Source: UNIVERSITY OF TOLEDO, THE submitted to NRP
GENETIC DETECTION AND GEOGRAPHIC ANALYSIS OF GREAT LAKES FISH INFECTION BY VIRAL HEMORRHAGIC SEPTICEMIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0222330
Grant No.
2010-38927-21048
Cumulative Award Amt.
$468,000.00
Proposal No.
2010-01479
Multistate No.
(N/A)
Project Start Date
Aug 1, 2010
Project End Date
Jul 31, 2015
Grant Year
2010
Program Code
[BB-J]- Viral Hemorrhagic Septicemia, OH
Recipient Organization
UNIVERSITY OF TOLEDO, THE
2801 W BANCROFT ST
TOLEDO,OH 43606-3390
Performing Department
University of Toledo-Lake Erie Center
Non Technical Summary
IThis ongoing research merges the powers of Molecular Genetic Technology and Geographic Information Systems (GIS) and to understand and combat a new disease outbreak in the Great Lakes region, viral hemorrhagic septicemia (VHS). A novel strain of VHS has sporadically broken out in various Great Lakes regions since 2006, causing massive fish die-offs that have threatened fisheries, the baitfish and aquaculture industries, economic development, tourism, and negatively affect public health perception. Our project objectives are to develop, test, and implement new molecular genetic and GIS tools to rapidly and accurately detect active, replicating VHS infection in fish and understand its disease pathways. We are collaborating with federal and state agencies and other universities to: (1) develop, optimize, and compare our newly developed Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) test to detect VHS and actively replicating VHS in infected fish tissues with cell culture/plaque assay "gold-standard" and other PCR based methods, (2) analyze fish population susceptibility and disease resistance, (3) assess the roles of fish immune responses to VHSv, and (4) profile the disease dynamics through niche and dispersal modeling. The resultant research product is being presented through geographic tools that integrate ecological, genetic, and environmental health data in a user-friendly format that is easily web-accessible by researchers, agencies, educators, policy-makers, and the general public (see http://www.utoledo.edu/as/lec/research/glgl/VHS/VHS_main.html). We presented our project results at student and professional annual conferences: Sigma Xi, Midwest Fish and Wildlife Conference, Walleye and Yellow Perch Task Groups of the Great Lakes Fishery Commission, the Lake Erie Millennium network, and International Association for Great Lakes Research, where we solicited feedback on our test design, validation, and implementation. We have been evaluating performance of our rapid StaRT-PCR test on samples from the laboratory and the field - including cell culture samples with quantitative amounts of virus, unknowns from "wild" field collected fish, and infected fish from laboratory challenge experiments (in collaboration with managers from the Ohio Departments of Natural Resources and Agriculture, and researchers from Michigan State University, University of Wisconsin Milwaukee, and U.S. Geological Service). Our primary output goal is to develop a marketable kit for rapid and accurate VHS detection to be used by lake and aquaculture facility managers. This test will aid the aquaculture, fishery, and baitfish industries, and reduce treatment costs for drinking water. Broader impacts are that improved natural resources and water quality enhance tourism and water recreation.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3110810110160%
3110810109010%
3030810108020%
3060320119010%
Knowledge Area
303 - Genetic Improvement of Animals; 311 - Animal Diseases; 306 - Environmental Stress in Animals;

Subject Of Investigation
0810 - Finfish; 0320 - Watersheds;

Field Of Science
1090 - Immunology; 1101 - Virology; 1080 - Genetics; 1190 - Limnology;
Goals / Objectives
Our continuing project objectives are to develop, test, and implement new molecular genetic and Geographic Information Systems (GIS) tools to rapidly and accurately detect active, replicating Viral Hemorrhagic Septicemia virus (VHSv) infection in fish and understand its disease pathways. We are optimizing our Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay to detect VHS in fish, and comparing these results with those from the current "gold- standard" traditional method of cell culture/plaque assay and alternate PCR methods from other laboratories. StaRT-PCR uniquely employs internal standardized controls, offering increased accuracy and quantification. Our specific project goals are to: (1) develop a high-resolution low-cost StaRT-PCR assay for rapid and accurate detection of VHSv and discrimination among strains, (2) analyze fish population susceptibility and disease resistance to VHSv, (3) assess the roles of innate immune signaling in mediating responses to VHSv, and (4) track the virus' geographic and environmental spread using GIS approaches. To date, our progress includes: (1) Primer sets and internal standards to detect all VHS strains, and strain IVb-specific, have been optimized and experimentally tested, showing a lower detection threshold of <10 molecules/reaction and a linear range of >11 orders of magnitude. We successfully extracted, reverse transcribed, and PCR amplified VHS IVb RNA from several fish species (white perch, smallmouth bass, yellow perch, muskellunge, and round goby). (2) We conducted VHSv-IVb immersion challenge experiments at various dosages using juvenile muskellunge, with Dr. Faisal and Dr. Kim at Michigan State University, and are comparing the results from StaRT-PCR with their cell-culture and RT-PCR assays. We tested 13 walleye unknowns in spring 2009 obtained from Ohio Department of Wildlife using StaRT-PCR in comparison with cell culture/plaque assay results from Ohio Department of Agriculture (ODA; Dr. Yan Zhang) and plan to implement our StaRT-PCR procedure for further testing at ODA in 2010-12. (3) Two cell lines, EPC and BF-2, have been infected in controlled laboratory experiments and used for StaRT-PCR test validation using a "blind" experimental design, whose results are leading to "fine-tuning" of our test. (4) New and positive outbreaks of VHS have been incorporated into a GIS database, for which we are currently aggregating data to construct and parameterize the model (using both VHSv outbreaks and VHSv non-outbreak sources). Dissemination output to date includes research presentations at annual conferences of Sigma Xi, Midwest Fish and Wildlife Association, Walleye and Yellow Perch Task Groups of the Great Lakes Fishery Commission, Ohio Fish and Wildlife Management Association, Lake Erie Millennium Network, and International Association for Great Lakes Research, and the April 2010 NOAA Ohio Sea Grant publication "Twine Line". Our primary project output target goal is to develop a marketable kit for VHS detection to be used by lake and aquaculture facility managers for rapid and accurate VHS identification.
Project Methods
(1) We are developing, optimizing, and testing our StaRT-PCR (Standardized Reverse Transcriptase Polymerase Chain Reaction) diagnostic test to detect viral hemorrhagic septicemia (VHS) strains in infected fish, and are comparing these results with those from the "gold-standard" of cell culture/plaque assay and other RT-PCR tests. Oligonucleotide native template and internal standard amplicons are size separated and their length, abundance, and amplicon copy numbers are quantified. Gene expression is determined and the number of copies measured per each target gene then are normalized. Additionally, StaRT-PCR methodology is being used to develop a novel method to detect whether VHSv is active and replicating in fish, i.e., is transmissible to others. Specifically, infected tissues are divided, RNA is extracted and reverse transcribed to cDNA for one sample, and the other sample is gene-specific reverse transcribed to the actively replicating strand. StaRT-PCR will measure the relative amount of positive strand transcription versus total transcription to determine the ratio of actively replicating viral copies/cell in comparison with the total number of viral copies. (2) StaRT-PCR is being tested on potentially infected field fish samples from federal and state agencies. We also are testing our assay using tissues from VHSv-IVb infected fishes (muskellunge and yellow perch) from immersion and injection challenge experiments to further optimize intrinsic quality control. We are statistically comparing our StaRT-PCR results with those obtained using standard plaque assay, other PCR tests, and quantitative real-time PCR; including positive versus negative determinations and number of viral copies. We are testing our StaRT-PCR quantification results using VHSv IVb infected cell lines (EPC and BF-2) in experiments using series of "unknowns" with varying levels of virus. (3) We are assessing the roles of innate immune signaling molecules in mediating responses by fish cells to VHSv and will identify viral inhibition of innate immune pathways upstream or downstream of interferon induction, which will be evaluated in relation to viral persistence, spread, and/or virulence. (4) To evaluate the potential habitat of VHSv, we are using ecological niche modeling, which incorporates geographic information system (GIS) layers of water quality and other environmental variables in comparison to the known outbreaks and absences of VHSv. To model VHSv dispersal, we are developing diffusion and spatial-interaction models that incorporate natural and human-mediated dispersal mechanisms, including: a) fish species movements, b) shipping, c) recreational boating traffic, and (d) bait trade network. Our primary project output target goal is to develop a marketable kit for VHS detection to be used by lake and aquaculture facility managers for rapid and accurate VHS identification, and will be conducting workshops with federal and state agency collaborators for training. We will be applying for APHIS approval.

