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
- Gutiérrez, G.; Rodríguez, S.M.; De Brogniez, A.; Gillet, N.; Golime, R.; Burny, A.; Jaworski, J.P.; Alvarez, I.; Vagnoni, L.; Trono, K.; et al. Vaccination against δ-Retroviruses: The Bovine Leukemia Virus Paradigm. Viruses 2014, 6, 2416–2427. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ladronka, R.M.; Ainsworth, S.; Wilkins, M.J.; Norby, B.; Byrem, T.M.; Bartlett, P.C. Prevalence of Bovine Leukemia Virus Antibodies in US Dairy Cattle. Vet. Med. Int. 2018, 2018, 33–36. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bartlett, P.; Michigan State University. Bovine Leukemia Virus in the U.S.: Impact and Options for Control #1 What Is It? Why Does It Matter? What Options for Control? Where to Start? What Can USAHA Do? Available online: https://blv.msu.edu/resources/USAHA-Oct-16-2017.pdf (accessed on 14 September 2021).
- Gao, A.; Kouznetsova, V.L.; Tsigelny, I.F. Bovine Leukemia Virus Relation to Human Breast Cancer: Meta-Analysis. Microb. Pathog. 2020, 149, 104417. [Google Scholar] [CrossRef] [PubMed]
- Buehring, G.C.; Delaney, A.; Shen, H.; Chu, D.L.; Razavian, N.; Schwartz, D.A.; Demkovich, Z.R.; Bates, M.N. Bovine Leukemia Virus Discovered in Human Blood. BMC Infect. Dis. 2019, 19, 297. [Google Scholar] [CrossRef]
- Aida, Y.; Murakami, H.; Takahashi, M.; Takeshima, S.N. Mechanisms of Pathogenesis Induced by Bovine Leukemia Virus as a Model for Human T-Cell Leukemia Virus. Front. Microbiol. 2013, 4, 328. [Google Scholar] [CrossRef] [Green Version]
- Kuczewski, A.; Mason, S.; Orsel, K.; van der Meer, F. Pilot Implementation of a Newly Developed Bovine Leukemia Virus Control Program on 11 Alberta Dairy Farms. J. Dairy Sci. 2021, 104, 4549–4560. [Google Scholar] [CrossRef]
- Ruiz, V.; Porta, N.G.; Lomónaco, M.; Trono, K.; Alvarez, I. Bovine Leukemia Virus Infection in Neonatal Calves. Risk Factors and Control Measures. Front. Vet. Sci. 2018, 5, 1–7. [Google Scholar] [CrossRef] [Green Version]
- Sajiki, Y.; Konnai, S.; Nishimori, A.; Okagawa, T.; Maekawa, N.; Goto, S.; Nagano, M.; Kohara, J.; Kitano, N.; Takahashi, T.; et al. Intrauterine Infection with Bovine Leukemia Virus in Pregnant Dam with High Viral Load. J. Vet. Med. Sci. 2017, 79, 2036–2039. [Google Scholar] [CrossRef] [Green Version]
- Watanuki, S.; Takeshima, S.N.; Borjigin, L.; Sato, H.; Bai, L.; Murakami, H.; Sato, R.; Ishizaki, H.; Matsumoto, Y.; Aida, Y. Visualizing Bovine Leukemia Virus (BLV)-Infected Cells and Measuring BLV Proviral Loads in the Milk of BLV Seropositive Dams. Vet. Res. 2019, 50, 1–12. [Google Scholar] [CrossRef] [Green Version]
- Mekata, H.; Sekiguchi, S.; Konnai, S.; Kirino, Y.; Honkawa, K.; Nonaka, N.; Horii, Y.; Norimine, J. Evaluation of the Natural Perinatal Transmission of Bovine Leukaemia Virus. Vet. Rec. 2015, 176, 254. [Google Scholar] [CrossRef]
- Juliarena, M.A.; Gutierrez, S.E.; Ceriani, C. Determination of proviral load in bovine leukemia virus–infected cattle with and without lymphocytosis. Am. J. Vet. Res. 2007, 68, 1220–1225. [Google Scholar] [CrossRef] [PubMed]
- Ruggiero, V.J.; Norby, B.; Benitez, O.J.; Hutchinson, H.; Sporer, K.R.B.; Droscha, C.; Swenson, C.L.; Bartlett, P.C. Controlling Bovine Leukemia Virus in Dairy Herds by Identifying and Removing Cows with the Highest Proviral Load and Lymphocyte Counts. J. Dairy Sci. 2019, 102, 9165–9175. [Google Scholar] [CrossRef] [Green Version]
