This report was prepared for the New York State Water Resources Institute (NYSWRI) and the Viral Hemorrhagic Septicemia Virus genetic diversity among invasive and native reservoir fishes of the Thousand Islands region, St. Lawrence River John M. Farrel l ( jmfarrel l@esf.edu) Anna L. Haws (alconkly@syr .edu) Rodman G. Getchel l ( rgg4@cornel l .edu) hn M. Farrel l ( jmfarrel l@esf.edu) Anna L. Haws (alconkly@esf.edu) Rodman G. Getchel l (RGG4 @esf.edu) PREPARED BY: John M. Farrell Professor/Director, Thousand Islands Biological Station SUNY College of Environmental Science and Forestry, Syracuse, NY Anna L. Haws (Conklyn) Ph.D. Candidate SUNY College of Environmental Science and Forestry, Syracuse, NY Rodman G. Getchell Assistant Research Professor, Aquatic Animal Health Program Cornell University College of Veterinary Medicine, Ithaca, NY PREPARED BY: John M. Farrell Professor/Director, Thousand Islands Biological Station SUNY College of Environmental Science and Forestry, This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health with support from the NYS Environmental Protection Fund 2022 \.\. U_N/ v~ l ~~ ""' to 0 lb ,tl>ED 1'·0• mailto:jmfarrell@esf.edu mailto:alconkly@syr.edu mailto:jmfarrell@esf.edu mailto:alconkly@esf.edu 2 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. › 02 Abstract and Summary › 02 Introduction › 02 Methods › 02 Results and Discussion › 02 Policy Implications › 02 Student Training › 02 References › 02 Appendices Abstract and Summary…………………………………………………………………………………..…………3 Policy Implications……………….……………………………………………………………………….…………3 Introduction………………………………………………………………………………………………….…………4 Methods……………………………………………………………………………….………………………………….6 Results and Discussion……………………………………………………………………………………………10 Student Training………………………………………………………………………………………………….….19 References……………………………………………………………………………………..……………………….19 Appendices…………………………………………………………………………………………………………….20 Contents VHSV-positive muskellunge mortality recovered from the St. Lawrence River near Waddington, NY during April 2022. This specimen was sequenced as part of this study. collected for this study. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 3 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Viral Hemorrhagic Septicemia Virus (VHSV), is a World Organization for Animal Health (OIE) reportable fish pathogen found across the northern hemisphere, and lineage VHSV-IVb was first detected in the Great Lakes basin following sizable fish kills in the mid-2000’s. The biological and environmental factors that promote VHSV persistence following establishment are not well understood. Therefore, this study is investigating annual, spatial, and host species patterns of viral genetic structure in the upper St. Lawrence River (USLR), an ecosystem where VHSV has persisted for 20 years. The USLR supports large populations of invasive round gobies, and these fish are critical to provide an environment suitable to rapid viral reproduction, evolution of new viral isolates, and dissemination of those isolates to the native community. Keywords Invasive Species, Pathogen, Conservation Policy Implications Foremost, this study contributes to assessing environmental considerations for a long-term sportfish restoration program targeted at identifying and protecting muskellunge spawning and nursery sites in the St. Lawrence River. This research also contributes to the greater knowledge regarding VHSV in the Laurentian Great Lakes watershed and provides additional evolutionary context to evaluate the epidemiology of this fish pathogen. Finally, this study represents the final chapter in Mrs. Haws’ dissertation to achieve her Doctor of Philosophy degree in Fish & Wildlife Biology and Management from the SUNY College of Environmental Science and Forestry. Executive Summary Summary Points of Interest 1. Invasive round gobies harbor VHSV, and are essential for the amplification, spread, and evolution of the virus in the St. Lawrence River. Current VHSV sequence types have caused mortality in the valuable muskellunge population. 2. The genetic composition of VHSV that exists in the St. Lawrence River is substantially diverged from the original reference strain, illustrating this virus’ capacity to exploit mutation to persist in the environment. 3. Genetic haplotypes are capable of sustaining infections for extended periods of time and dispersing throughout the St. Lawrence River. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 4 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Introduction Piscine novirhabdovirus, or Viral Hemorrhagic Septicemia Virus (VHSV), is a World Organization for Animal Health (OIE) reportable fish pathogen found in marine, estuarine, and freshwater ecosystems across the northern hemisphere (OIE, 2019). A novel lineage, VHSV- IVb, was detected in the Great Lakes basin following sizable fish kills in the mid-2000’s. Through IVb’s history in the watershed, researchers have documented more than 30 fish species vulnerable to infection, high rates of genetic mutation, recurrent outbreaks, and expanding geographic range (Getchell et al., 2019). While this pathogen is extremely generalist in its capacity to infect many species, sensitivity to infection is highly variable between fishes, and the biological and environmental factors that influence VHSV persistence following establishment are not well understood (Kim & Faisal, 2010). This research is investigating biotic and abiotic patterns of viral genetic diversity in the upper St. Lawrence River (USLR), an ecosystem where the virus has persisted for 20 years. The emergence of VHSV in this region coincided with dramatic declines of spotted muskellunge, an apex predatory fish in the SLR, making VHSV research critical to conservation efforts. Previous investigations in the USLR have emphasized consistently high prevalence in surveys of the virus, frequently observed fish kills since 2006 and enduringly suppressed populations of muskellunge (Cornwell et al. 2014, Farrell et al. 2017, Getchell et al. 2019). One dominant threat to their population recovery is invasion of nursery bays by large abundances of round gobies (Neogobius melanostomus). These invasive fish harbor VHSV and are considered essential to create an environment suitable to rapid viral reproduction, evolution of new viral isolates, and NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 5 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. dissemination of those isolates to the native community (Cornwell et al. 2012; Cornwell et al. 2014; Eckerlin et al. 2011; Getchell et al. 2019; Farrell et al. 2017; Stepien & Niner 2020). In-depth evolutionary analyses can improve ecosystem managers’ prediction capacity for transmission patterns, relative virulence, and host responses toward pathogens (Stepien et al., 2015). RNA viruses experience rapid genomic changes through their small genomes, lack of polymerase proofreading, and short generation times (Stepien et al., 2015). Genetic variation is expected to permit VHSV to further adapt to new environments and host species, as well as to evade host immune responses and facilitate long time maintenance in populations (Getchell et al., 2017; Stepien et al., 2015). Sequencing multiple genes is necessary to identify genetic changes that can be reflected in epidemiological behavior, and it’s been shown that just a few amino acid changes can result in altered VHSV virulence (Al-Hussinee et al., 2011; Getchell et al., 2017). Continual monitoring of VHSV types in fish populations across its range is fundamental to acknowledge changes to the virus’ genetic population structure and the relation to outbreak frequency and overall persistence (Garver et al., 2013). Previous investigations have shown that VHSV-IVb has diversified extensively following its first appearance in the Great Lakes (Cornwell et al. 2014; Thompson et al. 2011; Stepien et al. 2015). Genetic diversity analyses have largely used nucleotide sequences of the nucleoprotein (N) and glycoprotein (G) genes (Cornwell et al., 2012; Dadar, 2020). The N gene is the most abundantly transcribed genomic region, and the protein product closely associates with genomic RNA and facilitates interaction with viral polymerase (Kurath & Winton, 2008). Chimeric VHSV experiments have demonstrated that the NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 6 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. nucleoprotein gene also contains an important determinant of virulence in trout, thought to be the result of increased polymerase efficiency (Vakharia et al. 2019). The G protein performs the antigenic role that stimulates host immune responses, and genetic drift of this gene is expected