Progress 08/01/10 to 07/31/15

Outputs
Target Audience:Our target audiences are fishery managers, federal and state fishery agents, baitfish transporters, aquaculturespecialists, educators, and other university researchers. To make our VHSv project results publically available,we updated our project link on the Lake Erie Center website(http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html), which displays background information,current methodology, current projects, current progress, and past presentations. This website was constructed toreach the general public and the scientific community in order to develop awareness of VHSv and enhance otherresearch efforts on the virus. The website is updated regularly.This reporting period, our VHS team members gave project presentations at fourresearch conferences and venues,including the Ohio Research Review in Columbus Ohio and the Midwest Graduate Research Symposium in Toledo, OH, at a VHS ARS project meeting in Milwaukee WI, and at the USDA ARS facility in Leetown WV) to disseminate our findings on viral diagnostic detection, evolutionary patterns of the virus, and increasecollaborations with agencies and other universities. To furtherevaluate performance of our StaRT-PCR assay, we collaborated with Dr. Caird Rexroad III of the USDA ARS, Dr. Jeffrey Silverstein of the USDA ARS,Dr. Brian Shepherdof the USDA ARS, Dr. Allyn Spear of the USDA ARS, and Dr. Jhonatan Sepulveda-Villet of the University of Wisconsin Milwaukeeto utilize ourassay on the results of VHSv immersion and injection challenge experiments (published in two journal articles in 2013, inPLoS One and Journal of Virological Methods).We also have been working with Dr. Vikram Vakharia of University of Maryland on VHSv knock-out genes for furthercollaboration, and Dr. Stepien visited with him in Leetown WVto develop our collaboration, in January2015. Dr. Bossenbroek'slaboratory further integrated their VHSv modeling studies, and his PhD student graduated and defended her dissertation. They published a paper on the results in the journal Biological Invasions 2014: Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSv) by commercial shipping in the Laurentian Great Lakes, by Sieracki, Bossenbroek, and Faisal. Dr. Stepien has a new paper with her students and collaborators in press on the results in the prestigious journal PLOS One for 2015 publication: 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 by Stepien, Pierce, Leaman, Niner, and Shepherd. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A new Ph.D. student, Megan Niner, has worked on the VHSv project with Dr. Stepien, beginning in May 2014 through the present. Her projected Ph.D. is on, "Evolutionary Patterns of the VHSv Fish Virus", which will continue with other funding. Ms. Niner gave an oral and a poster presentation in 2015 on her early results from this project and also assisted in further data analyses for the final version of the PLOS One 2015 paper by Stepien et al.In summer 2015, an undergraduate researcher, Ms. Shelby Edwards, accompanied Ms. Niner on samplings of Great Lakes fishes across the Great Lakes to assess the geographic presence, distributions, and genetic types of any infections by VHSv. These samplings were in conjunction with fish samplings by a variety of federal and state agencies. This funding has provided important training and professional development for these two young women in science. Lindsey Pierce who successfully defended her Ph.D. dissertation, "The Evolution and Detection of the Fish ViralHemorrhagic Septicemia Virus (VHSv)" in August 2013 under Dr. Stepien, graduated in 2013 from theUniversity of Toledo, and is not a postdoctoral fellow at the Cleveland Clinic. This NIFA grant opportunitysignificantly aided the progress, output, and dissemination of her Ph.D. work. The funds allowed her to present ata wide variety of conferences, including an overseas conference in France. Pierce obtained three first-authoredpublications under the grant, with Stepien and other team members, and is the co-first author on another with Drs.Stepien andLeaman, and Niner, which is now in Press. Dr. Pierce's postdoctoral fellowship was a direct result and offshoot of thedissertation work on the evolutionary patterns of the pathogen, as she is not working on the evolutionary patternsof malaria. Also in Dr. Stepien's lab, postdoctoral fellow Dr. Amanda Haponski worked in 2014-2015to helpsample Lake Erie fishes for VHSv and to employ our assay to test for VHSv infection, together with new Ph.D. student Megan Niner. She elped to train newPh.D. student Megan Niner on the project, and assisted in fine-tuning the application of the VHSv assay. In Dr. Bossenbroek's lab, Jennifer Sieracki defended her Ph.D. dissertation, "Spatial modeling as adecision-making tool for invasive species management in the Great Lakes" in 2014. She has twofirst-authored publicationson the NIFA project results in the journal Biological Invasions, and PLoS One, both coauthoredwith Dr. Bossenbroek and both published in 2014.During the project, Dr. Sieracki developed her GIS and spatial modeling expertiseand was awarded a Sea Grant small grant to generalize her model and distribute it to state and federal agenciesthat work on managing aquatic invasive species. Dr. Sieracki presented project results to a working group of state and federal agencies managersassociated with a NOAA-CSCOR project in 2013. Dr. Sieracki is now working for the US Forest Service in Ft.Collins Colorado, and is doing similar modeling work. The work aided by the NIFA funding thus helped her launchher career. How have the results been disseminated to communities of interest?Ph.D. student Megan Niner presented an oral and poster presentation on the results in 2015, to the Ohio Research Review in Columbus OH and at the Midwest Graduate Research Symposium. Dr. Stepien is presenting a poster at the American Fisheries Society in Portland Oregon in August 2015. 2015 output also includes a new paper by Stepien et al. (2015) in the prestigious journal PLOS One, which is in press. Dr. Bossenbroek published two papers, both headed by his now-graduated Ph.D. student Jennifer Sieracki in 2014, in PLOS One and in Biological Invasions. Drs. Stepien and Leaman also presented updates on this project at the University of Wisconsin Milwaukee in January 2014 and at the USDA ARS in Leetown WV in January 2015. In addition, we have a website on the project. http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulentstrain - IVb -appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks insubsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved bythe USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. Wethus developed a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRTPCR)assay that incorporates synthetic competitive template internal standards for quality control to circumventfalse negative results. Results demonstrated high signal-to-analyte response and a linear dynamic range that spansseven orders of magnitude, ranging from 6 to 6,000,000 molecules. Infected fishes were found to harbor levels ofvirus that range to 1,200,000 VHSv molecules per 100,000 beta actin molecules, with 1,000 being a rough cut-offfor clinical signs of disease. Our new assay is rapid, inexpensive, and has significantly greater accuracy than otherpublished qRT-PCR tests and traditional cell culture diagnostics.We published two new papers in 2013 on our new VHSv Assay in the prestigious journals PLoS One and Journalof Virological Methods. This assay was developed and accomplished by the Stepien and Willey laboratories at theUniversity of Toledo, with Ph.D. student Lindsey Pierce and collaborators. We since have been employing the assay in new tests of the present extent of VHSv across the Great Lakes. Drs. Stepien and Leaman, with Ph.D. students Lindsey Pierce and Megan Niner also worked to sequence isolates of VHSv-IVbacross the Great Lakes, in order to understand the evolutionary diversification patterns of the virus. A new paperis in press in the prestigious journal PLOS One.Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus, which causes one of the world's most serious fish diseases, infecting >80 freshwater and marine species across the Northern Hemisphere. A new, novel, and especially virulent substrain - VHSv-IVb - first appeared in the Laurentian Great Lakes about a decade ago, resulting in massive fish kills. It rapidly spread and has genetically diversified. This study analyzes temporal and spatial mutational patterns of VHSv-IVb across the Great Lakes for the novel non-virion (Nv) gene that is unique to this group of novirhabdoviruses, in relation to its glycoprotein (G), phosphoprotein (P), and matrix (M) genes. Results show that the Nv-gene has been evolving the fastest (k=2.0x10-3 substitutions/site/year), with the G-gene at ~1/7 that rate (k=2.8x10-4). Most (all but one) of the 12 unique Nv- haplotypes identified encode different amino acids, totaling 26 changes. Among the 12 corresponding G-gene haplotypes, seven vary in amino acids with eight total changes. The P- and M- genes are more evolutionarily conserved, evolving at just ~1/15 (k=1.2x10-4) of the Nv-gene's rate. The 12 isolates contained four P-gene haplotypes with two amino acid changes, and six M-gene haplotypes with three amino acid differences. Patterns of evolutionary changes coincided among the genes for some of the isolates, but appeared independent in others. New viral variants were discovered following the large 2006 outbreak; such differentiation may have been in response to fish populations developing resistance, meriting further investigation. Two 2012 variants were isolated by us from central Lake Erie fish that lacked classic VHSv symptoms, having genetically distinctive Nv-, G-, and M-gene sequences (with one of them also differing in its P-gene); they differ from each other by a G-gene amino acid change and also differ from all other isolates by a shared Nv-gene amino acid change. Such rapid evolutionary differentiation may allow new viral variants to evade fish host recognition and immune responses, facilitating long-time persistence along with expansion to new geographic areas. Dr. Leaman's lab has worked on furthering our understanding of the mechanisms by which VHSv blocks hostinnate immune responses. Foremost among these studies is the incorporation of specificmutations into the intact virus through reverse genetic studies. A spatial database of VHSv positive and negative sites was created by Dr. Bossenbroek's lab, and a model thatidentifiedballast water as a vector of VHSv spread in the Great Lakes was completed. This model has beengeneralized to model the spread of multiple invasive species due to Great Lakes shipping model. The Bossenbroek lab published two papers on this in 2014, in the journals PLOS One and Biological Invasions, reporting on this work that was led by his Ph.D. student Jennifer Sieracki.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Stepien, C.A., L. Pierce, M. Niner, B. Shepherd, D. Leaman. 2014. Evolutionary Dynamics of the VHS Fish Virus: An Emerging Pathogen. VHS Project Symposium Presentation, University of Wisconsin, Milwaukee, WI. December 2014. Oral presentation by Stepien.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Stepien, C.A., L.R. Pierce, D. Leaman, M. Niner, B. Shepherd. 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 (Public Library of Science). In Press.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sieracki, J.L. J.M. Bossenbroek, and M. Faisal. 2014. Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) by commercial shipping in the Laurentian Great Lakes. Biological Invasions. 05/2014; 16(5). DOI: 10.1007/s10530-013-0556-2
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sieracki J.L., J.M. Bossenbroek, and W.L. Chadderton. 2014. A Spatial Modeling Approach to Predicting the Secondary Spread of Invasive Species Due to Ballast Water Discharge. PLoS ONE 9(12): e114217. doi:10.1371/journal.pone.0114217
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: C.A. Stepien, L. Pierce, M. Niner, and D. Leaman. 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. American Fisheries Society Annual Conference, Portland, Oregon, 2015. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Niner, M., L. Pierce, C. Stepien, D. Leaman. 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. Ohio Research Review, Columbus, OH. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Niner, M., L. Pierce, C. Stepien, D. Leaman 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. March 2015. Midwest Graduate Research Symposium. Toledo, OH Oral Research Presentation.