- Rola-Łuszczak, M.; Finnegan, C.; Olech, M.; Choudhury, B.; Kuźmak, J. Development of an Improved Real Time PCR for the Detection of Bovine Leukaemia Provirus Nucleic Acid and Its Use in the Clarification of Inconclusive Serological Test Results. J. Virol. Methods 2013, 189, 258–264. [Google Scholar] [CrossRef] [PubMed]
- Jimba, M.; Takeshima, S.; Matoba, K.; Endoh, D.; Aida, Y. BLV-CoCoMo-QPCR: Quantitation of Bovine Leukemia Virus Proviral Load Using the CoCoMo Algorithm. Retrovirology 2010, 7, 91. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jaworski, J.P.; Pluta, A.; Rola-Łuszczak, M.; McGowan, S.L.; Finnegan, C.; Heenemann, K.; Carignano, H.A.; Alvarez, I.; Murakami, K.; Willems, L.; et al. Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA. J. Clin. Microbiol. 2018, 56. [Google Scholar] [CrossRef] [Green Version]
- Hutchinson, H.C.; Norby, B.; Droscha, C.J.; Sordillo, L.M.; Coussens, P.M.; Bartlett, P.C. Bovine Leukemia Virus Detection and Dynamics Following Experimental Inoculation. Res. Vet. Sci. 2020, 133, 269–275. [Google Scholar] [CrossRef] [PubMed]
- Taxis, T.M.; DeJong, T.N.; Swenson, C.L.; Sporer, K.R.; Droscha, C.; Niles, D.; Bartlett, P.C. Reducing Bovine Leukemia Virus Prevalence on a Large Midwestern Dairy Farm by Using Lymphocyte Counts, ELISA Antibody Testing, and Proviral Load. Bov. Pract. 2020, 54, 136–144. [Google Scholar]
- John, E.E.; Droscha, C.; Cameron, M.; Stryhn, H.; Keefe, G.; McClure, A. Predict. Model for Bov. Leuk. Virus Proviral Load. J.T. Dev. in press.
- Yu, C.; Wang, X.; Zhou, Y.; Wang, Y.; Zhang, X.; Zheng, Y. Genotyping Bovine Leukemia Virus in Dairy Cattle of Heilongjiang, Northeastern China. BMC Vet. Res. 2019, 15. [Google Scholar] [CrossRef]
- Polat, M.; Takeshima, S.S.; Hosomichi, K.; Kim, J.; Miyasaka, T.; Yamada, K.; Arainga, M.; Murakami, T.; Matsumoto, Y.; Barra Diaz, V.; et al. A New Genotype of Bovine Leukemia Virus in South America Identified by NGS-Based Whole Genome Sequencing and Molecular Evolutionary Genetic Analysis. Retrovirology 2016, 13, 1–23. [Google Scholar] [CrossRef] [Green Version]
- Rola-Łuszczak, M.; Sakhawat, A.; Pluta, A.; Ryło, A.; Bomba, A.; Bibi, N.; Kuźmak, J. Molecular Characterization of the Env Gene of Bovine Leukemia Virus in Cattle from Pakistan with NGS-Based. Evid. Virus Heterog. 2021, 10, 910. [Google Scholar] [CrossRef]
- Taylor, T.L.; Volkening, J.D.; DeJesus, E.; Simmons, M.; Dimitrov, K.M.; Tillman, G.E.; Suarez, D.L.; Afonso, C.L. Rapid, Multiplexed, Whole Genome and Plasmid Sequencing of Foodborne Pathogens Using Long-Read Nanopore Technology. Sci. Rep. 2019, 9, 16350. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Green, M.R.; Sambrook, J. Isolation of High-Molecular-Weight DNA from Mammalian Blood Using Proteinase K and Phenol. Cold Spring Harb. Protoc. 2017, 2017. [Google Scholar] [CrossRef] [PubMed]
- Bharucha, T.; Oeser, C.; Balloux, F.; Brown, J.R.; Carbo, E.C.; Charlett, A.; Chiu, C.Y.; Claas, E.C.J.; de Goffau, M.C.; de Vries, J.J.C.; et al. STROBE-Metagenomics: A STROBE Extension Statement to Guide the Reporting of Metagenomics Studies. Lancet Infect. Dis. 2020, 20, e251–e260. [Google Scholar] [CrossRef]
- Goodwin, S.; Gurtowski, J.; Ethe-Sayers, S.; Deshpande, P.; Schatz, M.C.; McCombie, W.R. Oxford Nanopore Sequencing, Hybrid Error Correction, and de Novo Assembly of a Eukaryotic Genome. Genome Res. 2015, 25, 1750–1756. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, L.; Qu, L.; Yang, L.; Wang, Y.; Zhu, H. NanoReviser: An Error-Correction Tool for Nanopore Sequencing Based on a Deep Learning Algorithm. Front. Genet. 2020, 11, 900. [Google Scholar] [CrossRef] [PubMed]