to permit evasion of developed immunity responses of hosts (Panzarin et al., 2020; Vennerström et al., 2020). The G gene has presented high genetic diversity compared to other genes, and these sequences have been widely used to evaluate intraspecific phylogeography (Kuzmin et al., 2009). Recognizing how VHSV changes over time in geographic locations and host species is fundamental to predict future recurrence patterns and enhance our understanding of rapid evolution of pathogens (Domingo et al. 1998; Stepien et al. 2015). This study examines the hypothesis that VHSV genetic diversity is heterogenous across years, sites, and host species in the Thousand Islands region of the St. Lawrence River, and that both physical and biological structure of nursery bays influence the viral genetic diversity achieved in different areas. Methods SPECIMEN COLLECTION From 2018-2022, fish tissues were preserved for viral testing from round gobies in nursery bays of the Thousand Islands region (Figure 1). In 2020-2021, tissue collection in these sites was expanded to include potential native reservoirs (i.e., yellow perch, rock bass, brown bullhead, and Lepomis sunfish), and in 2022 a VHSV-positive muskellunge was recovered from the St. Lawrence River. A total of 1,520 fish were screened for VHSV infection as part of Anna’s dissertation research. From each fish, a pooled sample of liver kidney and spleen along with a separate sample of brain was collected and stored in 200 µL of RNALater and frozen at -20°C. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 7 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Viral RNA was extracted from 50 µL of homogenized tissue supernatant using a MagMax magnetic bead extraction and a MagMax-96 viral isolation kit following the protocols described in the kit and manufacturer’s extraction program AM1836_DW_50_V2. Fish were tested for VHSV using the qRT- PCR assay described by Hope et al. 2010, detecting a ~300bp fragment of the nucleoprotein gene of VHSV. Samples that tested positive for Figure 1 Map showing locations of nursery bays where wild fish were collected for this study. Esri, HERE, Garmin, (c) OpenStreetMap contributors, and the GIS user community Garlock Bay Cobb Sho~'Swan Bay Flynn Bay Blind ~ay o 0 Frinks Bay Rose Bay • Waddington 0 Peas Bay • Millens Bay 0 12.5 25 Legend Region 0 Alexandria Bay • Cape Vincent O Clayton - Grindstone O Waddington Ki lometers 50 Esri, HERE, Garmin, (c) OpenStreetMap contributors , and the GIS user community NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 8 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. VHSV infection with a concentration exceeding 1x103 N-gene copies/50 ng RNA were further pursued for genetic sequencing. REVERSE TRANSCRIPTION Reverse transcription was completed following the instructions in the SuperScript III First-Strand Synthesis System (Invitrogen) using random priming method. In each cDNA reaction, 0.5 μL cDNA Random Primer Mix (New England Biolabs) and 0.5 μL dNTPs (10 mM) were added to 5 μL total RNA, incubated at 65°C for 5 min, placed on ice and centrifuged briefly to collect reaction components. Then, 5 μL of cDNA synthesis mix (1 μL 5× buffer, 2 μL MgCl2, 1 μL DTT, 0.5 μL RNase OUT, 0.5 μL SuperScript III reverse transcriptase) was added and incubated at 50°C for 50 min, 85°C for 5 min, and placed on ice. Lastly, 1 μL RNase H was added followed by incubation at 37°C for 20 min to remove the original viral RNA from the new synthesized cDNA. A total of 11 μL of VHSV genome cDNA was synthesized from the RNA extracted with the MagMax-96 viral RNA isolation kit. POLYMERASE CHAIN REACTION (PCR) AND DNA PURIFICATION Two amplicons were synthesized in 35 cycles of PCR using OneTaq HotStart DNA Polymerase (New England Biolabs) and the corresponding primer sets. Amplification was conducted in a total volume of 20μL reactions using a Thermo Forma Hybaid PxE PCR thermal cycler. Reactions contained 1 μL cDNA, 4 μL of 5X OneTaq Standard PCR Buffer, 0.4 μL dNTPs (10mM), 0.1 μL OneTaq Hot Start Polymerase, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), and 13.7 μL nuclease-free water. Amplicons were produced using the following cycling program: 94°C for 30 s, followed by 35 cycles of 94°C for 25 s, 46°C for 45 s, 68°C for 1 min, with a final step of 68°C for 5 min. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 9 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. An 811 bp region of the N gene was amplified using: forward: 5′- GGGGACCCCAGACTGT-3’ reverse: 5′- TCTCTGTCACCTTGATCC-3’ A 914 bp region of the VHSV G gene was amplified using: forward: 5′- ACTACCTACACAGTGAC-3′ reverse: 5′-CAATTTGTCCCCGAATATCAT-3′ Amplicon quality was assessed by agarose gel electrophoresis running 2 μL on a 1% agarose gel. 15 μL of the remaining amplified DNA was purified using the Exo-CIP Rapid PCR Cleanup Kit (New England Biolabs) following manufacturer’s recommendations. DNA concentration was determined by fluorescence detection using a Qubit® Fluorometer (Invitrogen) and the Quant- iT™ dsDNA BR Assay kit (Life Technologies). The resulting amplicons were sequenced on a Sanger platform at the Biotechnology Resource Center, Cornell University. The Glycoprotein fragment was sequenced using the forward PCR primer, and the nucleoprotein fragment was completed with a primer that flanked the forward PCR primer, with the sequence 3’- GCAAGCACTGTCCGTACTTC-5’. BIOINFORMATICS ANALYSIS We define a haplotype as “a unique gene sequence that differs by one or more nucleotide substitutions” (Stepien et al. 2015). Sequence data Figure 2 Annotated VHSV genome map displaying functional genes and trailer regions. [ Nucleoprotein ] 3' UTR [Ph VHSV 0 3000 6000 9000 NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 10 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. were trimmed, aligned, checked, and edited manually using Geneious Prime software version 2023.1.2. The nucleoprotein consensus sequences were trimmed to 717 nt, corresponding to nt 406 to 1,122 of the full genome. Glycoprotein consensus sequences for each isolate were trimmed to 813 nt, corresponding to nt 3,348 to 4,160 of the full- length genomic sequence. Isolates with identical sequences were grouped together into ‘sequence types’ so that each unique haplotype was represented once in our trees. The 669 nt representing the central G region, corresponding to nt 3,409 to 4,077 of the full-length genomic sequence will be given a ‘universal sequence designator’ prior to publication. Amino acid substitutions were evaluated for each haplotype, with the Great Lakes index strain M103GL used as a reference (GenBank accession no. GQ385941). Multiple sequence alignments of the newly detected VHSV central G sequence types and the previously described sequences in the Laurentian Great Lakes were performed using ClustalOmega v.1.2.2. Phylogenetic analysis was conducted using the MrBayes v.3.2.6 plugin in Geneious Prime software. The analysis used the MCMC approach and was run for 1,100,000 generations and sampled every 200 cycles. The general-time- reversible (GTR) substitution model was employed. Tree drawing was performed using Geneious Prime v.2023.1.2. Results and Discussion SEQUENCE TYPES 93 nucleoprotein and 78 glycoprotein sequences were characterized from the St. Lawrence River in this study. Their collection dates, sites, and host species are detailed in appendix 1. The partial nucleoprotein sequences consisted of 13 unique sequence types with SNPs NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 11 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. identified at 14 locations. All N variants differed from the index strain by ≥3 nt changes (Figure 3). All nucleoprotein isolates possessed a cytosine at position 1057, and while this residue agrees with the vcG001 type specimen MI03, it represents a nonsynonymous substitution from the vcG002 reference strain originating from a St. Lawrence River round goby collected in a 2006 outbreak (GenBank accession no. KY359355.2). Figure 3 Nucleoprotein genetic differences among 13 unique sequence types identified in the St. Lawrence River isolates. (A) N Fragment represents the 714 bp N protein region sequenced, corresponding to nt 406-1,122 Of the 11,184 nt full- length genome sequence of Great Lakes VHSV strain MI03. Small vertical lines indicate sites of genetic differences as a composite including all variants, depicting synonymous and nonsynonymous as black or red, respectively. (B) Specific sequence differences in each sequence type shown relative to MI03 as a reference sequence. Boxed nt indicate non- synonymous changes that result in amino acid (AA) changes in predicted N protein sequence. For sites in