Progress 08/01/13 to 07/31/14

Outputs
Target Audience: Our target audiences are fishery managers, federal and state fishery agents, baitfish transporters, aquaculture specialists, educators, and other university researchers. To make our VHSv project results publically available, we updated our project link on the Lake Erie Center website (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html), which displays background information, current methodology, current projects, current progress, and past presentations. This website was constructed to reach the general public and the scientific community in order to develop awareness of VHSv and enhance other research efforts on the virus. The website is updated regularly. This reporting period, our VHS team members gave project presentations at several research conferences and venues, including the American Fisheries Society (Little Rock, Arkansas),the International Conference on Marine Invasive Species: Management of Ballast Water and Other Vectors (Muscat, Oman), and the Joint Aquatic Sciences Meeting (Portland, Oregon) to disseminate our findings on viral diagnostic detection, evolutionary patterns of the virus, and increase collaborations with agencies and other universities. We continued research collaborations with Dr. Mohamed Faisal and Dr. Robert Kim of Michigan State University, where we conducted VHSv immersion challenges on fish to StaRT-PCR assay (we published in two journal articles in 2013 on these results, in PLoS One and Journal of Virological Methods). To further evaluate performance of our StaRT-PCR assay, we collaborated with Dr. Brian Shepherd of the USDA ARS, Dr. Frederick Goetz of the University of Wisconsin Milwaukee (now at NOAA)and Dr. James Winton of USGS in Seattle to utilize our assay on the results ofVHSv immersion and injection challenge experiments (published in two journal articles in 2013, in PLoS One and Journal of Virological Methods). We also have been working with Dr. Vikram Vakharia of University of Maryland on VHSv knock-out genes for further collaboration, and he visited us to present on his research, and develop our collaboration, in April 2014. Dr. Bossenbroek’s laboratory further integrated their VHSv modeling studies, and is working to develop a user-friendly interface that will enable others, such as state and federal managers, to use this tool in ArcGIS. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Lindsey Pierce successfully defended her Ph.D. dissertation, “The Evolution and Detection of the Fish Viral Hemorrhagic Septicemia Virus (VHSv)” in August 2013 under Dr. Stepien, graduated in December 2013 from the University of Toledo, and is not a postdoctoral fellow at the Cleveland Clinic. This NIFA grant opportunity significantly aided the progress, output, and dissemination of her Ph.D. work. The funds allowed her to present at a wide variety of conferences, including an overseas conference in France. Pierce obtained three first-authored publications under the grant, with Stepien and other team members, and is the co-first author on another with Drs. Stepien and Leaman, which is in review. Dr. Pierce’s postdoctoral fellowship was a direct result and offshoot of the dissertation work on the evolutionary patterns of the pathogen, as she is not working on the evolutionary patterns of malaria. Also in Dr. Stepien’s lab, postdoctoral fellow Dr. Amanda Haponski has worked in summer 2014 to help sample Lake Erie fishes for VHSv and to employ our assay to test for VHSv infection. She has helped to train new Ph.D. student Megan Niner on the project. In Dr. Bossenbroek’s lab, Jennifer Sieracki just recently defended her Ph.D. dissertation, “Spatial modeling as a decision-making tool for invasive species management in the Great Lakes”. She has a first-authored publication on the NIFA project results in the journal Biological Invasions, and another in review in PLoS one, both co-authored with Dr. Bossenbroek. During the project, Dr. Sieracki developed her GIS and spatial modeling expertise and was awarded a Sea Grant small grant to generalize her model and distribute it to state and federal agencies that work on managing aquatic invasive species, which is being completed by a new masters student, Stephanie Nummer. Dr. Sieracki presented project results to a working group of state and federal agencies managers associated with a NOAA-CSCOR project in 2013. Dr. Sieracki is now working for the US Forest Service in Ft. Collins Colorado, and is doing similar modeling work. The work aided by the NIFA funding thus helped her launch her career. Also in Dr. Bossenbroek's lab, undergraduate researcher Lindsay Vollmar graduated in December 2012 from the business school and developed her undergraduate thesis through this project. She developed a survey to determine anglers’ willingness-to-pay for bait that has been certified free of VHSv. Her preliminary results suggest that people with higher incomes are more likely to pay more for VHSv free bait. A manuscript based on her survey results is currently in review, with Dr. Bossenbroek and Dr. McIntosh, an economist from the University of Minnesota Duluth, as co-authors. Dr. Leaman's lab has trained an undergraduate researcher on this project over the past year, and he will return for the 2014/2015 academic year. This student, Tyler Popil, used the VHSv work as the basis for his honors research. A doctoral student in the lab, Qi Ke, has taken the lead on the project, which will make up a portion of his dissertation work on host-virus interactions. Qi Ke gave a poster presentation on his VHSv work in spring 2014. How have the results been disseminated to communities of interest? Ph.D. students Lindsey Pierce and Jennifer Sieracki both presented departmental seminars/dissertation defenses on their dissertation work results from the project, which were well attended in August 2013 and June 2014, respectively. During the project period, Dr. Pierce gave a departmental seminar on the results at the Cleveland Clinic in November 2013Dr. Stepien presented on the project results at the American Fisheries Society annual conference in Little Rock Arkansas in September 2013, at the International Conference on Marine Invasive Species: Management of Ballast Water and other Vectors in Oman in February 2014, and at the Joint Aquatic Sciences Meeting in Portland Oregon in May 2014. Dr. Leaman’s doctoral student Qi Ke gave a poster presentation on his VHSv work in spring 2014. Project results for this year also were published in the journals PLoS One (Stepien, Pierce, Willey, Leaman, and other collaborators) and Biological Invasions (Bossenbroek, Sieracki, and others). Three other publications from our project now are in review. Our 2013-4 output this year also included a classroom exercise for high school students on the virus, which were implemented in classrooms through the Lake Erie Center’s high school program that involves graduate students (by Lindsey Pierce). This is detailed on the program website (http://www.utoledo.edu/nsm/lec/gk12_grant/Lesson_Plans.html). What do you plan to do during the next reporting period to accomplish the goals? Dr. Stepien’s laboratory is planning to further test fishes from Lake Erie and Lake Michigan for VHSv infection. We now are processing those we collected in early summer 2014, and will repeat collections in spring-summer 2015 before the project ends. So far, indications are that we found no signs of VHSv in Lake Erie fishes in 2014. We also are planning to sequence for the entire genome the variant VHS-IVb isolates we did for selected genes (G, Nv, P, and M) that are in review in our most recent paper. We are beginning to work on the N gene variants (which will be done by new Ph.D. student Megan Niner and postdoctoral fellow Amanda Haponski, in 2014-5). Then we will pursue whole genome sequencing of all positive samples. Variants of particular interest include two 2012 Lake Erie fish specimens, which had unique sequences for several genes. Dr. Leaman's laboratory is planning to finish the current work on the VHSv M protein and move on to Nv and P proteins. Although work on M will continue, the P studies will allow his lab to better define overall mechanisms of viral inhibition of host responses. The Nv studies may identify specific ways in which the host detects the virus prior to viral shutdown of host response. As such, the Nv gene (or gene product) may allow the lab to develop new stimulatory molecules to “prime” virus-infected cells to mount more robust antiviral responses. Such adjuvant-like effects could be useful in the development of live, attenuated VHSv vaccines. Dr. Bossenbroek’s laboratory is planning to finalize the publications of Jennifer Sieracki and Lindsay Vollmar. They further plan to explore the landscape genetic relationships between the patterns of spread of VHSv in the Great Lakes and the population genetic analysis conducted by Dr. Stepien’s lab. Also the ArcGIS tool to predict invasive species spread via ballast water in the Great Lakes will be introduced to state and federal resource managers.

Impacts
What was accomplished under these goals? Viral Hemorrhagic Septicemia virus (VHSv) is one of the world’s most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb –appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus developed a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response and a linear dynamic range that spans seven orders of magnitude, ranging from 6 to 6,000,000 molecules. Infected fishes were found to harbor levels of virus that range to 1,200,000 VHSv molecules per 100,000 beta actin molecules, with 1,000 being a rough cut-off for clinical signs of disease. Our new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics. We published two new papers in 2013 on our new VHSv Assay in the prestigious journals PLoS One and Journal of Virological Methods. This assay was developed and accomplished by the Stepien and Willey laboratories at the University of Toledo, with Ph.D. student Lindsey Pierce and collaborators. Drs. Stepien and Leaman, with Ph.D. student Lindsey Pierce also worked to sequence isolates of VHSv-IVb across the Great Lakes, in order to understand the evolutionary diversification patterns of the virus. A new paper is in review. This new, novel, and especially virulent substrain –IVb– first appeared in the Laurentian Great Lakes a decade ago, causing massive fish kills in 2005 and 2006, spreading rapidly, and genetically diversifying. VHSv and other novirhabdoviruses infect only fishes and are distinguished by a novel non-virion (Nv) gene region, whose evolutionary variability largely was unknown prior to this investigation. We analyze the temporal and spatial courses of VHS-IVb mutational patterns, comparing substitutions in Nv, along with the glycoprotein (G), phosphoprotein (P), and matrix (M) genes. Our results for VHSv-IVb sequences reveal that the Nv-gene evolved the fastest of the gene regions examined, with 49 substitutions; 61% were non-synonymous. G-gene substitutions occurred at ~1/10 that rate, with the second most numerous substitutions (14), of which 57% (8) were non-synonymous. The P and M genes were more conserved, having just 4 and 6 sequence haplotypes, and evolving at ~1/13 and ~1/15 of the Nv gene’s rate. Conclusions were that some VHSv-IVb variants appeared to display roughly congruent patterns of divergence among different genes, whereas others lacked any obvious relationship. Greatest overall viral diversity was found in the lower Great Lakes (Lakes Erie and Ontario), which may be related to geographic proximity to strain IVb’s hypothesized evolutionary origin in the northwestern Atlantic Ocean. New VHSv-IVb variants appeared after a large 2006 outbreak that spanned much of the Great Lakes; this evolution may have been in response to fish populations developing resistance to the virus following the large die-offs. We describe two recent (collected in 2012) and very different haplotypes from central Lake Erie, which were isolated from fishes that were swimming erratically but lacked classic VHSv symptoms. Rapid evolutionary diversification of VHS may allow new viral variants to evade fish host recognition and immune responses, facilitating long-time persistence in populations along with expansion to new geographic areas. Dr. Leaman's lab has worked on furthering our understanding of the mechanisms by which VHSv blocks host innate immune responses. His work has continued to focus primarily on the VHSv M protein. This protein can block host protein expression, and studies in the lab have uncovered a likely mechanism for how M functions in this regard. Investigations aimed at finalizing this mechanism and translating the results back to a better understanding of VHSv virulence are in progress. Foremost among these studies is the incorporation of specific mutations into the intact virus through reverse genetic studies. The Leaman lab has reverse engineered a point mutation into the VHSv M gene that potently blocked the anti-host function of this gene while leaving its viral replication function intact. Future studies will aim to incorporate other mutations into M and Nv genes to, hopefully, allow us to further uncouple viral replication and virulence functions. A spatial database of VHSv positive and negative sites was created by Dr. Bossenbroek's lab, and a model that identifies ballast water as a vector of VHSv spread in the Great Lakes was completed. This model has been generalized to model the spread of multiple invasive species due to Great Lakes shipping model. The group currently is developing a user-friendly interface for this model that will enable others, such as state and federal managers, to use this tool in ArcGIS.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Pierce, L.R. The Evolution and Detection of the Fish Viral Hemorrhagic Sepicemia Virus (VHSv). Ph.D. Dissertation, University of Toledo, December 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pierce, L.R., J.C. Willey, E.L. Crawford, V.V. Palsule, B.S. Shepherd, and C.A. Stepien. 2013. Accurate detection and quantification of the fish Viral Hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay. PLoS One. 8(8): e71851. doi:10.1371/journal.pone.0071851 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071851
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Qi Ke, A. Pore, J. Wildschutte, T. Williams, and D. Leaman. 2014. Viral Hemorrhagic Septicemia virus M protein represses host protein expression. University of Toledo Graduate Research Symposium, Toledo, Ohio.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Stepien, C.A., L.R. Pierce, D. Leaman, J. Willey. Accurate Detection and Quantitation of the Fish Viral Hemorrhagic Septicemia Virus (VHSv) With a New Real-Time PCR Assay. American Fisheries Society Annual Conference. Little Rock, Arkansas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Stepien, C.A., L.R. Pierce, and D. Leaman. Evolutionary Diversification and Spread of the VHSv Fish Virus in the Great Lakes and Beyond. International Conference on Marine Invasive Species: Management of Ballast Water and Other Vectors. Muscat, Oman. Feb. 17, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Stepien, C.A., L.R. Pierce, and D. Leaman. Evolutionary Dynamics of the VHS Fish Virus: An Emerging Pathogen. Joint Aquatic Sciences Meeting, Molecular Ecology Session. Portland Oregon. May 22, 2014.
  • Type: Other Status: Published Year Published: 2013 Citation: Pierce, L.R. The Evolution and Detection of the Fish Viral Hemorrhagic Septicemia virus (VHSv). Department Seminar at the Cleveland Clinic. Cleveland, Ohio. November 20, 2013.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Sieracki, J.L., J.M. Bossenbroek, and W.L. Chadderton. Spatial Modeling Approach to Predicting the Secondary Spread of Invasive Species Due to Ballast Water Discharge. Submitted to PLOS One.
  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Vollmar, L., C.R. McIntosh, J.M. Bossenbroek. Anglers responses to bait certification regulations: the case of virus-free bait demand. Submitted to Journal of Environmental Management.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Sieracki, J.L., J.M. Bossenbroek, and M. Faisal. 2013. Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) by commercial shipping. Biological Invasions. DOI 10.1007/s10530-013-0556-2 in the Laurentian Great Lakes
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Sieracki, J. L. 2014. Spatial modeling as a decision-making tool for invasive species management in the Great Lakes. Ph.D. Dissertation, University of Toledo, Toledo, Ohio. August 2014.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pierce, L.R., J.C. Willey, E.L. Crawford, D.W. Leaman, V.V. Palsule, M. Faisal, R.K. Kim, B.S. Shepherd, and C.A. Stepien. 2013. A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): Enhanced quality control with internal standards. Journal of Virological Methods. 189:129-142. http://dx.doi.org/10.1016/j.jviromet.2013.01.006
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Stepien, C.A., L.R. Pierce, D. Leaman, B. Shepherd. Gene diversification in an emerging quasispecies: A decade of mutation in fish viral hemorrhagic septicemia (VHS) across the Laurentian Great Lakes.


