A Dissertation Entitled Evolutionary Patterns and Occurrences of The
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A Dissertation entitled Evolutionary Patterns and Occurrences of the fish Viral Hemorrhagic Septicemia Virus in the Laurentian Great Lakes by Megan Denise Niner Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology (Ecology) ___________________________________________ Dr. Carol A. Stepien, Committee Chair ___________________________________________ Dr. Douglas Leaman, Committee Member ___________________________________________ Dr. Daryl Moorhead, Committee Member ___________________________________________ Dr. Vikram Vakharia, Committee Member ___________________________________________ Dr. William Sigler, Committee Member ___________________________________________ Dr. Cyndee Gruden, Dean College of Graduate Studies The University of Toledo August 2019 Copyright © 2019, Megan Denise Niner Chapters 1 and 4 of this document are copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Evolution and Presence of Viral Hemorrhagic Septicemia Genogroup IVb in the Great Lakes by Megan Denise Niner Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology (Ecology) The University of Toledo August 2019 Studying naturally occurring viruses outside of the lab can provide insight into the different evolutionary pathways between hosts and pathogens, aiding future predictions for their spread and levels of severity. One such virus is the focus of this dissertation: Viral Hemorrhagic Septicemia virus (VHSV). This global pathogen has four major genogroups, I-IV, among which, our main focus, IVb emerged in the Laurentian Great Lakes in the early 2000s, killing >30 fish species in large outbreaks from 2005-2010. Few detections were reported between 2010 and 2016, however, two minor outbreaks occurred in 2017. Following recovery of 21 new isolates from sampling between 2015- 2017, we compared the genetic composition of these isolates with historical sequences using a combined approach of phylogenetic analysis with population genetics to examine spatial and temporal trends in IVb based on the sequence of the glycoprotein, or G-gene, along with a concatenated five-gene dataset using a reduced number of isolates. The 185 G-gene sequences revealed 36 unique haplotypes, whose relationships evolved from two apparent original haplotypes, designated “a” and “b”. Most haplotypes differed from those by single to a few nucleotides, with “a” and “b” diverging by just one. Prior to iii 2011, “a” and “b” were prevalent, buy have gradually become less frequent over time. The 14 haplotype descendants of “a” primarily occurred in the Upper and Central Great Lakes, and the 22 “b” variants mostly were found in the Lower Great Lakes. Significant genetic divergences characterized the geographic distributions of haplotypes among the Upper, Central, and Lower Great Lakes, and those from the later time period significantly differed from the early and middle time periods. Patterns of nucleotide substitutions corresponded to geographic divergences, whereas amino acid changes characterized temporal trends. We thus found that VHSV-IVb has continued to diversify in the Great Lakes, following spatial and temporal with distinct spatial and temporal patterns. Using genomics, we conducted a phylogenetic comparison of 46 whole IVb genomes, along with comparisons to the North American and Asian VHSV-IVa, and the European I/III genogroups. A total of 253 nucleotide substitutions were found in IVb, with 85 resulting in amino acid changes. Most substititutions occurred in the non-coding region and Nv- genes, which also was true for the IVa and I/III genomes. IVa was estimated to have the fastest substitution rate (2.01x10-3), followed by IVb (6.64x10-5) and I/III (4.09x10-5). Phylogenetic analysis of the novirhabdoviruses based on full genomes agreed with past results that were based on partial genes. We also investigated possible origins of the Nv- gene in VHS, whose results showed sister relationships but were homoplastic beyond that level. Cell culture experiments compared three isolates collected from fish hosts in 2016 to the original 2003 isolate for viral production and host immune response suppression. The 2016 isolates displayed lower virulence and less suppression of the host antiviral response, which suggested that changes in the genome have phenotypic consequences. iv Acknowledgements I would like to thank all those who put their time and energy into this project. To Dr. Stepien for providing me the opportunity and resources to work on this fascinating project. To Dr. Moorhead for handling the complicated logistics of my mostly-absent committee. To Dr. Leaman for letting me work