M.L. 2013 Minnesota Aquatic Invasive Species Research Center Subproject Abstract For the Period Ending June 30, 2019 SUBPROJECT TITLE: MAISRC Subproject 7.2: Developing eradication tools for invasive species Phase II: Virus Discovery and evaluation for use as potential biocontrol agents SUBPROJECT MANAGER: Dr. Nicholas Phelps AFFILIATION: University of Minnesota Department of Fisheries, Wildlife and Conservation Biology MAILING ADDRESS: 2003 Upper Bufford Circle CITY/STATE/ZIP: St. Paul, MN 55108 PHONE: 612-624-7450 E-MAIL: phelp083@umn.edu WEBSITE: http://www.maisrc.umn.edu FUNDING SOURCE: Environment and Natural Resources Trust Fund (ENRTF) LEGAL CITATION: M.L. 2013, Chp. 52, Sec. 2, Subd. 06a SUBPROJECT BUDGET AMOUNT: $455,210 AMOUNT SPENT: $424,946 AMOUNT REMAINING: $20,264 Overall Subproject Outcome and Results One possible component to an effective integrated pest management plan for aquatic invasive species would be through the introduction or promotion of species-specific pathogens. This high-risk, high-reward approach must be carefully assessed with thorough investigation and scientifically justified risk assessment. In Phase II of this long-term effort, we characterized the virome invasive and native fish species and zebra mussels. We achieved our ultimate goal of this project and identified a candidate virus (koi herpes virus) that caused high mortality in common carp and was not detected in native fish species – this virus will be the focus of Phase III. We also identified many other novel and undescribed viruses in health and dead fish, however the implications of these results are unknown and warrant additional research to better understand the threat to native species and/or potential as biocontrol agents. The virome of zebra mussels was also interesting with lower viral diversity than the fish species investigated; however, no viruses emerged as potential zebra mussel biocontrol candidates from field samples or laboratory trials. This study emphasized the value of advanced molecular approaches to unbiased viral discovery and diagnostics. The methods we developed and optimized for sample collection, processing, and sequence analysis (all together called a ‘pipeline’), have informed testing protocols at the Minnesota Veterinary Diagnostic Laboratory. We have also elevated awareness among managers that viral diversity is much higher than currently known and deserves more attention as early indicators of potential threats. The project team spent considerable time during Phase II engaging with managers, scientists, and the public in multiple formats. It is important that this type of research is transparent and understandable to all stakeholders. To that end, we held formal in person meetings, attended local-national-international scientific conferences, published a peer-review manuscript, networked with internationally-renowned experts, produced two videos, and provided interviews for print, radio and TV media. Subproject Results Use and Dissemination We had learned during Phase 1 of this project (MAISRC Sub Project 7.1) that communication, outreach and transparency were very important for this type of project. To that end, the project team has spent considerable time engaging with managers, scientists, and the public in multiple formats. This has included formal in person meetings, local-national-international scientific conferences, peer-review publication, networking with

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internationally-renowned experts, video production, and print, radio and TV media. A summary of this is listed below: Formal in-person meetings: Great Lakes Fish Health Committee, MN DNR Koi Herpes Virus Working Group. Scientific conferences: American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop, MAISRC showcase (x3), International Conference on Aquatic Invasive Species, Minnesota Veterinary Diagnostic Laboratory, Aquatic Invaders Summit III, Freshwater Mollusk Conservation Society, International Symposium on Aquatic Animal Health. NOTE: Most of these conferences were supported by non-LCCMR funding. Peer-review publication: Padhi, S. K., I. E. Tolo, M. McEachran, A. Primus, S. K. Mor, N. B. D. Phelps. In press. Koi herpesvirus and carp edema virus: Infections and coinfections during mortality events of wild common carp in the United States. Journal of Fish Disease. Several other publications are in progress. Networking with experts: Dr. Ken McColl, Dr. Tom Waltzek, Dr. Mikolaj Ademek, and others. Video production: Video 1 (viewed 822 times as of 8/8/19), Video 2 (viewed 96 times as of 8/8/19). Media: New York Times, KSTP 5, KARE 11, Star Tribune, Minnesota Daily, MN DNR Press release, MAISRC newsletters.