Progress 08/01/12 to 07/31/13

Outputs
Target Audience: Our target audiences are fishery managers, federal and state fishery agents, baitfish transporters, aquaculture specialists, educators, and other university researchers. To make our VHSv project results publically available, we updated our project link on the Lake Erie Center website (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html), which displays background information, current methodology, current projects, current progress, and past presentations. This website was constructed to reach the general public and the scientific community in order to develop awareness of VHSv and enhance other research efforts on the virus. The website is regularly updated. Our VHS team gave project presentations at several research conferences and venues, including The American Fisheries Society, Ecological Society of America, Eco Summit, Evolution, International Association of Great Lakes Research, International Conference on Aquatic Invasive Species, Lake Erie Fishery Genetics and Stock Structure Workshop for Managers, Lake Erie Inland Waters Annual Research Review, Lucas County League of Women Voters Annual meeting, Midwest Graduate Symposium, Ohio Department of Agriculture Aquatic Animal Health conference, Ohio Fish and Wildlife Managers Association, Sigma Xi Scientific Honorary Society Annual Conference, United States Department of Agriculture Annual Progress Meeting, Walleye and Yellow Perch Task Group Meetings of the Great Lakes Fishery Commission, to increase awareness of current viral diagnostic detection methods and collaborations with other federal agencies and universities. We developed research collaborations with Dr. Mohamed Faisal and Dr. Robert Kim of Michigan State University to conduct VHSv immersion challenges on muskellunge to compare our newly developed StaRT-PCR assay to currently approved and developing detection diagnostics. To further test our StaRT-PCR assay, we developed collaborations with Dr. Frederick Goetz of the University of Wisconsin Milwaukee and Dr. James Winton of USGS in Seattle to conduct VHSv immersion and injection challenge experiments. Changes/Problems: We request a one-year no-cost exension in order to complete our study and allow the two dissertations on the project to be finished and published. We also seek to implement our test with the Ohio Department of Agriculture, and to futher evaluate it using additional fish samples and possible invertebrate vectors. We thus request an extension through July 31, 2014. What opportunities for training and professional development has the project provided? This USDA NIFA grant has allowed Ph.D. student Lindsey Pierce from Dr. Stepien’s lab to expand her dissertation by including the development and implementation of a rapid and reliable test to detect and quantify the VHS virus. Pierce and Stepien published “Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv)” in Molecular Phylogenetics and Evolution 2012. They, with other team members, published “A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): Enhanced quality control with internal standards” in the Journal of Virological Methods 2013. The team also has another manuscript “Accurate detection and quantification of the fish Viral Hemorrhagic Septicemia virus (VHSv) with a 2-color fluorometric real-time PCR assay” in review for PLOS One that focuses on the transition of the StaRT-PCR test into a real-time approach. Ms. Pierce is planning to defend her Ph.D. dissertation in summer 2013. Ms. Vrushalee Palsule, a research Technician from Dr. Stepien’s lab, has been working on this project and has given several oral and poster presentations on our StaRT-PCR assay, as well as being a co-author on the two papers for the VHSv assay. Dr. Leaman’s lab has trained three undergraduate researchers on this project over the past year, all of whom graduated in May. Two of the students, Tyler Williams and Daniel Hettel, will attend medical school in the fall. Daniel used the VHSv work as the basis for his honors thesis, and was awarded both the outstanding Biological Sciences student this year, as well as outstanding student of the College of Natural Sciences and Mathematics. The third undergraduate student, Jacob Blandford, will work for a year and then is interested in attending graduate school to study invasive fish species. In addition, a postdoctoral fellow, Julia Wildschutte, worked on the project for 9 months, and at this time Qi Ke, a doctoral student in the lab, has taken over her portion of the project, which will make up a portion of his dissertation work on host-virus interactions. In Dr. Bossenbroek’s lab, Ph.D. candidate Jennifer Sieracki and undergraduate researcher Lindsay Vollmar have done most of the work on this project. Jennifer has developed her GIS and spatial modeling expertise and has one manuscript in review based on this work. She also will be submitting a Sea Grant proposal, which if funded will enable her to generalize her model and distribute it to state and federal agencies that work on managing aquatic invasive species. Linsday Vollmar graduated in December 2012 from the business school and developed her undergraduate thesis through this project. She developed a survey to determine anglers willingness-to-pay for bait that has been certified free of VHSv. Her preliminary results suggest that people with higher incomes are more likely to pay more for VHSv free bait. We are currently analyzing the full survey results with the help of an economist from the University of Minnesota Duluth. How have the results been disseminated to communities of interest? To disseminate our findings to the community: (1) We updated our VHS webpage on our Lake Erie Center website to include current findings of VHSv detection and quantification (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/startpcr.html), presentations (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/Presentations.html), and publications (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/publications.html). (2) Ph.D. student Lindsey Pierce presented project results at the Ohio Fish and Wildlife Managers Association in Columbus Ohio in 2011, the International Evolution Conference in Ottawa, Canada, the Evolutionary Biology Meeting in Marseille, France, the Midwest Graduate Research Symposium in Toledo, OH, the Eco Summit in Columbus, OH, the Ohio Fish and Wildlife Managers Association in Columbus, OH all in 2012, and the Midwest Graduate Research Symposium in Toledo, OH in 2013. (3) DNA technician Ms. Vrushalee Palsule presented project results at Ohio Department of Agriculture Aquatic Animal Health Committee Annual Meeting in Reynoldsburg, OH in 2011, 51st Annual Ohio Fish and Wildlife Managers Association in Columbus, OH, and 4th International EcoSummit Ecological Sustainability in Columbus, OH in 2012, Ohio Annual Research Review Meeting in Columbus, OH, 52nd Annual Ohio Fish and Wildlife Managers Association in Columbus, OH and 18th International Conference on Aquatic Invasive Species in Niagara Falls Ontario, Canada in 2013. (4) Dr. Stepien presented project results at the American Fisheries Society in Seattle, WA in September 2011, the EcoSummit in Columbus, Ohio in 2012, and while on sabbatical in Abu Dhabi, Poland, and Australia (in Melbourne and Tasmania), and the International Conference on Aquatic Invasive Species in Niagara Falls Ontario, Canada in April 2013. Dr. Leaman gave a public seminar on his work at Bowling Green State University in October, and his student Daniel Hettel gave a seminar in April at the departmental honors symposium. In addition, an article on the overall project is posted on the college website (http://utnews.utoledo.edu/index.php/11_02_2012/interdisciplinary-team-swims-together-to-track-fish-killer) and was profiled in the 2013 issue of “UT Discovers” that highlights health-related research on both University of Toledo campuses. Ph.D. candidate Jennifer Sieracki has presented project results at International Associational of Great Lakes Research Conference in Duluth, MN in 2011, and the International Association for Landscape Ecology Symposium in Newport, RI, and American Fisheries Society annual meeting in St. Paul, MN in 2012. Dr. Bossenbroek presented project results at the Ecological Society of America conference in Austin, TX in 2011. What do you plan to do during the next reporting period to accomplish the goals? Dr. Stepien and her laboratory members are planning to further evaluate field samples for VHSv occurrence, using the new assay described in the PLOS One article. They plan to test a variety of fish samples as well as possible invertebrate vectors. She also is planning to work with Dr. Yan Zhang of the Ohio Department of Agriculture to implement the new assay for the state of Ohio. Dr. Stepien's Ph.D. student Lindsey Pierce is currently evaluating the evolutionary trajectory of four VHSv genes - G, M, P, and Nv - in the Great Lakes in a paper to be submitted in summer 2013. Ms. Pierce is planning to defend her Ph.D. dissertation in summer 2013. Dr. Leaman’s laboratory is planning to finish the current work on the VHSv M protein for publication this summer, and then move on to work on P protein. Although work on M will continue, the P studies will allow his lab to better define overall mechanisms of viral inhibition of host responses, and to provide information on avenues for potential therapeutic intervention to either ameliorate the impact of the virus on host function, or to interfere with viral replication. In particular, we have identified a region of the M protein that is involved in anti-host activity, and developing small peptides spanning this region might allow us to inhibit the adverse affects of M on cellular function. The goal is to initiate these types of “translational” studies after completing the papers in progress. Ph.D. student Jennifer Sieracki from Dr. Bossenbroek's lab is editing her manuscript according to review comments, and will resubmit it for publication by July 29, 2013. She also will submit a Sea Grant small grant proposal to futher develop her spread models and develop a user-friendly interface that will enable others, such as state and federal managers, to use her tool in ArcGIS.