with his lab and helping me understand the cell culture related portions of this project. To my other committee members, Dr. Vakharia and Dr. Sigler, for agreeing to be involved with this project and their support. To Dr. Krishnamurthy for allowing me a space in her lab. To Shelby Edwards, for countless hours in the field, lab and on the road. To my fiancé, parents, sister, coworkers at the LEC and the main campus for not letting me give up. v Table of Contents Abstract……………………………………………………………………………………iii Acknowledgements………………………………………………………………………..v Table of Contents………………………………………………………………………….vi List of Tables………………………………………………………………………………x List of Figures…………………………………………………………………………...xiii Preface……………………………………………………………………………………xx 1. Introduction…………………………………………………………………………….1 2. Evolutionary trajectory of the fish Viral Hemorrhagic Septicemia virus across its history in the Laurentian Great Lakes: Temporal and spatial patterns……………….10 2.1. Abstract…………………………………………………………………..10 2.2. Introduction………………………………………………………………11 2.2.1. VHS evolution, outbreaks, and hosts…………………………….13 2.2.2. Aim and objectives………………………………………………16 2.3. Materials and Methods…………………………………………………..16 2.3.1. Sampling…………………………………………………………16 2.3.2. RNA extraction and reverse transcription………………………..17 2.3.3. qPCR tests for VHSV-IVb and quantification…………………...18 2.3.4. Preparation of historic isolates in cell culture……………………19 2.3.5. Sequencing VHSV isolates………………………………………19 2.3.6. Genetic data analyses…………………………………………….20 2.4. Results ……………………………………………………………………23 2.4.1. VHSV-IVb detections between 2015-2017……………………...23 vi 2.4.2. Evolutionary patterns from the G-gene…………………………..24 2.4.3. Patterns from concatenated gene analyses……………………….26 2.4.4. Population genetic trends………………………………………...27 2.5. Discussion………………………………………………………………..29 2.5.1. VHS-IV occurrences and evolutionary trajectory………………..29 2.5.2. Evolutionary patterns across space and time.……………………30 2.5.3. Substitution rates, types, and patterns..…………………………..32 2.5.4. Gene specific variation…………………………………………..34 2.5.5. Host species generality, specificity, and infection……………….37 2.5.6. Selection and co-evolution……………………………………….41 2.5.7. Summary and conclusions……………………………………….42 2.6. Acknowledgements………………………………………………………42 3. Genomic and immunogenic changes across the evolutionary history of the VHSV-IVb fish virus (Piscine novirhabdovirus) in the Laurentian Great Lakes………………….75 3.1. Abstract…………………………………………………………………..75 3.2. Introduction………………………………………………………………76 3.3. Materials and Methods…………………………………………………...81 3.3.1. Sample nomenclature…………………………………………….81 3.3.2. Virus isolation……………………………………………………81 3.3.3. Full genome sequencing…………………………………………82 3.3.4. Genetic analyses………………………………………………….83 3.3.5. Evaluating evolution and selection………………………………84 3.3.6. Cell lines and cell culture experiments…………………………..85 vii 3.4. Results……………………………………………………………………88 3.4.1. Genomic and genic changes……………………………………...88 3.4.2. Evolutionary relationships……………………………………….91 3.4.3. Differences in cytopathicity and immune response……………...94 3.4.4. IFN and virus production and expression of mRNAS…………...94 3.5. Discussion………………………………………………………………..95 3.5.1. Evolutionary trends………………………………………………95 3.5.2. Phylogenetic patterns: novirhabdoviruses……………………….98 3.5.3. Phylogenetic Patterns: VHSV-IVb………………………………99 3.5.4. Evolutionary Perspectives on the Nv-gene……………………..103 3.5.5. Differences in cytopathogenicity……………………………….103 3.5.6. Summary and conclusions……………………………………...105 3.6. Acknowledgments………………………………………………………105 4. Discussion…………………………………………………………………………...131 4.1. General Conclusions……………………………………………………131 4.1.1. Presence and distribution of VHSV-IVb 2015 to 2017………...132 4.1.2. Recent changes and population genetic trends of IVb………….133 4.1.3. Genomic trends of IVb, with comparisons to IVa and I/III…….135 4.1.4. Genomic characterization of novirhabdoviruses and the origin of the Nv-gene……………………………………………………..136 4.1.5. In vitro comparison of 2016 isolates versus the original 2003 isolate…………………………………………………………...137 4.2. Future Research and Recommendations………………………………..138 viii 4.2.1. Continued surveillance with narrowed focus…………………...138 4.2.2. Resolving the relationship between genogroups Ia and III……..139 4.2.3. Genomics: sequencing of additional samples…………………..139 4.2.4. Pathogenicity of new isolates and changes in host immune suppression……………………………………………………...140 References………………………………………………………………………………141 ix List of Tables Table 2-1 VHSV positives from our collections in 2012, 2015, and 2016. .....................44 Table 2-2 Primers for PCR and sequencing per gene region, with name, reference, sequence, annealing temperature, and extension