Environment and Natural Resources Trust Fund (ENRTF) M.L. 2013 Minnesota Aquatic Invasive Species Research Center Sub-Project Work Plan Final Report

Date of Report: August 16, 2019

Final Report

Date of Work Plan Approval: February 1, 2017

Sub-Project Completion Date: June 30, 2019

Project Completion Date: June 30, 2019

SUB-PROJECT TITLE: MAISRC Sub-Project 7-2: Developing eradication tools for invasive species Phase II: Virus Discovery and evaluation for use as potential biocontrol agents Sub-Project Manager: Dr. Nicholas Phelps

Organization: University of Minnesota – Minnesota Aquatic Invasive Species Research Center

Mailing Address: 135 Skok Hall, 2003 Upper Bufford Circle

City/State/Zip Code: St. Paul, MN 55108

Telephone Number: (612) 624-7450

Email Address: phelp083@umn.edu

Web Address: http://www.maisrc.umn.edu Location: Statewide

Total ENRTF Sub-Project Budget: Sub-Project Budget: $445,210

Amount Spent: $424,946

Balance: $20,264

Legal Citation: M.L. 2013, Chp. 52, Sec. 2, Subd. 06a Appropriation Language: $4,350,000 the first year and $4,350,000 the second year are from the trust fund to the Board of Regents of the University of Minnesota to develop and support an aquatic invasive species (AIS) research center at the University of Minnesota that will develop new techniques to control aquatic invasive species including Asian carp, zebra mussels, and plant species. This appropriation is available until June 30, 2019, by which time the project must be completed and final products delivered.

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I. SUB-PROJECT TITLE: MAISRC Sub-Project 7-2: Developing eradication tools for invasive species: Phase II: Virus Discovery and evaluation for use as potential biocontrol agents II. SUB-PROJECT STATEMENT: Rationale: Although ambitious, eradication of aquatic invasive species is the ultimate goal of many aquatic invasive species. One possible approach would be through the introduction or promotion of species-specific pathogens. This high-risk, high-reward approach must be carefully assessed with thorough investigation and scientifically justified risk assessment. Phase I (Years 1-2.5) of the long-term project provided initial baseline data on viruses of carp species in the region. Phase II (Years 2.5-6) will build upon this work for carp species and now include zebra mussels to utilize newly developed techniques to more strategically identify viral biocontrol candidates for control of invasive carp and zebra mussels. More specifically, Phase II will 1a) Collect apparently healthy invasive carp and mussel species in the Midwest region; 1b) Collect samples from mortality events of native and invasive fish and mussel populations in the Midwest region; 2) Conduct virus discovery by next generation sequencing and culture potential pathogens; 3) Determine the disease causing potential of two selected viruses, one for native and invasive fish and the other for native and invasive mussels; and 4) Communicate findings to scientific, management, and public stakeholders. This will provide the scientific foundation to begin to evaluate specific pathogens for invasive species control. Furthermore, understanding the virome of invasive species will serve as a potential early indicator for the movement and distribution of pathogens that may threaten native species. Phase II will largely be basic research (60%) generating baseline data on the virome diversity of invasive and native species. Significant effort will also be in applied research (40%), whereby diagnostic and disease challenge findings will be used to inform the health management of fish populations. III. SUB-PROJECT STATUS UPDATES: Sub-Project Status as of July 31, 2017: During the first part of the project, we have focused our efforts on sample collection. We have collected samples from six fish kill events of invasive and native fish. Koi Herpes Virus (KHV) was identified from a large common carp mortality event in Lake Elysian. This is a significant finding since this is the first report of KHV in wild fish in Minnesota and the candidate biocontrol agent for common carp in Australia. We are working with the MN DNR and hope to conduct follow up surveys in the coming months to estimate viral persistence, mortality rates and prevalence in surrounding lakes. Sampling of healthy and sick/dead fish and mussels will continue in the coming months. We have made changes within our personnel category due to the promotion of Dr. Sunil Kumar Mor. Dr. Mor is now an Assistant Professor with the Minnesota Veterinary Diagnostic Laboratory and head of the Molecular Development section. Although his percent effort will be lower, the capacity and value he brings with this new position will be highly beneficial to the project. In addition, the official start dates of Dr. Mor and Dr. Alex Primus has been delayed to 7/1/17. With the cost savings we have hired Dr. Soumesh Kumar Padhi to be a full time post doctoral associate starting in August 2017. We have also hired Dr. Todd Knutson, a bioinformatics specialist to assist part-time with the project. Lastly, we have added Isaiah Tolo to the team. Isaiah received the competitive University of Minnesota Diversity Scholars Fellowship for the 2017-2018 academic year and will be at no cost to the project until Year 2. The descriptions in Column A of the budget spreadsheet have been updated accordingly. These changes in personnel do not affect the overall budget, but have delayed spending, hence a full balance on this budget line. In the meantime, Meg Thompson has provided assistance on the project by collecting and processing samples. She is currently being paid from a non-ENRTF source of funds.