Impacts
What was accomplished under these goals? Our new VHSv assay (submitted to PLOS-One by Pierce, Willey, Palsule, Yeo, Shepherd, and Stepien in April 2013), which was developed by the Stepien and Willey laboratories, is VHSv specific and detects a single VHSv molecule (100% accuracy with 5 molecules). In comparison, 10-64% false negatives characterize standard RT-PCR assays and 46-73% false negatives occur with cell culture assays.Results demonstrate high signal-to-analyte response (slope = 1.00 ± 0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules, with ~ 1,000being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published real-time PCR tests and traditional cell culture diagnostics. The Stepien laboratory is planning next to partner with Dr. Yan Zhang of the Ohio Department of Agriculture to implement thisnew procedure and help develop its potential for export to other states’ labs, with the goal of acommercially available kit – leading to Ohio jobs; as well as protecting our fisheries,aquaculture, and tourism industries. Dr. Leaman’s lab has worked on furthering our understanding of the mechanisms by which VHSv blocks host innate immune responses. Although his studies have implicated several viral proteins in this process, they focused primarily on the VHSv M protein over the past year. This protein can block host protein expression, and studies in the lab have uncovered a likely mechanism for how M functions in this regard. Investigations aimed at finalizing this mechanism and translating the results back to a better understanding of VHSv virulence are in progress. They hope to integrate the sensitive VHSv PCR test (see above) into these studies as they correlate changes in M sequences with host perturbation and viral replication analyses. In addition to these studies, they have cloned and sequenced VHSv P protein from a variety of VHSv strains in anticipation of work on this protein in the coming year. A spatial database of VHSv positive and negative sites has been created by Dr. Bossenbroek's lab, and a model that identifies ballast water as a vector of VHSv spread in the Great Lakes has been completed. This model will be used to develop a generalizable Great Lakes shipping model that can aid in the predicting of the spread of other invasive species.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Stepien, C.A., Pierce, L.R., Blandford, J., Leaman, D. (2013) Evolutionary and biogeographic patterns of an emerging quasispecies: the fish Viral Hemorrhagic Septicemia Virus (VHSv). 18th International Conference on Aquatic Invasive Species, Niagara Falls, ON Canada. April, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Pierce, L.R., Willey, J.C., Palsule, V.V., Yeo, J., Crawford, E.L., Stepien, C.A. (2013) Accurate detection and quantification of the fish Viral Hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay. Midwest Graduate Student Symposium, Toledo, OH. April, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Hettel, D., Wildschutte, J., Williams, T., Pore, A., Leaman, D. (2013) Viral Hemorrhagic Septicemia virus M protein represses host protein expression. University of Toledo Department of Biological Sciences Undergraduate Honors Symposium, Toledo, OH. April, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Palsule, V.V., Pierce, L.R., Willey, J.C., Yeo, J., Crawford, E.L., Stepien, C.A. (2013) An accurate, rapid, and low-cost test to diagnose the Viral Hemorrhagic Septicemia virus (VHSv). 52nd Annual Ohio Fish and Wildlife Managers Association, Columbus, OH. February, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Palsule, V.V., Pierce, L.R., Willey, J.C., Yeo, J., Crawford, E.L., Stepien, C.A. (2013) A new PCR-based assay to detect and quantify the fish Viral Hemorrhagic Septicemia virus (VHSv): a tool for fishery managers. Ohio Annual Research Review Meeting, Columbus, OH. January, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Stepien, C.A. (2013) Fisheries Resources and Invasive Species: Insights from Genetic Studies. Environment Agency - Abu Dhabi - conference to develop joint research with University of Toledo, Abu Dhabi, United Arab Emirates. January, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Leaman, D. (2012) Proteins regulating innate immune responses to RNA viruses in humans, mice and fish. Department of Biology Seminar, Bowling Green State University, Bowling Green OH. October, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Palsule, V., Pierce, L.R., Stepien, C.A. (2012) Does viral load of VHS virus change over the course of infection in immersion challenged and injection challenged yellow perch? International Ecosummit, Columbus, OH. October, 2012
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Leaman, D., Stepien, C.A. (2012) Detection of fish Viral Hemorrhagic Septicemia virus (VHSv) using StaRT-PCR: enhanced quality controls with internal standards. International Ecosummit, Columbus, OH. October, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Stepien, C.A., Pierce, L.R. (2012) Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). EcoSummit, Columbus, OH. October, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pierce, L.R., Stepien, C.A. (2012) Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Evolutionary Biology Meeting, Marseille, France. September, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Sieracki, J.L., Bossenbroek, J.M., Faisal, M. (2012) Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) via commercial shipping in the Laurentian Great Lakes. 142nd Annual Meeting of the American Fisheries Society. St. Paul, MN. August, 2012
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pierce, L.R., Stepien C.A. (2012) Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). International Evolution Conference, Ottawa, Ontario, Canada. July, 2012. Pierce, L.R. (2012) A rapid test to detect the fish Viral Hemorrhagic Septicemia virus (VHSv). Toledo, OH. April, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Sieracki, J.L., Bossenbroek, J.M., Faisal, M. (2012) Modeling the spread of viral hemorrhagic septicemia virus (VHSV) via ballast water discharge in the Laurentian Great Lakes. 27th Annual Landscape Ecology Symposium. Newport, RI. April, 2012
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pierce, L.R., and Stepien, C.A. (2012) Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Midwest Graduate Symposium, Toledo, OH. March, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pierce, L.R., Willey, J., Crawford, E., Leaman, D., and Stepien, C.A. (2012) Detection of fish Viral Hemorrhagic Septicemia virus (VHSv) using StaRT-PCR: enhanced quality control with internal standards. Ohio Fish and Wildlife Managers Association, Columbus, OH. February, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Palsule, V., Pierce, L.R., and Stepien, C.A. (2012) Does viral load of VHS viral change over the course of infection in immersion and injection challenged yellow perch? Ohio Fish and Wildlife Managers Association, Columbus, OH. February, 2012.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Pore, A.J. (2012) Studies on Host-Virus interaction for Viral Hemorrhagic Septicemia virus (VHSv). Masters Thesis Defense, The University of Toledo, Toledo, OH. April, 2012.
  • Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Pore, Adam (2012) Studies on host-virus interaction for Viral Hemorrhagic Septicemia virus (VHSv). University of Toledo Masters Thesis, Department of Biology. (Dr. Douglas Leaman, advisor)
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Pierce, L.R. (2013) Evolutionary history and detection of the Viral Hemorrhagic Septicemia virus (VHSv). Ph.D. Dissertation University of Toledo, Department of Environmental Science (to be defended summer 2013).
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Sieracki, J. (2013) Modeling the secondary spread of invasive species in the Great Lakes. Ph.D. Dissertation University of Toledo, Department of Environmental Science (to be defended fall 2013).
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Pierce, L.R., and Stepien, C.A. (2012) Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Molecular Phylogenetics and Evolution, 63:327-341.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Sepulveda Villet, O.J. and Stepien, C.A. (2011) Fine-scale population genetic structure of Lake Erie yellow perch Perca flavescens in Lake Erie. Canadian Journal of Fisheries and Aquatic Sciences 68:1-19. doi:10.1139/F2011-077
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Grzybowsi, M., Sepulveda Villet, O.J., Stepien, C.A., Rosauer, D., Binkowksi, F., Klaper, R., Shepherd, B., and Goetz, F. (2010) Genetic variation of 17 wild yellow perch populations from the Midwest and East coastal United States using microsatellites. Transactions of the American Fisheries Society. 139:270-287.
  • Type: Websites Status: Published Year Published: 2013 Citation: Project Website (2013): http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html
  • Type: Websites Status: Published Year Published: 2012 Citation: Nowak, C. (2012) Interdisciplinary Team Swims Together to Track Fish Killer: http://utnews.utoledo.edu/index.php/11_02_2012/interdisciplinary-team-swims-together-to-track-fish-killer.
  • Type: Websites Status: Published Year Published: 2011 Citation: Hilton, J. (2011) UT Studies Fish Virus in Lake Erie. The Independent Collegian. http://www.independentcollegian.com/ut-studies-virus-in-lake-erie-1.2 464335
  • Type: Websites Status: Published Year Published: 2010 Citation: Brannan, S. (2010) VHS Test Fishes for Faster Answers. Ohio Sea Grant Communications. http://www.utoledo.edu/as/lec/pdfs/vhs_twineline_spsum2010.pdf
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Leaman, D. (2011) Proteins regulating innate immune responses to RNA viruses in humans, mice and fish. Department of Medical Microbiology Seminar, Toledo, OH. October, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Stepien, C.A., Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Faisal, M., Kim, R. (2011) A rapid genetic test for the VHS Fish virus and viral load from laboratory challenge experiments. American Fisheries Society, Seattle, WA. September, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Bossenbroek, J., Sieracki, J. (2011) Modeling the spread of viral hemorrhagic septicemia virus (VHSv) via Great Lakes shipping. Ecological Society of America, Austin, TX. August, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Stepien, C.A., Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Faisal, M., Kim, R. (2011) A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. American Fisheries Society, Seattle, WA. August, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Pierce, L.R., Willey, J, Faisal, M., Kim, R., Stepien, C.A. (2011) A rapid genetic test for the VHS Fish virus and viral load from laboratory challenge experiments. International Association of Great Lakes Research, Duluth, MN. May, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Sieracki, J., Bossenbroek, J. (2011) Modeling the spread of viral hemorrhagic septicemia virus (VHSv) by within Great Lakes shipping. International Association of Great Lakes Research, Duluth, MN. May, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Stepien, C.A., Willey, J., Leaman, D., Bossenbroek, J. (2011) Genetic detection and geographic analysis of Great Lakes fish infection by Viral Hemorrhagic Septicemia virus (VHSv). Ohio Department of Agriculture, Aquatic Animal Health Committee Meeting, Reynoldsburg, OH. April, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Palsule, V., Pierce, L.R., Crawford, E.C., Pore, A., Willey, J., Leaman, D., Stepien, C.A. (2011). Progress in rapid PCR testing for Viral Hemorrhagic Septicemia virus (VHSv). Ohio Dept. of Agriculture Aquatic Animal Health Care Necropsy Clinic, Reynoldsburg, OH. April, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Pierce, L.R., Willey, J.M., Faisal, M., Kim, R., Stepien, C.A. (2011) A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. Lake Erie Inland Waters Annual Research Review, Columbus, OH. April, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Pierce, L.R., Palsule, V., Willey, J., Faisal, M., Kim, R., Stepien, C.A. (2011) A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. Annual Midwest Graduate Symposium, Toledo, OH. April, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Rominski, R., Pore, A., Leaman, D. (2011) Studies of host-virus interaction for the Viral Hemorrhagic Septicemia Virus phosphoprotein and nonvirion protein. University of Toledo Department of Biological Sciences Undergraduate Honors Symposium, Toledo, OH. April, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Pierce, L. R., Willey, J.M., Crawford, E.L., Leaman, D., Stepien, C.A. (2010) Unique RT-PCR test for replicating viral hemorrhagic septicemia virus, with internal controls. American Fisheries Society Annual Conference, Pittsburg, PA. September, 2010.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Stepien, C.A., Pierce, L. R., Willey, J.M., Crawford, E.L., Leaman, D. (2010) Unique RT-PCR test for replicating viral hemorrhagic septicemia virus, with internal controls. The International Conference on Aquatic Invasive Species, San Diego, CA. September, 2010.
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Pierce LR, Willey JC, Palsule VV, Yeo J, Crawford EL, Shepherd BS, Stepien CA (In review) Accurate detection and quantification of the fish Viral Hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay. Plos One.
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Sieracki, J.L., Bossenbroek, J.M., and Faisal, M. (In revision) Modeling the Secondary Spread of Viral Hemorrhagic Septicemia Virus (VHSV) by Commercial Shipping in the Laurentian Great Lakes. Biological Invasions.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pierce LR, Willey JC, Crawford EL, Palsule VV, Leaman DW, Faisal M, Kim RK, Shepherd BS, Stanoszek LM, Stepien CA (2013) A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): Enhanced quality control with internal standards. Journal of Virological Methods, 189-129-142.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Pierce, L.R., Palsule, V., Willey, J., Faisal, M., Kim, R., Stepien, C.A. (2011) A rapid genetic Test for the VHS fish virus and viral load from laboratory challenge experiments. 2nd Annual Midwest Graduate Symposium, Toledo, OH. March, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Pierce, L.R., Willey, J., Crawford, E., Leaman, D., Stepien, C.A. (2011) Results of a unique RT-PCR test for detecting VHS viral infections in fish. Ohio Fish and Wildlife Managers Association, Columbus, OH. February, 2011.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Pierce, L.R., Willey, J.M., Crawford, E.L., Leaman, D., Stepien, C.A. (2010) Unique RT-PCR test with internal controls to detect, quantify and distinguish active VHSv in fish. Lake Erie Fishery Genetics and Stock Structure Workshop for Managers, Toledo, OH. December, 2010.