which an AA change occurred, the first letter shows AA in the MI03 sequence type and the second letter indicates AA in the variant sequence type. A) N Fragment 400 VHSV 0 B) 600 3000 II II II II II II II 11 11 II 11 11 6000 Position II AA / Nonsynonymous / Synonymous 9000 Variant N 442 N 527 N 547 N 557 N 709 N 793 N 812 N 889 N 893 N 905 N 937 N 973 N 1024 N 1054 N 1057* C ~ ~ C C 4 G C A C 6 C C 7 A C 8 C 9 T C 10 ~ A C 11 C T C 12 ~ ~ C 13 A C AA change ~ ~ ~ L-F* No. nt diffferent No. of from MI03 isolates 0 40 1 33 8 NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 12 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Only four of the described nucleoprotein haplotypes encoded nonsynonymous substitutions, all generating alanine to threonine amino acid changes. Three variants contained one nonsynonymous change, and one variant contained two AA changes. All other variants only exhibited synonymous substitutions and likely have the same functional Figure 4 Glycoprotein genetic differences among 13 unique sequence types identified in the St. Lawrence River isolates. (A) Horizontal line represents the 813 bp G protein region sequenced, corresponding to nt 3,409–4,077 Of the 11,184 nt full-length genome sequence of Great Lakes VHSV strain MI03. Small vertical lines indicate sites of genetic differences as a composite including all variants, and asterisks indicate at which genetic heterogeneity observed within single isolates. (B) Specific sequence differences in each sequence type shown relative to MI03 as a reference sequence. Boxed nt indicate non-synonymous changes that result in amino acid (AA) changes in predicted G protein sequence. For sites in which an AA change occurred, the first letter shows AA in the MI03 sequence type and the second letter indicates AA in the variant sequence type. Shaded columns and dotted lines represent overlap with the 669 nt central glycoprotein partial sequence used to apply a universal sequence designation (USD). VHSV 0 3000 Position AA / Nonsynonymous / Synonymous B) No. nt diffferent No. of G3359 G3397 G3536 G3557 G3650 G3671 G3698 G3746 G3816 G3829 G3849 G3887 G3890 G3989 G4007 G4061 G4111 G4154 fromMI03 isolates USD --- A G A G A G G A G G A C G A G G C A G A G A G A G A C C A G I G A G A G A G A G A G A C G A G A C G A G IK-E R-K 1-V G G ~ G C G ~ G C G C G ~ G C G § G C C C G C G C 4 7 vcG047 5 2 vcG066 6 22 vcG076 6 12 Haws-4• 7 22 Haws-5* 7 2 Haws-6* 7 2 Haws-7* 7 4 Haws-8* 8 1 Haws-9• 8 vcG076 8 1 Haws- 11 * 8 1 Haws-12* 8 1 Haws-13* NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 13 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. phenotype as the type strain MI03. The most abundant haplotype in these isolates had no nucleotide changes in reference to MI03 based on the partial N sequences. The second most abundant haplotype had a single nonsynonymous change at nt 527 (Figure 3). The partial glycoprotein sequences contained 13 unique sequence types with SNPs recorded at 18 locations (Figure 3). Among the 13 unique sequences, 12 central G types were represented (Figure 4). Three of these vcG types (vcG047, vcG066 and vcG076) were previously detected in the St. Lawrence River, and nine of these types have not been previously described (See Appendix 2) (Cornwell et al. 2014, Getchell et al. 2019). All isolates were different from the index strain by ≤4 nucleotides, and every sequence contained the C-G mutation at nt 4007 (cG pos. 599, appendix 2) that was first described to distinguish vcG002. Two nonsynonymous substitutions were found outside of the central G region, both generating glycine to aspartic acid changes. The G-A change at nt 3397 occurred in every isolate, and this residue also matches the vcG002 type strain. The AA change at nt 3359 only occurred in a single isolate, and its central G sequence corresponded to vcG079 (Figure 4). Four nonsynonymous substitutions were observed in the central G region at nt 3816, 3829, 3849, and 3989 (cG pos. 406, 421, 441, and 581, Appendix 2). Each isolate had a G-A substitution at position 3829, creating an Arginine to Lysine AA change. This SNP was first described in vcG014 by Cornwell et al. 2012. 