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We learned from Phase I of this project (MAISRC SubProject 7-1) that an increased communication effort was needed to generate collaboration on sample collection. We have presented at the joint meeting of the American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop and the Great Lakes Fish Health Committee to present on this project. The presentations were titled: “Investigating fish kills: Looking back, looking deep and looking forward” and “Understanding the virome of invasive carp: What it could mean for biocontrol”. These presentations resulted in an active discussion on the potential use of viruses for biocontrol, interest to submit samples for the project and potential collaborations for future research efforts related to this project. In addition, we have invited a world leader on the use of viruses for biocontrol, Dr. Ken McColl (Commonwealth Scientific and Industrial Research Organization, Australia), to present at the 2018 iCOMOS meeting to be held at the University of Minnesota, more information: http://icomos.umn.edu. We expect that as part of Dr. McColl’s visit, we will host meetings with members of state and federal agencies to socialize this approach and generate ideas for future research needs. Sub-Project Status as of January 29, 2018 The project is progressing as expected. Dr. Soumesh Kumar Padhi has joined the project as post-doctoral associate on September 11, 2017. The last quarter of this project was focused on healthy common carp and silver carp sampling along with fish kill events of native and invasive species. We have also collected zebra mussels from different lakes in Minnesota. A work flow, starting from sample homogenization, sample pooling, nucleic acid extraction by targeting viral particle concentration, removal of host genome contamination in the NGS process, detection of KHV, SVCV and CEV from samples using qPCR are currently being optimized. Based on these optimized protocols we will process all the sampled tissue for virus analysis. The communications efforts were increased by giving presentations at the MAISRC showcase and Minnesota Veterinary Diagnostic Laboratory. The project was also presented at the 20th International Conference on Aquatic Invasive Species entitled “Understanding the Carp Virome: What Could It Mean for the Control of Invasive Carp?”. Amendment request (1/29/18): We are requesting to move the moderate cost savings from the capital equipment purchase to a new service category for shipping samples from collaborating labs. We expect no additional expenses related to capital equipment. We could now use the extra funds to improve sample collection for fish kill events from other states. This amendment request does not change the scope of the project, timeline or overall budget. Activity 1 ($69,500 to $69,782; net = $282)

- Create new service category for shipping, $282 Activity 2 ($8,000 to $7,718; net = -$282)

- No additional expenses are expected in this category, balance should be $0 Amendment Approved: 02/06/18 Sub-Project Status as of July 31, 2018: We have made significant progress in the last six months and are on schedule. All fish kill and healthy fish tissue samples from the 2017 season were processed and screened for the presence of KHV, CEV and SVCV. We confirmed that all carp kills investigated as part of this project were associated with KHV. This is a major finding and getting international attention. Interestingly, we have detected CEV in two different lakes, co-infected with KHV. This is a very unique infection and the first time CEV has been detected in wild common carp in Minnesota and the second time in the USA. The thymidine kinase and partial p4a genes were amplified by conventional PCR from KHV and CEV positive tissues, respectively. Sanger sequencing was performed to get the nucleotide sequences of these amplified genes and determine the relationship of KHV and CEV present in MN to the other international variants. These results are still pending but promise to provide an understanding of genotypic distribution of KHV and CEV viral populations in the region. Sampling of ongoing fish kills continues for the 2018 field season – as of this report, 15 mortality events have been investigated, with results pending.

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A more complete picture of the viral communities present in healthy carp, fish kills and zebra mussels is moving at a good peace. The viral RNAs were eluted using a newly developed and optimized RNA extraction protocol from all the tissues collected in 2017 field season. These RNA samples were submitted to University of Minnesota Genomics Centre (UMGC) for RNA-Hiseq next generation sequencing. The optimization of DNA-NGS protocol is under process. Based on our research and consultation with others, we have decided move forward with the investigation of KHV as a potential biocontrol agent in our experimental challenge study. We have received two specific cell lines required for isolation of KHV and are currently growing those cells for subsequent in vitro culture. Members of our research team presented at the Eastern Fish Health Workshop, Aquatic Invader Summit and a special meeting of the Freshwater Mollusk Conservation Society focused on the health of native and invasive mussels. The presentations were all well received and garnered significant interest by attendees. We are preparing a manuscript on the KHV and CEV outbreaks we observed during the 2017 season. Dr. Ken McColl from Commonwealth Scientific and Industrial Research Organization, Australia has visited MAISRC in 3rd May 2018 during iCOMOS-2018 to present “Use of virus as a biocontrol agent”. Our research group had a meeting with him to discus the different approaches and future research needs towards the development of current biocontrol projects. Amendment request (7/17/18): We are requesting to move funds between categories and activities due to cost savings in Activity 1 and additional needs in Activity 2. This amendment request does not change the scope of the project, timeline or overall budget. Activity 1 ($69,782 to $57,162; net = -$12,620) We have saved travel funds by collaborating with other researchers and leveraging their ability to collect samples and use their own supplies.

- $500 reduction to in-state travel - $1,500 reduction to domestic travel - $10,620 reduction to lab and field supplies

Activity 2 ($259,828 to $272,448; net = $12,620) We have had better than expected success collecting samples and are consequently underbudget on two service contracts and supplies. Additional funds would ensure all samples can be processed and analyzed.