Progress 08/01/11 to 07/31/12

Outputs
OUTPUTS: Outputs: Project objectives are to develop, test, and implement new molecular genetic and Geographic Information System tools to rapidly and accurately detect Viral Hemorrhagic Septicemia virus (VHSv) in fish and understand its disease pathways. Specific outputs include: (1) Our new StaRT-PCR (Standardized Reverse Transcriptase Polymerase Chain Reaction) assay was developed, tested, and optimized, for which a manuscript is in review in the journal Marine Biotechnology. StaRT-PCR uniquely uses internal standards to improve accuracy and prevent false negatives. Our results show that StaRT-PCR is VHSv specific to a single VHSv molecule (100% accurate at six molecules), and has no false negatives. In comparison, false negatives ranged from 14-58% in SYBR green real time qRT-PCR tests, and 46-75% in cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. VHSv molecules quantified using StaRT-PCR ranged from 1-1.2x105 VHSv/106 actb1 molecules in wild caught fishes and 1-8.4x105 in laboratory challenged specimens. In the latter, muskellunge with lesions had a greater mean number of viral molecules (1.9x104) than those without (1.1x103). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our StaRT-PCR test thus accurately and reliably detects and quantifies VHSv. (2) Spatial models for identifying the role shipping played in the secondary spread of VHSv were created and optimized. Models that included ballast discharge locations performed better than all other models, with model results having agreement with actual documented occurrences. Montreal was identified as a potential introduction site for VHSv. (3) We assessed the roles of VHSv N, G, P, NV, and M genes in perturbing fish innate immune signaling pathways. We assessed their abilities to individually impact both virus detection and the interferon response pathway, and used a variety of experimental approaches to identify which proteins inhibit these pathways. We identified that the M gene blocks mRNA export from the nucleus to the cytoplasm. We are moving on to evaluate the P gene. Project outputs that reach people include a variety of conference talks and poster presentations. Project goals/outputs to be completed during the no-cost extension are: (1) Convert our StaRT-PCR assay to a more-widely usable real-time PCR platform for dissemination to other diagnostic labs, including the Ohio Department of Agriculture, (2) publish the results of the spread model, and (3) Test the function of the P gene, in comparison to other genes. We thus request a no-cost extension to complete these goals. In addition, a high school classroom exercise on viral-host interactions to the VHS fish virus is being developed by Ph.D. student Lindsey Pierce, which will include a You-Tube video, as part of output. Project website is http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html PARTICIPANTS: Participants: (1) Erin Crawford, Lab. manager: Assisted to develop and optimize primers and internal standards for fish reference genes actb1, ef1a, 18srRNA, and target gene VHSv (N-gene) for StaRT-PCR, as well as reference gene actb1 and target gene VHSv (N-gene) for the real time assay. (2) Lindsey Pierce, Ph.D. candidate: Developed and optimized primers and internal standards for fish reference genes and target genes with Crawford, interfaced with muskellunge challenge program at MSU for data analysis and comparison, worked to detect and quantify VHSv from fish tissues from challenge experiments, collaborated with the Ohio DNR to test fish for VHSv, presented 6 VHS project presentations, made updates to the VHS webpage, wrote a NSF DDIG grant with Dr. Stepien, and published a paper on VHSv evolution with Stepien. (3) Vrushalee Palsule, Technician: Assisted to develop and optimize primers and internal standards with Crawford and Pierce, worked to detect and quantify VHSv from fish tissue samples, gave 2 project presentations at scientific conferences, and assisted Ph.D. student Pierce in testing wild caught samples. (4) Douglas Murphy, Technician (retired in April 2012): Assisted Pierce with VHSv fieldwork, equipment, and ordering of supplies. (5) Dr. Carol Stepien, PI: Attended and developed contacts at 4 scientific conferences and gave 2 VHS presentations. Stepien collaborated with Dr. Faisal of MSU and Dr. Goetz of UWM in laboratory challenges of muskellunge and yellow perch, participated in USDA-APHIS phone conference calls for StaRT-PCR development and ongoing communications. Dr. Stepien also oversaw all grant writing and reports, plus all scientific papers and communications. (6) Dr. James Willey, coPI: Participated in StaRT-PCR development conference calls with USDA-APHIS and mentored Pierce in PCR troubleshooting. (7) Dr. Jonathan Bossenbroek, co-PI: Supervised Ph.D. student Jennifer Sieracki and presented project results at the Ecological Society of American in 2011. (8) Jennifer Sieracki, Ph.D. student: Presented VHSv project results at the 3rd Midwest Graduate Research Symposium and the annual conference for the U.S. Regional Association of the International Association of Landscape Ecology. Sieracki also interfaced with Dr. Faisal of MSU for VHSv geographic coordinates. (9) Dr. Douglas Leaman, coPI: Provided VHSv-IVb infected EPC and BF-2 cell pellets to the Willey lab, trained Pierce in using real-time PCR, and oversaw the experiments. (10) Adam Pore, M.S. student: Completed his VHSv master's work on innate immune function and defended his thesis in May 2012. (11) Robert Rominski, Undergraduate student researcher: Assisted Pore to complete master's thesis research. TARGET AUDIENCES: Target Audiences: We updated our VHS research project link on the Lake Erie Center website (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS _main.html) that displays background information, current methodology, current projects, current progress, and past presentations. This website was constructed to reach the general public for awareness, and the scientific community in order to enhance other research efforts. The website is regularly updated. Our VHS team has presented project presentations at several conferences (American Fisheries Society, Annual League of Women Voters, Annual Midwest Graduate Symposium, Ecological Society of America, Eco Summit, Evolution, International Association of Great Lakes Research, International Conference on Aquatic Invasive Species, Lake Erie Fishery Genetics and Stock Structure Workshop for Managers, Lake Erie Inland Waters Annual Research Review, Ohio Department of Agriculture Aquatic Animal Health, Ohio Fish and Wildlife Managers Association, Sigma Xi Scientific Honorary Society Annual Conference, United States Department of Agriculture Annual Progress Meeting, Walleye and Yellow Perch Task Group Meetings) to increase awareness of current DNA diagnostic detection methods and collaborations with other federal agencies and universities. We developed research collaborations with Dr. Mohamed Faisal and Dr. Robert Kim of MSU to conduct VHSv immersion challenges on muskellunge to compare our newly developed StaRT-PCR assay to currently approved and developing detection diagnostics. To further test our StaRT-PCR assay, we developed collaborations with Dr. Frederick Goetz of the University of Wisconsin Milwaukee and Dr. James Winton of USGS in Seattle to conduct VHSv immersion and injection challenge experiments. PROJECT MODIFICATIONS: One of the original goals for this project was to modify our StaRT-PCR test so that it would detect only the positive strand of VHSv, allowing us to determine if the virus is actively replicating inside the fish (i.e., is transmissible). Over the past year, we conducted extensive work towards this project goal and pursued several possible avenues, to arrive at the conclusion that we need to adjust our project direction. We consulted with Dr. Maureen Purcell from UGSG in Seattle, WA, who also worked towards developing similar tests for other RNA viruses, but experienced similar difficulties that thwarted development of a positive strand test. Thus, our research team has come to the conclusion that developing a reliable specific test for the VHSv positive strand will require much more time and resources than are available through this grant award. We therefore would like to adjust the one goal of this project towards achieving a very positive and promising outcome that would be readily utilizable by a wider variety of diagnostic laboratories. We have completed the development of our StaRT-PCR VHSv test (funded by this grant), which incorporates an expensive Agilent equipment technology that is not readily available in most fish diagnostic laboratories. The laboratory of coPI Dr. James Wiley has been translating StaRT-PCR tests for a variety of human diseases into the more-commonly used and less-expensive ABI7500 real-time PCR platform (Applied Biosystems, Inc.). Most fish diagnostic laboratories have ABI real-time PCR equipment. We thus propose to redirect work from developing a VHSv positive strand specific test to implementing our StaRT-PCR VHSv assay into a real-time 2-color fluorometric assay for use on commonly used real-time platforms, such as the ABI7500. We project that this test will be easy to implement in university, diagnostic, and commercial laboratories, as most of these facilities already have real-time technology. The inclusion of our internal standards in this assay via StaRT-PCR will greatly aid baitfish and aquaculture industries, as other real-time assays do not include these standards and may likely report false negatives. We have already developed and optimized our primers, internal standards, and colored probes for this assay and are currently in the process of testing performance characteristics. Completion of this real time test is projected as late summer.