9/13 sequence variants experienced the AA change at nt 3849 first identified in vcG047 (Cornwell et al. 2014), 6/13 had the nt 3989 change described in vcG070 (Getchell et al. 2019), and only one variant had the nonsynonymous change at nt 3819, novel to this study’s haplotype Haws-9. The two haplotypes that were the most abundant sequence types in NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 14 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. these isolates, vcG076 and Haws-5, differ only by a single synonymous change at nt 3671 (Figure 4). PHYLOGENETIC ANALYSIS Phylogenetic analysis of central G sequence types observed in this study displays expanding genetic diversity in the St. Lawrence River, descending from variants previously described in genetic sequencing efforts. Relationships in the central G tree show the sequence types detected in this study were most closely grouped to vcG014, described by Cornwell et al. 2012. (Figure 5). SPATIAL ASSOCIATIONS Multiple sequence types were found in each sub-region of the USLR. The primary 3 regions possessed similar VHSV G-gene richness, with seven haplotypes observed in the Cape Vincent and Alexandria Bay isolates, and six in the Clayton-Grindstone samples (Figure 6A). Six of the G-gene haplotypes only occurred in one of the geographic regions with an ephemeral occurrence in 1-2 isolates (Haws- 6, 7, 9, 11, 12, and 13). Four haplotypes (vcG076, Haws-4, Haws-5, Haws-8) occurred across the three sub-regions of the study area, and they all also occurred in multiple regions simultaneously during a single sampling year. Three previously described sequence types identified by Cornwell et al. 2014 (vcG047), and Getchell et al. 2019 (vcG069 and vcG076), appeared among the isolates in this study. vcG076 was one of the most abundant sequence types across geographic regions, but both vcG069 and vcG047 were only detected again in isolated incidents. vcG069 was both described and reobserved in Cape Vincent, while vcG066 was initially described from Clayton in 2017, but found in Alexandria Bay thereafter. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 15 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Figure 5. Phylogenetic relationships among the St. Lawrence River 2019-2022 sequence types and the previously described partial glycoprotein (G) sequence types (669 nt) from the Laurentian Great Lakes. Labels for all branches indicate individual virus isolate designations. Labels with an asterisk indicates sequences reported in this study. Posterior probability values are shown at the branch nodes and the scale bar indicates the number of substitutions per nt site. 0.95 vcG008 vcG011 vcG010 vcG007 vcG006 vcG00S vcG004 vcG003 --------------------ovcG009 vcG051 vcG0S0 vcG048 vcG045 vcG032 vcG016 vcG015 vcG002 0.9 vcG017 0.93 vcG018 vcG014 0.9 0.96 vcG049 vcG046 --------------------0 Haws-9* vcG047* 0.65 0.51 .-----o vcG066* --------o Haws-8* L-----..;; 0;.;..·7..;;9~-------o Haws-5* vcG076* -------a Haws-13* 1--------0 Haws-12* L_ ________ ....:0::.:.-:::.:98:.v-----o Haws-1 1 * Haws-7* Haws-4* NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 16 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Spatial trends display the connectivity of the St. Lawrence ecosystem, and that the VHSV genetic structure contains both geographically widespread and locally isolated sequences. Sequence types that are sustained across multiple seasons are capable of persisting in the same location and dispersing throughout the ecosystem. YEAR ASSOCIATIONS Several central G haplotypes were detected during each of the sampling years. Among the most dominant haplotypes, vcG079 presented continuous presence during every sampling year between 2017-2022, but the relative proportion of this haplotype declined between 2021-2022. Haws-5 was first identified among the 2020 isolates and increased in relative proportion until 2022. Haws-4 also appeared in the 2020 isolates and maintained similar relative proportions throughout the study period. Haws-8 occurred in 2021 isolates and was observed again in 2022 (Figure 6). vcG047 was first described in samples collected during 2011 (Cornwell et al. 2014), and was reobserved in 2017, and again 2022. vcG066 was first observed in 2017 samples (Getchell et al. 2019) and appeared again in a single isolate from 2020. Ephemeral haplotypes were also observed during each of the study years. 