- $2,500 increase to service contract with the Minnesota Veterinary Diagnostic Laboratory - $4,000 increase to service contract with the UMN Genomics Center - $6,120 increase to lab and field supplies

Sub-Project Status as of January 31, 2019: We have continued to make good progress in the previous six months of the project period. We are nearly complete with collection of fish kills and healthy common carp – we plan to work with commercial fishermen in the coming months to collect common carp from the final two lakes. We have spent considerable time this project period working to process, sequence, analyze and finalize the results for viral discovery. While still in progress, we have already confirmed the detection of 11 novel viruses from common carp mortality events and five novel viruses from mortality events of native fish species. Results for healthy common carp, silver carp and zebra mussels are still pending. We have also confirmed six additional lakes positive for KHV, two lakes with CEV, and two lakes with both KHV and CEV. These results continue a trend of detections that first started in 2017 of

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this project. We are currently finalizing the phylogenetics to better determine the origin of the viral strains detected in Minnesota. Culture of the KHV remains a challenge for our project team (and other researchers around the world). We continue to discuss with collaborators and are working to modify and optimize are methods to improve isolation. However, we have begun experiments to grow the virus in vivo (in live fish) and are hopeful this strategy will prove effective in the coming months. Lastly, we have continued to communicate project progress at scientific conferences and with local/federal stakeholders. Amendment Request May 21, 2019: We request to move funds between budget categories within Activity 2 to conduct MinION (Oxford Nanopore Technology) sequencing of 240 DNA samples in our own lab, extracted from fish tissues throughout this project. The DNA HiSeq sequencing turnaround time at the University of Minnesota Genomics Centre (UMGC) is taking longer than expected and we are concerned we will not be able to complete all samples as proposed. Therefore, we have decided to do the DNA sequencing at our lab. This amendment request does not change the scope of the project, timeline, or overall budget. Activity 2 - Funding allocated for UMGC and VDL will be moved to purchase supplies to perform DNA sequencing in our own lab instead of outsourcing to the service contracts. This change will have no impact on the overall budget allocated to Activity 2 and will result in the following changes: Professional/Technical Services – decrease from $93,870 to $75,781; net -$18,089

- $1,823 reduction to Diagnostic services at the Minnesota Diagnostic Laboratory - $16,266 reduction to Sequencing services at University of Minnesota Genomics Center

Supplies-lab and field – increase from $11,092 to $29,181; net +$18,089

- $18,089 increase to lab supplies

Amendment Approved by LCCMR: 06/18/2019 Overall Sub-Project Outcomes and Results: One possible component to an effective integrated pest management plan for aquatic invasive species would be through the introduction or promotion of species-specific pathogens. This high-risk, high-reward approach must be carefully assessed with thorough investigation and scientifically justified risk assessment. In Phase II of this long-term effort, we characterized the virome invasive and native fish species and zebra mussels. We achieved our ultimate goal of this project and identified a candidate virus (koi herpes virus) that caused high mortality in common carp and was not detected in native fish species – this virus will be the focus of Phase III. We also identified many other novel and undescribed viruses in health and dead fish, however the implications of these results are unknown and warrant additional research to better understand the threat to native species and/or potential as biocontrol agents. The virome of zebra mussels was also interesting with lower viral diversity than the fish species investigated; however, no viruses emerged as potential zebra mussel biocontrol candidates from field samples or laboratory trials. This study emphasized the value of advanced molecular approaches to unbiased viral discovery and diagnostics. The methods we developed and optimized for sample collection, processing, and sequence analysis (all together called a ‘pipeline’), have informed testing protocols at the Minnesota Veterinary Diagnostic Laboratory. We have also elevated awareness among managers that viral diversity is much higher than currently known and deserves more attention as early indicators of potential threats. The project team spent considerable time during Phase II engaging with managers, scientists, and the public in multiple formats. It is important that this type of research is transparent and understandable to all stakeholders. To that end, we held formal in person meetings, attended local-national-international scientific conferences,