Impacts
Outcomes/Impacts: Our project outcomes/impacts are a new highly accurate assay to diagnose and quantify VHSv, which is a significant improvement over the existing months-long process of cell culture/plaque assay and other qRT-PCR methods developed to date. Cell culture/plaque assay and other qRT-PCR tests lack the internal standards that give our StaRT-PCR assay 100% accuracy. A publication on the StaRT-PCR results was submitted to the journal Marine Biotechnology in June 2012, which demonstrates that our new StaRT-PCR assay is rapid and accurate under all conditions, including in the face of possible PCR inhibitors, and unlike other available VHSv assays does not result in false negatives. Findings to date are detailed and disseminated on our project website: http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html. We now are in the process of transforming our StaRT-PCR assay to a real-time PCR platform in order to facilitate cost-effective adoption of our method by federal and state diagnostic labs, with Ohio Department of Agriculture and Michigan State University serving as our first test implementers. Several possible fish infections of VHSv were analyzed for the Ohio DNR and for Michigan DNR, which the new publication details, reporting on both whether the fish had VHSv, and quantified the virus for positive cases. Collaborations with several other VHSv research groups, and donations of test samples included: Michigan State University's Aquatic Animal Health Laboratory, USGS Western Fisheries Research Center in Seattle WA, Cornell University's College of Veterinary Medicine, and University of Wisconsin, Milwaukee (WATER Institute). We partnered with Dr. Mohamed Faisal of Michigan State University in a series of laboratory VHSv challenge experiments involving juvenile muskellunge, in which we quantified the amount of virus and reported on our findings in the publication. We also assayed quantities of VHSv in yellow perch challenge experiments, working with Dr. James Winton of the USGS Western Fisheries Research Center and Dr. Frederick Goetz of University of Wisconsin. Our project has resulted in a M.S. thesis (Mr. Adam Pore), and Ms. Lindsey Pierce and Ms. Jennifer Sieracki are each continuing to work on their respective Ph.D. dissertations. Other employees and students who have been trained and mentored as part of the project include Ms. Vrushalee Palsule (VHSv laboratory technician), Ms. Erin Crawford (head technician, Dr. Willey), Mr. Shane Woolwine (Dr. Stepien), and undergraduate researcher Robert Rominski (Dr. Leaman). Many project presentations have been given, at national, regional and local levels. Pierce and Stepien were awarded a National Science Foundation Doctoral Dissertation Improvement Grant (NSF DDIG) to investigate the evolutionary patterns of VHSv, for which they published a 2012 paper in the journal Molecular Phylogenetics and Evolution. We anticipate that our new assay will significantly improve diagnostics of the VHSv disease, aiding the fishery, aquaculture, transport, and tourism industries.

Publications

  • Bossenbroek, J., Sieracki, J. (2011) Modeling the spread of Viral Hemorrhagic Septicemia virus (VHSv) via Great Lakes shipping. Ecological Society of America, Austin, TX.
  • Leaman, D. 2011. Proteins regulating innate immune responses to RNA viruses in humans, mice and fish. Department of Medical Microbiology Seminar, Toledo, OH.
  • Stepien, C.A., Willey J., Leaman, D., Bossenbroek, J. (2011) Genetic detection and geographic analysis of Great Lakes fish infection by Viral Hemorrhagic Septicemia virus (VHSv). Ohio Dept. of Agriculture Aquatic Animal Health Care Committee Meeting, Reynoldburg, OH. (oral presentation given by Palsule and Pierce)
  • Pierce, L.R., Crawford, E.C., Palsule, V., Pore, A., Willey, J., Leaman, D., Stepien, C.A. (2011). Progress in rapid PCR testing for Viral Hemorrhagic Septicemia virus (VHSv). Ohio Dept. of Agriculture Aquatic Animal Health Care Necropsy Clinic, Reynoldburg, OH. (presentation given by Palsule)
  • Stepien, C.A., Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Faisal, M., Kim, R. (2011) A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. American Fisheries Society, Seattle, WA. (poster presentation by Stepien)
  • Pierce, L.R., Palsule, V., Willey, J., Faisal, M., Kim, R., Stepien, C.A. (2011) A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. Annual Midwest Graduate Symposium, Toledo, OH. (oral presentation by Pierce)
  • Publications: 
 Pierce, L.R., Stepien, C.A. (2012). Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Molecular Phylogenetics and Evolution, 63:327-341.
  • Pore, Adam (2012) Studies on host-virus interaction for Viral Hemorrhagic Septicemia virus (VHSv). University of Toledo Masters Thesis, Department of Biology. (Dr. Douglas Leaman, advisor)
  • Pierce, L.R., Willey, J.C., Crawford, E.L., Palsule, V.V., Leaman, D.W., Faisal, M., Kim, R.K., Shepherd, B.S., Stanoszek, L.M., Stepien, C.A. (2012) A new StaRT-PCR approach to detect the fish Viral Hemorrhagic Septicemia virus (VHSv): Enhanced quality control with internal standards. Marine Biotechnology (submitted June 2012).
  • Sieracki, J., Bossenbroek, J. (2012) Modeling the secondary spread of Viral Hemorrhagic Septicemia Virus (VHSv) in the Great Lakes by commercial shipping (to be submitted in fall 2012).
  • Pierce, L.R. (2013) Evolutionary history and detection of the Viral Hemorrhagic Septicemia virus (VHSv). Ph.D. Dissertation University of Toledo, Department of Environmental Science (to be defended spring 2013).
  • Sieracki, J. (2013) Modeling the secondary spread of invasive species in the Great Lakes. Ph.D. Dissertation University of Toledo, Department of Environmental Science (to be defended fall 2013).
  • Project Website (2012): http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html
  • Newsletters: Hilton, J. 2011. UT Studies Fish Virus in Lake Erie. The Independent Collegian. http://www.independentcollegian.com/ut-studies-virus-in-lake-erie-1.2 464335
  • Abstracts and Conference Presentations: Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Leaman, D., Stepien, C.A. (2012) Detection of fish Viral Hemorrhagic Septicemia virus (VHSv) using StaRT-PCR: Enhanced quality controls with internal standards. Eco Summit, Columbus, OH. (submitted for Sept. 2012)
  • Palsule, V., Pierce, L.R., Stepien, C.A. (2012) Does viral load of VHS virus change over the course of infection in immersion challenged and injection challenged yellow perch Eco Summit, Columbus, OH. (submitted for Sept. 2012)
  • Stepien, C.A., Pierce, L.R. (2012) Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Eco Summit, Columbus, OH. (submitted for Sept. 2012)
  • Pierce, L.R., Stepien, C.A. (2012) Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Evolution, Ottawa, Canada. (submitted for July 2012)
  • Pierce, L.R. (2012) A rapid test to detect the fish Viral Hemorrhagic Septicemia virus (VHSv). Annual League of Women Voters, Oregon, OH. (oral outreach presentation)
  • Pore, A.J. (2012) Studies on Host-Virus interaction for Viral Hemorrhagic Septicemia virus (VHSv). Masters Thesis Defense, The University of Toledo, Toledo, OH.
  • Pierce, L.R., Stepien, C.A. (2012) Evolution and biogeography of an emerging quasispecies: Diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv). Annual Midwest Graduate Symposium, Toledo, OH. (oral presentation)
  • Palsule, V., Pierce, L.R., Stepien, C.A. (2012) Does viral load of VHS virus change over the course of infection in immersion and injection challenged yellow perch Ohio Fish and Wildlife Managers Association, Columbus, OH. (oral presentation)
  • Pierce, L.R., Willey, J., Crawford, E., Leaman, D., Stepien, C.A. (2012) Detection of fish Viral Hemorrhagic Septicemia virus (VHSv) using StaRT-PCR: Enhanced quality control with internal standards. Ohio Fish and Wildlife Managers Association, Columbus, OH. (poster)