- Magi, A.; Giusti, B.; Tattini, L. Characterization of MinION Nanopore Data for Resequencing Analyses. Brief. Bioinform. 2017, 18, 940–953. [Google Scholar] [CrossRef]
- Pluta, A.; Willems, L.; Douville, R.N.; Kuźmak, J. Effects of Naturally Occurring Mutations in Bovine Leukemia Virus 5′-ltr and Tax Gene on Viral Transcriptional Activity. Pathogens 2020, 9, 836. [Google Scholar] [CrossRef]
- Mcgirr, K.M.; Buehring, G.C. Tax and Rex Sequences of Bovine Leukaemia Virus from Globally Diverse Isolates: Rex Amino Acid Sequence More Variable than Tax. J. Vet. Med. B Infect. Dis. Vet. Public Health 2010, 52, 8–16. [Google Scholar] [CrossRef]
- Tajima, S.; Aida, Y. The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers. J. Virol. 2000, 74, 10939–10949. [Google Scholar] [CrossRef] [Green Version]
- Pluta, A.; Blazhko, N.V.; Ngirande, C.; Joris, T.; Willems, L.; Kuźmak, J. Analysis of Nucleotide Sequence of Tax, Mirna and Ltr of Bovine Leukemia Virus in Cattle with Different Levels of Persistent Lymphocytosis in Russia. Pathogens 2021, 10, 246. [Google Scholar] [CrossRef] [PubMed]
- Moratorio, G.; Fischer, S.; Bianchi, S.; Tomé, L.; Rama, G.; Obal, G.; Carrión, F.; Pritsch, O.; Cristina, J. A Detailed Molecular Analysis of Complete Bovine Leukemia Virus Genomes Isolated from B-Cell Lymphosarcomas. Vet. Res. 2013, 44, 19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, J.; Wang, H.; Mao, L.; Yu, H.; Yu, X.; Sun, Z.; Qian, X.; Cheng, S.; Chen, S.; Chen, J.; et al. Rapid Genomic Characterization of SARS-CoV-2 Viruses from Clinical Specimens Using Nanopore Sequencing. Sci. Rep. 2020, 10, 17492. [Google Scholar] [CrossRef] [PubMed]
- Einsiedel, L.; Woodman, R.J.; Flynn, M.; Wilson, K.; Cassar, O.; Gessain, A. Human T-Lymphotropic Virus Type 1 Infection in an Indigenous Australian Population: Epidemiological Insights from a Hospital-Based Cohort Study. BMC Public Health 2016, 16, 266–281. [Google Scholar] [CrossRef] [Green Version]
- Beyter, D.; Ingimundardottir, H.; Oddsson, A.; Eggertsson, H.; Bjornsson, E.; Jonsson, H.; Atlason, B.; Kristmundsdottir, S.; Mehringer, S.; Hardarson, M.; et al. Long Read Sequencing of 3,622 Icelanders Provides Insight into the Role of Structural Variants in Human Diseases and Other Traits. Nat Genet. 2021, 53, 779–786. [Google Scholar] [CrossRef]
- Lamb, H.J.; Hayes, B.J.; Nguyen, L.T.; Ross, E.M. The Future of Livestock Management: A Review of Real-Time Portable Sequencing Applied to Livestock. Genes 2020, 11, 1478. [Google Scholar] [CrossRef]
- Franco-Sierra, N.D.; Díaz-Nieto, J.F. Rapid Mitochondrial Genome Sequencing Based on Oxford Nanopore Sequencing and a Proxy for Vertebrate Species Identification. Ecol. Evol. 2020, 10, 1–17. [Google Scholar] [CrossRef]
- Li, H. Minimap2: Pairwise Alignment for Nucleotide Sequences. Bioinformatics 2018, 34, 3094–3100. [Google Scholar] [CrossRef]
- Grubaugh, N.D.; Gangavarapu, K.; Quick, J.; Matteson, N.L.; de Jesus, J.G.; Main, B.J.; Tan, A.L.; Paul, L.M.; Brackney, D.E.; Grewal, S.; et al. An Amplicon-Based Sequencing Framework for Accurately Measuring Intrahost Virus Diversity Using PrimalSeq and IVar. Genome. Biol. 2019, 20, 1–19. [Google Scholar] [CrossRef] [Green Version]
- Edge, P.; Bansal, V. Longshot Enables Accurate Variant Calling in Diploid Genomes from Single-Molecule Long Read Sequencing. Nat. Commun. 2019, 10, 1–10. [Google Scholar] [CrossRef] [Green Version]
- Edgar, R.C. MUSCLE: A Multiple Sequence Alignment Method with Reduced Time and Space Complexity. BMC Bioinform. 2004, 5, 113. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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