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published a peer-review manuscript, networked with internationally-renowned experts, produced two videos, and provided interviews for print, radio and TV media. IV. SUB-PROJECT ACTIVITIES AND OUTCOMES: ACTIVITY 1: Sample collection – Collect apparently healthy invasive carp and mussel species and collect samples from native and invasive fish and mussel mortality events in the Midwest region. Description: Investigation into the viral communities of apparently healthy animals, in particular invasive species, is rare, yet critically important to the long-term goals of this project. In addition, the opportunistic collection of animals found moribund or freshly dead as a result of a mortality event will provide diagnostic evidence of pathogenic viruses (or other microbes). While these events are occasionally investigated, communication is limited and diagnostic quality and approaches are highly variable. These limitations will be overcome in this project by conducting a standardized and thorough diagnostic exam. The Project Team will opportunistically collect fish in coordination with local DNRs, commercial fishermen, and researchers to maximize collection success and minimize costs. The collaborators will determine the specific sites and collection times; however, the goal is to collect common carp from ten Minnesota lakes during the project period. In addition, silver carp will be collected from four locations in the Illinois and Mississippi River systems. At each collection, 60 fish will be collected to ensure a statistically valid sample, assuming a 5% pathogen prevalence and 95% confidence in the diagnostic assays. In total, the sample collection goals are 600 common carp and 240 silver carp. The collection of zebra mussels from 20 locations has/will occur as part of an ongoing MAISRC project (Project Manager: Dr. Michael Sadowsky) and shared with this project. Opportunistic sampling of naturally occurring mortality events of native fish and mussels, as well as invasive carp and zebra mussels, will also be included in this activity. All species of fish and mussels will be included from wild, farm-raised, and laboratory sources. This is important to evaluate disease causing potential and species specificity of novel viruses. Samples from mortality events across the Midwest region will be accepted, but focus will be given to invasive carp and mussel populations in Minnesota. Aquatic animal health experts from the region will be provided with a collection protocol, submission form, and other information related to this project offering a no-cost diagnostic exam. This will help ensure consistent and appropriate samples and observational data are collected. A minimum of five fish or mussels with clinical lesions consistent with the mortality event will be targeted from each event. The number of fish mortality events in Minnesota alone is estimated at approximately 500; however, the number that will be reported and have quality samples collected is unknown. Our goal is to include 20 events per year in this project.

Summary Budget Information for Activity 1: ENRTF Budget: $57,162 Amount Spent: $55,559 Balance: $1,603

Activity Completion Date: Outcome Completion Date 1. Collect 600 common carp from 10 locations in Minnesota December 2018 2. Collect 240 silver carp from 4 locations in the Illinois and Mississippi Rivers December 2018 3. Collect 1,200 zebra mussels from collaborating researchers December 2018 4. Collect samples from 40 fish or mussel mortality events in the Midwest region December 2018

Activity Status as of July 31, 2017:

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The initial focus of this project has been to collect samples from healthy and sick/dead fish to identify viruses in the fish populations. We have collected samples from two carp mortality events: Lake Jonathan (6/13/17) and Lake Elysian (6/28/17). Standard water quality and diagnostic testing, including bacteriology, parasitology, and virus isolation did not yield significant findings for either case. However, Lake Elysian was confirmed positive for Cyprinid Herpes Virus-3 (aka Koi Herpes Virus, KHV) by molecular diagnostics. The population was re-sampled (7/10/17) and confirmed the diagnosis. This is a significant finding since this is the first report of KHV in wild fish in Minnesota and the candidate biocontrol agent for common carp in Australia. We are working with the MN DNR and hope to conduct follow up surveys in the coming months to estimate viral persistence, mortality rates and prevalence in surrounding lakes. Samples have also been collected from four mortality events of native species (i.e., bluegill, black crappie, muskellunge): Schwanz Lake (5/13/17), Lake Nokomis (6/7/17), Forrest Lake (6/13/17) and Maple Lake (7/14/17). Standard diagnostic and water quality testing did not yield significant findings. Tissue samples (e.g., gill, spleen, kidney) for all mortality events have been stored for next generation sequencing (Activity 2). Sampling of healthy and sick/dead populations will continue in the coming months. An increased effort, in collaboration with other MAISRC researchers, to sample invasive zebra mussels and native mussels will be a priority as this has proved challenging so far. Activity Status as of January 29, 2018 A total of 300 common carp were collected from five different lakes in Minnesota, including Long Lake, Lake Gervais, Lake Typo, Lake Albert Lea and Benton Lake (Table 1). Necropsies were performed at the Minnesota Veterinary Diagnostic Laboratory to collect brain, gill, spleen and kidney tissues and pooled in groups of five fish according to tissue type and location. Similarly, we have collected 120 silver carp from two different locations of Illinois River in collaboration with the Illinois DNR. These fish tissues were sampled as mentioned above. Zebra mussels were collected from 14 different lakes in collaboration with Dr. Michael Sadowsky’s Lab (Table 2), with 20 to 50 individual mussels per location. Lastly, we responded to or were sent samples from 14 fish kills events, including common carp and other native fish species in 2017, and collected 130 tissue samples (Table 3). All samples from carp, zebra mussels and mortality events were stored at -80°C for downstream analysis (Activity 2). Table 1. Healthy carp sampling

ID Lake Name Species Total fish number

Brain samples

Gill samples

Kidney samples

Spleen samples

Total number of tissues

pools 1 Long Lake Common carp 60 12 12 12 12 48 2 Gervais Common carp 60 12 12 12 12 48 3 Typo Lake Common carp 60 12 12 12 12 48 4 Albert Lea Common carp 60 12* 12* 12 5 Benton Lake Common carp 60 12 12 12 12 48

6 Starved Rock Pool- Illinois

Silver carp 60 12 12 12 12 48

7 Illinois River Silver carp 60 12 12 12 12 48 * Tissues submitted pre-processed from USFS Table 2. Zebra mussel sampling * Samples are currently frozen, exact numbers to be determined during necropsy.