Progress 08/01/10 to 07/31/11

Outputs
OUTPUTS: Outputs: Our continuing objectives are to develop, test, and implement new molecular genetic and Geographic Information Systems (GIS) tools to rapidly and accurately detect active, replicating Viral Hemorrhagic Septicemia virus (VHSv) infection in fish and understand its disease pathways. Progress includes: (1) Primer and internal standards for housekeeping genes beta-actin (ACTB), EF1-alpha, 18S RNA, and VHSv strains spanning the N gene were developed, optimized, and used to assay analytical performance of our standardized mixture of internal standards (SMIS). Primers are specific for VHSv as no amplification was seen for human viruses, vesicular stomatitis virus, and encephalomyocarditis virus or for fish virus infectious hematopoietic necrosis virus. Sensitivity testing showed a lower detection threshold of 6 molecules/reaction (rx) with 100% accuracy. RT (reverse transcription) and PCR efficiency were measured various amounts of fish RNA/RT. A larger amount of 30ug fish RNA/RT rx showed significant decrease (p=0.035) compared to a 1og standard. PCR efficiency per EDTA/rx showed no difference in number of VHSv molecules/10e6 ACTB, with decrease in amplification signal from 100 fluorescent light units at 1.0mm EDTA/rx, to 30 at 1.3mm, and 0 at 1.4mm. (2) Positive strand primers for ACTB and VHSv-IVb were selected for gene-specific reverse transcription to detect active virus. An alien tag sequence was added to the VHSv-IVb R2 primer to use in a strand-specific reverse transcription reaction. VHSv-IVb F2 primer and a primer corresponding to the tag sequence were used for PCR amplification and detection. In vitro transcription was used to generate RNA fragments of the plus and minus strands of VHSv-IVb to use as controls for the development of the active test. Specificity and sensitivity experiments are ongoing. (3) We cloned three of the VHSv IVb proteins into eukaryotic expression vectors for expression in fish cells. We acquired expression plasmids for fish IFN and MAVS to study the role of these pathways in cellular detection and resistance to VHSv. We assessed the abilities of several VHSv-IVb proteins to perturb innate immune response pathways. We demonstrated that IFN pretreatment of cells fully blocked VHSv replication. To assess the potential roles of VHSv M, P and NV proteins in this process, we performed a variety of cellular assays in which these viral proteins were expressed in conjunction with activators of the innate immune response (either MAVS or IFN), and downstream responses monitored by target promoter/luciferase constructs or endogenous gene expression. Results suggested that both M and P proteins inhibited cellular responses, while NV appeared to augment innate immune response. (4) Obtained geographic coordinates of VHSv occurrences in the Great Lakes from Dr. Mohamed Faisal. Construction of best-fit model to evaluate VHSv pathways is currently underway. (5) Conducted phylogenetic analyses of the relationships among VHSv strains and substrains using G, N, and NV gene sequences, to be related to the evolutionary diversification of IVb, and cellular response to variants. PARTICIPANTS: Participants: 1) Erin Crawford, Lab. manager: Developed and optimized primers and internal standards for fish housekeeping genes beta actin, EF1-alpha, 18s RNA, RPL1, and target gene VHSv; Conducted work on active VHSv test. 2) Lindsey Pierce, Ph.D. candidate: Developed and optimized primers and internal standards for fish housekeeping genes and target gene with Crawford; Interfaced with muskellunge challenge program at Michigan State University (MSU) for data analysis and comparison; Worked on detection and quantification of VHSv in tissues from MSU's immersion challenge; Collaborated with Michigan DNR 2010 to sample and test gizzard shad for potential VHS outbreak; Tested walleye from Lake Erie fish kills for Ohio DNR; Presented VHS project results at AFS 2010, Lake Erie Fishery Genetics and Stock Structure Workshop for Managers 2010, Ohio State Annual Research Review 2011, Annual Ohio Fish and Wildlife Management Conference 2011, Annual Midwest Graduate Symposium 2011, and the International Association for Great Lakes Research 2011; Updated VHS webpage. 3) Vrushalee Palsule, Technician: Assisted to develop and optimize primers and internal standards with Crawford and Pierce; Worked on detection and quantification of VHSv from challenge experiment tissues; Helped to present VHS project results at the Ohio Department of Agriculture Aquatic Animal Health meeting and led a presentation to them in Sept. 2011; Assisted Ph.D. student Pierce with wild caught samples. 4) Adam Pore, M.S. student: Co-authored a presentation with undergraduate Robert Rominski from Dr. Leaman's lab. 5) Robert Rominski, Undergraduate student: Presented VHS project results at the Univ.of Toledo's Department of Biological Sciences Undergraduate Honors Symposium 2011. 6) Douglas Murphy: Assisted Pierce with fieldwork and ordering. 7) Dr. Carol Stepien, PI: Organized and hosted the Lake Erie Fishery Genetics and Stock Structure Workshop for Managers that included VHS project work 2010; Gave oral VHS presentation at the International Conference on Aquatic Invasive Species 2010 and poster presentation at AFS 2011; Collaborated with Dr. Mohamed Faisal of MSU in immersion challenge experiment and on project design; Continued collaborations with Univ. of Wisconsin Milwaukee and USGS Seattle for yellow perch immersion challenge;Conference calls with USDA-APHIS ; Oversaw all grant writing and reports, plus all scientific papers and communications. 8) Dr. James Willey, coPI: Developed the actively replicating test and supervised lab work. 9) Dr. Douglas Leaman, coPI: provided VHSv-IVb infected EPC and BF-2 cell pellets to the Willey lab; trained Pierce in real-time PCR, and oversaw the experiments; Began work to develop yellow perch cell line; 10) Dr. Jonathan Bossenbroek, co-PI: supervised Ph.D. student Jennifer Sieracki. (11) Jennifer Sierlacki, Ph.D. student: presented VHS project results at the International Association of Great Lakes Research 2011 and developed contacts with Dr. Mohamed Faisal for VHSv geographic coordinates. TARGET AUDIENCES: Target Audiences: 
We have updated our VHS research project link on the Lake Erie Center website (http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_main.html) that displays background information, current methodology, current projects, current progress, and future considerations. This website was constructed to reach the general public for awareness, and the scientific community in order to enhance other research efforts. The website is regularly updated. Our VHS team has presented project presentations at several meetings (International Association of Great Lakes Research, Ohio Department of Agriculture Aquatic Animal Health, University of Toledo's Department of Biological Sciences Undergraduate Honors Symposium, 2nd Annual Midwest Graduate Symposium, Ohio Fish and Wildlife Managers Association, Lake Erie Inland Waters Annual Research Review, Lake Erie Fishery Genetics and Stock Structure Workshop for Managers, American Fisheries Society, and the International Conference on Aquatic Invasive Species) to increase awareness of current DNA diagnostic detection methods and collaborations with other federal agencies and universities. We developed ongoing research collaborations with Dr. Mohamed Faisal and Dr. Robert Kim of Michigan State University for VHSv immersion challenges on muskellunge, comparing our StaRT-PCR assay results with other detection diagnostics. To further test our StaRT-PCR assay, we collaborated with Dr. Fredrick Goetz of the University of Wisconsin Milwaukee and Dr. James Winton of USGS in Seattle who conducted VHSv immersion and injection challenge experiments, for which we are assaying the results using StaRT-PCR. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Outcomes/Impacts: 
Impacts to date include: (1) Dr. Stepien organized and hosted the Lake Erie Fishery Genetics and Stock Structure Workshop for Managers that included VHS project work in December 2010, gave an oral VHS presentation and developed contacts with other VHSv researchers at the International Conference on Aquatic Invasive Species, Aug. 2010 in San Diego CA, presented at the American Fisheries Conference in Seattle in Sept. 2011, collaborated with Dr. Mohamed Faisal of Michigan State University in immersion challenge experiment with muskellunge, continued collaboration with the University of Wisconsin Milwaukee for yellow perch immersion challenge, and participated in USDA-APHIS phone conference calls for StaRT-PCR development and ongoing communications. (2) Dr. Stepien's Ph.D. student Lindsey Pierce presented VHS platform presentations, at the American Fisheries Society Sept. 2010 where she was selected to compete for best student paper, receiving honorable mention for best oral presentation, Lake Erie Fishery Genetics and Stock Structure Workshop for Managers Dec. 2010, Ohio State Annual Research Review Jan. 2011, 51st Annual Ohio Fish and Wildlife Management Conference 2011, 2nd Annual Midwest Graduate Symposium 2011 where she was awarded 2nd place best oral presentation and Best Woman in STEMM award, and the International Association for Great Lakes Research June 2011. (3) Adam Pore, a master's student studying VHSv immune pathways in Dr. Leaman's laboratory (UT) co-authored a presentation with undergraduate Robert Rominski at the University of Toledo's Department of Biological Sciences Undergraduate Honors Symposium 2011. (4) Robert Rominski, undergraduate student (Dr. Leaman) presented VHS project results at the University of Toledo's Department of Biological Sciences Undergraduate Honors Symposium 2011 and was awarded 2nd place best oral presentation. (5) Vrushalee Palsule, project technician, presented project results to the Ohio Dept. of Agriculture Aquaculture Program in Sept. 2011. (6) We updated our VHS research project link on the Lake Erie Center website, which describes background information, current methodology, current projects, current progress, and future considerations. http://www.utoledo.edu/nsm/lec/research/glgl/VHS/VHS_ main.html. (7) We completed an immersion challenge experiment and are currently working on tissue processing in collaboration with Dr. Mohamed Faisal of Michigan State University to test our StaRT-PCR results. (8) We completed an immersion and injection challenge experiment in collaboration with Dr. Fredrick Goetz of the University of Wisconsin Milwaukee and are processing the samples. (9) Used newly developed assay to test for VHS gizzard shad die-off in Harrison Michigan Dec. 2010 (negative result) and walleye from Lake Erie fish kill at Davis Besse and Port Clinton for Ohio DNR (negative result). (10) Dr. Jon Bossenbroek's Ph.D. student Jennifer Sieracki presented VHS results at the International Association of Great Lakes Research May 2011 and developed contacts with Dr. Mohamed Faisal for additional VHS outbreak sites in the Great Lakes region.

Publications

  • Pierce, L. R., Willey, J.M., Crawford, E.L., Leaman, D., Stepien, C.A. 2010. Unique RT-PCR test for replicating viral hemorrhagic septicemia virus, with internal controls. American Fisheries Society Annual Conference, Pittsburg, PA. September, 2010.
  • Stepien, C.A., Pierce, L. R., Willey, J.M., Crawford, E.L., Leaman, D. 2010. Unique RT-PCR test for replicating viral hemorrhagic septicemia virus, with internal controls. The International Conference on Aquatic Invasive Species, San Diego, CA. September, 2010.
  • Publications: Sepulveda Villet, O.J. and Stepien, C.A. (2011) Fine-scale population genetic structure of Lake Erie yellow perch Perca flavescens in Lake Erie. Canadian Journal of Fisheries and Aquatic Sciences 68:1-19. doi:10.1139/F2011-077
  • Pierce, L.R., Stepien, C.A. (2011) Evolution and Biogeography of an Emerging Quasispecies: Patterns of the Fish Viral Hemorrhagic Septicemia Virus (VHSv) from the G, N, and Nv Genes (Submitted September 2011).
  • Grzybowsi, M., Sepulveda Villet, O.J., Stepien, C.A., Rosauer, D., Binkowksi, F., Klaper, R., Shepherd, B., and Goetz, F. (2010) Genetic variation of 17 wild yellow perch populations from the Midwest and East coastal United States using microsatellites. Transactions of the American Fisheries Society. 139:270-287.
  • Newsletters: Hilton, J. 2011. UT Studies Fish Virus in Lake Erie. The Independent Collegian. http://www.independentcollegian.com/ut-studies-virus-in-lake-erie-1.2 464335 Brannan, S. 2010. VHS Test Fishes for Faster Answers. Ohio Sea Grant Communications. http://www.utoledo.edu/as/lec/pdfs/vhs_twineline_spsum2010.pdf
  • Abstracts and Conference Presentations: Stepien, C.A., Pierce, L.R., Crawford, E.L., Palsule, V., Willey, J., Faisal, M., Kim, R. 2011. A rapid genetic test for the VHS Fish virus and viral load from laboratory challenge experiments. American Fisheries Society, Seattle, WA. September, 2011. (presented by Stepien).
  • Bossenbroek, J., Sieracki, J. 2011. Modeling the spread of viral hemorrhagic septicemia virus (VHSv) via Great Lakes shipping. Ecological Society of America, Austin, TX. August, 2011
  • Pierce, L.R., Willey, J, Faisal, M., Kim, R., Stepien, C.A. 2011. A rapid genetic test for the VHS Fish virus and viral load from laboratory challenge experiments. International Association of Great Lakes Research, Duluth, MN. May, 2011.
  • Sieracki, J., Bossenbroek, J. 2011. Modeling the spread of viral hemorrhagic septicemia virus (VHSv) by within Great Lakes shipping. International Association of Great Lakes Research, Duluth, MN. May, 2011.
  • Stepien, C.A., Willey, J., Leaman, D., Bossenbroek, J. 2011. Genetic detection and geographic analysis of Great Lakes fish infection by Viral Hemorrhagic Septicemia virus (VHSv). Ohio Department of Agriculture, Aquatic Animal Health Committee Meeting, Reynoldsburg, OH. April, 2011.
  • Pierce, L.R., Willey, J.M., Faisal, M., Kim, R., Stepien, C.A. 2011. A rapid genetic test for the VHS fish virus and viral load from laboratory challenge experiments. Lake Erie Inland Waters Annual Research Review, Columbus, OH. April, 2011.
  • Rominski, R., Pore, A., Leaman, D. 2011. Studies of host-virus interaction for the Viral Hemorrhagic Septicemia Virus phosphoprotein and nonvirion protein. University of Toledo Department of Biological Sciences Undergraduate Honors Symposium, Toledo, OH. April, 2011.
  • Pierce, L.R., Palsule, V., Willey, J., Faisal, M., Kim, R., Stepien, C.A. 2011. A rapid genetic Test for the VHS fish virus and viral load from laboratory challenge experiments. 2nd Annual Midwest Graduate Symposium, Toledo, OH. March, 2011.
  • Pierce, L.R., Willey, J., Crawford, E., Leaman, D., Stepien, C.A. 2011. Results of a unique RT-PCR test for detecting VHS viral infections in fish. Ohio Fish and Wildlife Managers Association, Columbus, OH. February, 2011.
  • Pierce, L.R., Willey, J.M., Crawford, E.L., Leaman, D., Stepien, C.A. 2010. Unique RT-PCR test with internal controls to detect, quantify and distinguish active VHSv in fish. Lake Erie Fishery Genetics and Stock Structure Workshop for Managers, Toledo, OH. December, 2010.