SNP Position | Sample ID | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Daughter 1_CS | Dam 1_CS | Daughter 8_CS | Daughter 2_CS | Dam 2_CS | Daughter 3_CS | Dam 3_CS | Daughter 4_CS | Dam 4_CS | Daughter 5_CS | Dam 5_CS | Daughter 6_CS | Dam 6_CS | Daughter 7_CS | Dam 7_CS | ||
G4 (26-105) | 71 | D | N | D | D | D | D | D | D | D | D | D | D | D | D | D |
GAG PRO POLY PROTEIN (31-1417) | 465 | W | W | W | W | W | W | W | W | W | R | R | W | W | W | W |
642 | I | I | I | I | I | I | I | I | I | I | I | I | I | I | V | |
1162 | E | G | E | E | E | E | E | E | E | E | E | E | E | E | E | |
1173 | G | G | G | R | R | G | G | G | G | G | G | G | G | G | G | |
REX (1-56) | 48 | S | S | S | S | S | S | S | S | S | S | S | S | F | S | F |
140 | L | R | L | L | L | L | L | L | L | L | L | L | R | L | R | |
R3 (1-44) | No SNPS | |||||||||||||||
EPP (1-515) | 31 | I | I | I | I | I | I | I | I | I | I | I | T | I | I | I |
475 | H | H | H | P | P | H | H | H | P | P | P | H | H | H | H | |
479 | L | L | V | L | L | L | L | V | L | L | L | L | L | V | L | |
TAX 1-309 | 182 | T | T | T | T | T | A | A | T | T | T | T | T | T | T | T |
AS1 1-87 | 3 | P | S | P | P | P | P | P | P | P | P | P | P | P | P | P |
31 | G | G | G | E | G | G | G | G | G | G | G | G | G | G | G |
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Pavliscak, L.A.; Nirmala, J.; Singh, V.K.; Sporer, K.R.B.; Taxis, T.M.; Kumar, P.; Goyal, S.M.; Mor, S.K.; Schroeder, D.C.; Wells, S.J.; et al. Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome. Pathogens 2021, 10, 1191. https://doi.org/10.3390/pathogens10091191
Pavliscak LA, Nirmala J, Singh VK, Sporer KRB, Taxis TM, Kumar P, Goyal SM, Mor SK, Schroeder DC, Wells SJ, et al. Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome. Pathogens. 2021; 10(9):1191. https://doi.org/10.3390/pathogens10091191
Chicago/Turabian StylePavliscak, Laura A., Jayaveeramuthu Nirmala, Vikash K. Singh, Kelly R. B. Sporer, Tasia M. Taxis, Pawan Kumar, Sagar M. Goyal, Sunil Kumar Mor, Declan C. Schroeder, Scott J. Wells, and et al. 2021. "Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome" Pathogens 10, no. 9: 1191. https://doi.org/10.3390/pathogens10091191
APA StylePavliscak, L. A., Nirmala, J., Singh, V. K., Sporer, K. R. B., Taxis, T. M., Kumar, P., Goyal, S. M., Mor, S. K., Schroeder, D. C., Wells, S. J., & Droscha, C. J. (2021). Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome. Pathogens, 10(9), 1191. https://doi.org/10.3390/pathogens10091191