ID Lake Name Species Total number of zebra mussels*

1 Vadnais Zebra Mussels 20-50 2 Lower Prior Zebra Mussels 20-50 3 Victoria Zebra Mussels 20-50

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4 Le Homme Dieu Zebra Mussels 20-50 5 Miltona Zebra Mussels 20-50 6 Carlos Zebra Mussels 20-50 7 Cowdry Zebra Mussels 20-50 8 Praire Zebra Mussels 20-50 9 Pelican Zebra Mussels 20-50

10 Ossawinnamakee Zebra Mussels 20-50 11 Gull Zebra Mussels 20-50 12 Zumbro Zebra Mussels 20-50 13 Superior Zebra Mussels 20-50 14 Pike Zebra Mussels 20-50

Table 3. Fish kill sampling

ID Lake Name Species Brain samples

Gill samples

Liver samples

Kidney samples

Spleen samples

1 Schwanz Lake Bluegill 1 1 1 1 Largemouth

Bass 1

2 Lake Nokomis Tiger Muskie 1 Black Crappie 3 1

3 Lake Jonathan Common Carp 1 2 2 1 4 Forest Lake Yellow Perch 1 1 1 1

Black Crappie 1 1 1 Pumpkinseed 1 1 1 1

Bluegill 1 1 1 1 5 Lake Elysian Common Carp 6 6 6 6 6 Duck Lake Bluegill 1 1 1 1 7 Maple Lake Yellow perch 1 1 1 1

Bluegill 1 1 1 1 Pumpkinseed 1 1 1 1

Bullhead * * * * 8 Lake Independence Yellow perch 1 1 1 1

Bullhead 1 1 1 1 9 Clear Lake Unknown 1 1 1 1 10 Lake Totonka Common Carp 1 1 1 1 11 Pymatuning Reservior, PA Common Carp 4 5 5 4 12 Lake Washington Common Carp 1 1 1 1 13 Kegonsa Lake, WI Common Carp 1 1 1 1

Waubesa Lake, WI Common Carp 1 1 1 1 Monona Lake, WI Common Carp 1 1 1 1

14 Cottonwood Common Carp 3 3 3 3 Tiger Muskie 1 1 1 1

Activity Status as of July 31, 2018: The healthy fish and fishkill samples collected in last season were screened for presence of koi herpes virus (KHV), Carp edema virus (CEV) and spring viremia of carp virus (SVCV). A duplex probe base qPCR has been optimized to detect both KHV and CEV simultaneously and for SVCV, a single probe base qPCR was designed. The common carp fish kills were all confirmed KHV positive through real time PCR (Table 4). Interestingly a co-infection of KHV and CEV has been diagnosed in Lake Jonathon, Lake Cottonwood and Lake Kegnosa. This is the first time that CEV has been detected in wild population of carp in the region – only one other confirmed outbreak has occurred in the USA (New Jersey in 2016).

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In 2018, four outbreaks of apparent disease have occurred in wild common carp in MN, two mortality events occurred in lake Byllesby and Fish Lake which have been confirmed as KHV. The collection of fish kill and healthy fish tissues for the 2018 field season are ongoing (Table 5). The collected tissues are processed and then stored at -80°C. Additionally PCR and Sanger sequencing of the thymidine kinase gene of KHV were carried out to determine the genetic relationship between MN and international KHV. This analysis revealed that all samples of KHV in Minnesota, Pennsylvania and Wisconsin originating from mortality events of wild common carp in 2017 were virtually identical to one another and belonged to the European genotype, suggesting a recent introductions form a similar source. Table 4. Real time detection of KHV and CEV from 2017 collected tissue

Lake Name Fish Species Koi herpes virus Carp edema virus Spring viremia of carp virus

Long Lake Healthy carp Negative Negative Negative Gervais Healthy carp Negative Negative Negative

Typo Lake Healthy carp Negative Negative Negative Albert Lea Healthy carp Negative Negative Negative

Benton Lake Healthy carp Negative Negative Negative Starved Rock Pool-

Illinois Healthy carp Negative Negative Negative

Illinois River Healthy carp Negative Negative Negative Schwanz Lake Native fishkill Negative Negative Negative Lake Nokomis Native fishkill Negative Negative Negative

Forest Lake Native fishkill Negative Negative Negative Duck Lake Native fishkill Negative Negative Negative

Maple Lake Native fishkill Negative Negative Negative Lake Independence Native fishkill Negative Negative Negative

Clear Lake Native fishkill Negative Negative Negative Lake Totonka Carp Fishkill Positive Negative Negative

Pymatuning Reservior, PA

Carp Fishkill Positive Negative Negative

Lake Washington Carp Fishkill Positive Negative Negative Kegonsa Lake, WI Carp Fishkill Positive Positive Negative Waubesa Lake, WI Carp Fishkill Positive Negative Negative Monona Lake, WI Carp Fishkill Positive Negative Negative