2020 presented three unique haplotypes, 2021 had one, and 2022 had two. This data shows that genetic haplotypes are capable of sustaining infections in the St. Lawrence River for extended periods of time, with the oldest detected type dated back to at least 2011. Each outbreak season also produced new genetic variation, and only a limited amount of the overall diversity observed persisted between seasons. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 17 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Figure 6. Distribution of identified VHSV central G sequence types isolated among (A) different sub- regions in the upper St. Lawrence River, (B) different isolation years, and (C) different host species. A Cape Vincent C _o Clayton - Grindstone +-' ro (.) Alexandria Bay 0 _J Waddington I USO 0 10 20 30 40 ■ vcG047 B ■ vcG066 2022 ■ vcG076 ■ Haws-4 2021 ■ Haws-5 s.... ro 2020 ■ ~ Haws-6 2019 I ■ Haws-7 2017 ■ ■ Haws-8 ■ Haws-9 0 10 20 30 40 ■ Haws-11 C ■ Haws-12 CJ) Muskellunge I ■ Haws-13 (l) ·u Lepomis sp. (l) a. CJ) +-' Yellow perch CJ) 0 I Round goby 0 20 40 60 No. of isolates ' NEW YORK STATE WATER ~ ~ RESOURCES INSTITUTE Cornell University 18 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. HOST SPECIES ASSOCIATIONS Round goby sequences comprised the majority of isolates that were successfully sequenced in this study. All six of the unique ephemeral sequences occurred in a round goby host, and all sequences found in other host species were also detected in round gobies. Two of the previously described haplotypes, vcG047 and vcG069, were also only observed in round gobies. For 21 individual hosts, both the brain and pooled organ samples were amplified and sequenced, and 7/21 of these fish possessed different haplotypes between the brain and pooled organ isolations. Sequence heterogeneity was observed in infected round gobies, pumpkinseed, and muskellunge. The muskie mortality contained a mixture of the two most abundant haplotypes, vcG079 in its pooled organ sample, and Haws-5 in the brain. Round gobies both sustain abundant and widespread haplotypes and generate new genetic variation. Our data suggests that native hosts are exposed to the genetic structure contained in round goby infections, and that the most abundant haplotypes are likely to be those present in infected native fishes. Conclusions Increased surveillance for VHSV in free- ranging fish and active assessment of risk factors to prevent further virus impacts are major components of the action items promoted by USDA-APHIS in the Laurentian Great Lakes (Gustafson et al. 2018; Thompson et al. 2011). Routine and spatially robust genetic typing of field isolates is necessary to track the genetic diversity and epidemiology of VHSV through evolutionary time, across the basin. Isolates with the same genetic sequences are most likely epidemiologically linked, and therefore using NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 19 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. SNPs as genetic tags is useful to investigate viral movement and evolutionary patterns (Thompson et al. 2011). This research shows the widespread and continued presence of VHSV in the upper St. Lawrence River and highlights the continued need to prevent the spread of new VHSV variants into additional water bodies. The genetic composition of VHSV that exists in the St. Lawrence River is substantially diverged from the original reference strain, illustrating this virus’ capacity to exploit mutation to persist in the environment. Monitoring the viral composition of round gobies provides a comprehensive illustration of the genetic diversity present in the overall fish community at a location and time. Student Training One Ph.D. level graduate student, Mrs. Anna Haws, was trained in genetic sequence preparation and analysis during this project. She conducted all reverse transcription and amplification reactions, PCR product cleaning, DNA quantity/quality evaluation, and genetic data preparation and analysis. This work will be the final chapter in her doctoral dissertation for a degree in Fish & Wildlife Biology & Management from SUNY ESF. Anna in the field holding a round goby NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 20 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Publications/ Presentations Haws, A.L., Getchell, R.G., Farrell, J.M. (2023). Viral Hemorrhagic Septicemia Virus genetic diversity in the Upper St. Lawrence River. Manuscript in preparation. Haws, A.L. “INTRODUCTIONS, INTERACTIONS, AND IMPACTS: ROUND GOBY AND VIRAL HEMORRHAGIC SPETICEMIA VIRUS IN THE UPPER ST. LAWRENCE RIVER”. PhD dissertation, SUNY College of Environmental Science and Forestry, in preparation. Haws, A.L., Getchell, R.G., Farrell, J.M. (July 24-27 2023). Influence of Round Gobies on VHSV Disease Ecology in the Upper St. Lawrence River. [Conference presentation]. 2023 meeting of the AFS Fish Health Section. Haws, A.L., Getchell, R.G., Farrell, J.M. (September 24-26). Influence of Round Gobies on VHSV Disease Ecology in the Upper St. Lawrence River. [Conference presentation]. 2023 NYS Invasive Species Expo. Haws, A.L., Getchell, R.G., Farrell, J.M. (TBD). Viral Hemorrhagic Septicemia Virus genetic diversity in the Upper St. Lawrence River. [Conference presentation]. 2024 meeting of the New York chapter of the American Fisheries Society. 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(2014). Round gobies are an important part of VHS genotype IVb ecology in the St. Lawrence River and eastern Lake Ontario. Journal of Great Lakes Research, 40(4), 1002– 1009. Domingo, E. (1998). Quasispecies Structure and Persistence of RNA Viruses. Emerging Infectious Diseases, 4(4), 521–527. Eckerlin, G. E., Farrell, J. M., Casey, R. N., Hope, K. M., Groocock, G. H., Bowser, P. R., & Casey, J. (2011). Temporal Variation in Prevalence of Viral Hemorrhagic Septicemia Virus Type IVb among Upper St. Lawrence River Smallmouth Bass. Transactions of the American Fisheries Society, 140(3), 529–536. Farrell, J. Getchell, R., Kapuscinski, K., LaPan, S. (2017). Long-Term Trends of St. Lawrence River Muskellunge: Effects of Viral Hemorrhagic Septicemia and Round Goby Proliferation Creates Uncertainty for Population Sustainability. American Fisheries Society Symposium. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 21 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Getchell, R. G., E. J., Bogdanowicz, S. M., Andrés, J. A., Schulman, A. T., Kramer, J., Marquis, H. (2019). Investigation of round goby viral haemorrhagic septicaemia outbreak in New York. Journal of Fish Diseases, 42(7), 1023–1033. Gustafson, L. L., Remmenga, M. D., Gardner, I. A., Hartman, K. H., Creekmore, L. H., Goodwin, A. E., Whaley, J. E., Warg, J. V., Gardner, S. L., & Scott, A. E. (2014). Viral hemorrhagic septicemia IVb status in the United States: Inferences from surveillance activities and regional context. Preventive Veterinary Medicine, 114(3–4), OIE (Office of International Epizooties) (2019). Manual of diagnostic test for aquatic animals: chapter 2.3.10 viral haemorrhagic septicaemia. Edited by World Organization of health. Stepien, C. A., Pierce, L. R., Leaman, D. W., Niner, M. D., Shepherd, B. S., & Chen, T. Y. (2015). Gene diversification of an emerging pathogen: A decade of mutation in a novel fish Viral Hemorrhagic Septicemia (VHS) substrain since its first appearance in the Laurentian Great Lakes. PLoS ONE, 10(8), 1–26. Stepien, C. A., & Niner, M. D. (2020). Evolutionary trajectory of fish Piscine novirhabdovirus (=Viral Hemorrhagic Septicemia Virus) across its Laurentian Great Lakes history: Spatial and temporal diversification. Ecology and Evolution, 10(18), 9740– 9775. Vakharia, V. N., Li, J., McKenney, D. G., & Kurath, G. (2019). The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout. Journal of Virology, 93(18), e00382-19. Appendices Appendix 1. Summary of isolates used in this study and sequences obtained. Appendix 2. Summary of previously described USD variants in Laurentian Great Lakes used in phylogenetic analyses. Appendix 3. Methods addendum. Disclaimer The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the opinions or policies of the funding organizations or agencies. Mention of trade names or commercial products does not constitute their endorsement by these organizations. NEW YORK STATE WATER RESOURCES INSTITUTE Cornell University 22 This report was prepared for the New York State Water Resources Institute (NYSWRI) and the NYS DEC Bureau of Invasive Species and Ecosystem Health. Appendix 1. Summary of isolates used in this study and sequences obtained. 17-GETCHELL­ TIBS1 17-GETCHELL· TIBS3 17-GETCHELL­ TIBSS 17-GETCHELL­ TIB57 19-3B 19-13P 19-16P 19-18B 20-459P 20-4UP 20-471P 20-652P 20-826B 20-843P 20-888P 20·889P 20-893-P 20-898B 20-902B 20-902P Dato Location Collect<>d Frinks Bay 5/><><>