Cottonwood Carp Fishkill Positive Positive Negative Lake Jonathan Carp Fishkill Positive Positive Negative

Lake Elysian Carp Fishkill Positive Negative Negative

Table 5. Fishkill event sample collection till 30th June 2018

Lake Name Species Tissue type Brain Gill Kidney Spleen

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Cornelia Common Carp 1 1 Gold fish 1 1 1 Bullhead 1 1 1 1

Pamela Common Carp 1 2 1 1 Gizzard Shad 1 1 1

Minnetonka Blue Gill 1 1 1 1 Goodners Black Crappie 1 1 1 1 Bullantyne Common Carp 1 1 1 1

Gervais Black Crappie 1 1 1 1 Nokomis Common Carp 1 1 1 1 long lake Common Carp 1 1 1 1 Traverse White bass 1 1 1 1 Byllesby Common Carp* 4 4 4 4 Fish lake Common Carp* 5 5 5 5 Waconia Blue Gill 1 1 1 1

Black Crappie 1 1 1 1 Yellow Perch 1 1 1 1 Pumpkinseed 1 1 1 1

*Confirmed positive for KHV. Tests pending for all other samples. Activity Status as of January 31, 2019: Tissues have now been sampled from an additional 228 carp, 15 goldfish, and 120 silver carp. We sampled more lakes than attempted in 2017 in order to gather samples from locations near where KHV outbreaks had occurred previously. Additionally, since KHV may also be carried by goldfish and common carp hybrids we included these fish in samples taken from East Lake, which had populations of carp, goldfish and potential hybrids. However due to the difficulty in obtaining complete samples of 60 fish from each location sampled we will work to sample two more lakes for common carp by winter seining. Additionally, we collected zebra mussels samples from an in-lab stress trial in collaboration with Dr. Michael Sadowsky’s Lab. Lastly, we added samples from 19 fish kills events, including common carp and other native fish species in 2018, and collected 121 tissue sample pools (Table 7). All samples from carp, zebra mussels and mortality events were stored at -20°C for downstream analysis (Activity 2). The collected tissues were processed and then stored at -20°C. PCR screening was used to identify additional KHV, CEV and coinfections in MN (Table 8). We had previously reported the important finding of koi herpes and carp edema virus coinfections and genetic subtyping. From subsequent large-scale carp mortality events, we found an additional six locations to be KHV positive, two locations to be CEV positive, and two locations to have coinfections. This included a lake that was previously tested negative for KHV which may indicate a low prevalence of the virus in Long lake or recent introduction. Additionally, Lake Elysian was found to have a high prevalence of KHV (4 of 7 fish sampled) even a year after outbreak in the lake as well as coinfection with both viruses in one individual sampled. Table 6. Healthy carp sampling

ID Lake name Species Total fish number

Brain samples

Gill samples

kidney samples

Spleen samples Blood

1 Long Lake Common carp 60 12 12 12 12 0 2 Gervais Common carp 60 12 12 12 12 0 3 Typo Lake Common carp 60 12 12 12 12 0 4 Albert Lea Common carp 60 0 0 12* 12* 0 5 Benton Lake Common carp 60 12 12 12 12 0

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6 Starved Rock Pool- Illinois Silver carp 60 12 12 12 12 0

7 Illinois River Silver carp 60 12 12 12 12 0

8 East Common carp and Hybrids 45 9 9 9 9 9

9 East Goldfish 15 2 2 2 2 3 10 Cannon Common carp 12 12 12 12 12 12 11 Stieger Common carp 60 12 12 12 12 0 12 Crystal Common carp 25 5 5 5 5 5 13 Long Lake Common carp 25 0 5 0 0 5 14 Elysian Common carp 11 11 11 11 11 7 15 Jefferson Common carp 25 5 5 5 5 5

16 Starved Rock Pool- Illinois Silver carp 30 6 6 6 6 0

17 Stratton Park Silver carp 90 18 18 18 18 0 Table 7. Fish kill sampling

ID Lake Name Species Brain

samples Gill

samples Muscle Kidney

samples Spleen

samples

1 Cornelia

Common carp 0 1 0 1 0 Gold fish 1 1 0 0 0 Bullhead 1 1 0 1 1

2 Pamela Common carp 1 2 0 1 1 3 Sweeney Gizzard shad 1 1 0 0 1 4 Minnetonka Blue gill 1 1 0 1 1 5 Goodners Black crappie 1 1 0 1 1 6 Ballantyne Common carp 1 1 0 1 1 7 Gervais Black crappie 1 1 0 1 1 8 Nokomis Black crappie 1 1 0 1 1 9 Long Common carp 1 2 1 1 1

10 Traverse White bass 1 1 0 1 1 11 Byllesby Common carp 4 4 0 4 4 12 Fish Common carp 5 5 0 5 5

13 Waconia

Blue gill 1 1 0 1 1 Yellow Perch 1 1 0 1 1 Pumpkinseed 1 1 0 1 1 Black crappie 1 1 0 1 1

14 Freeborn Common carp 1 1 0 1 1

15 Morehouse

Park Common carp 1 1 0 1 1 16 Currant Common carp 1 1 0 1 1 17 Clear Common carp 1 1 0 1 1 18 Spring Black Crappie 1 1 0 1 1 19 Scotch Common carp 0 2 0 0 0

Table 8. Real time detection of KHV and CEV from 2018 collected tissue

Lake Name Fish Species Koi herpes virus

Carp edema virus

Spring viremia of carp virus

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Long Lake Healthy carp Negative Negative Negative Gervais Healthy carp Negative Negative Negative

Typo Lake Healthy carp Negative Negative Negative Albert Lea Healthy carp Negative Negative Negative

Benton Lake Healthy carp Negative Negative Negative Starved Rock Pool-

Illinois Healthy carp Negative Negative Negative

Illinois River Healthy carp Negative Negative Negative Schwanz Lake Native fishkill Negative Negative Negative Lake Nokomis Native fishkill Negative Negative Negative

Forest Lake Native fishkill Negative Negative Negative Duck Lake Native fishkill Negative Negative Negative

Maple Lake Native fishkill Negative Negative Negative Lake Independence Native fishkill Negative Negative Negative

Clear Lake Native fishkill Negative Negative Negative Lake Totonka Carp Fishkill Positive Negative Negative Pymatuning

Reservior, PA Carp Fishkill Positive Negative Negative

Lake Washington Carp Fishkill Positive Negative Negative Kegonsa Lake, WI Carp Fishkill Positive Positive Negative Waubesa Lake, WI Carp Fishkill Positive Negative Negative Monona Lake, WI Carp Fishkill Positive Negative Negative

Cottonwood Carp Fishkill Positive Positive Negative Lake Jonathan Carp Fishkill Positive Positive Negative

Lake Elysian Carp Fishkill Positive Negative Negative Cornelia Native and Carp fishkill Negative Negative Negative Pamela Carp Fishkill Negative Negative Negative

Sweeney Native fishkill Negative Negative Negative Minnetonka Native fishkill Negative Negative Negative

Goodners Native fishkill Negative Negative Negative Ballantyne Carp Fishkill Negative Negative Negative

Gervais Native fishkill Negative Negative Negative Nokomis Native fishkill Negative Negative Negative long lake Diseased carp Positive Negative Negative Traverse Native fishkill Negative Negative Negative Byllesby Carp Fishkill Positive Negative Negative Fish lake Carp Fishkill Positive Negative Negative Waconia Native fishkill Negative Negative Negative Freeborn Carp Fishkill Negative Positive Negative

Morehouse Park Carp Fishkill Positive Negative Negative Currant Carp Fishkill Positive Positive Negative

Clear Carp Fishkill Positive Negative Negative Spring Native fishkill Negative Negative Negative Scotch Carp Fishkill Positive Negative Negative

East Healthy carp Not tested Not tested Not tested East Healthy carp Not tested Not tested Not tested

Cannon Healthy carp Negative Not tested Not tested Stieger Healthy carp Not tested Not tested Not tested

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Crystal Healthy carp Not tested Not tested Not tested Long Lake Healthy carp Not tested Not tested Not tested

Elysian Healthy and diseased carp Positive Not tested Not tested Jefferson Healthy carp Not tested Not tested Not tested

Activity Status as of July 31, 2019: All the healthy fish and zebra mussels survey sample collection proposed in phase II of the project was completed before 31st January 2019. There were no fish mortality events during past 6 month that were investigated. Final Report Summary: The goal of phase II project was to find potential viral pathogen which should be highly specific to the invasive fish species in state Minnesota. To fulfill this goal, we have collected a large number of samples representing both healthy and diseased invasive fish, and diseased native fish. We were able to leverage a MAISRC collaboration (Subproject #2) to obtain the zebra mussels. In the last two years, we collected samples from nearly as many lakes/rivers as we had hoped, with a very good distribution across the state and region. All of the fish and mussels were necropsied, tissues processed and submitted for viral screening. Figure. Example of common carp collected following a mortality event. Gross pathology of freshly dead wild carp from Cottonwood Lake with KHV and CEV coinfection. Loss of skin and scales on the surface of the body (a). Necrosis in the gill filaments (b). Hemorrhages and loss of scales on the surface of the body (c). Enopthalmos (d).

Table . Overall summary of samples collected for virus screening during the project period. Details included in activity updates above.

Number of lakes

sampled Number of individuals

collected

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Surveys of healthy animals Common carp 12 503 Silver carp 4 240 Zebra mussels 14 ~700 Surveys of mortality events Common carp 20 137 Native fish 16 108 Zebra mussels Held in laboratory 29 pooled samples

All fish samples collected during this project were tested for three important viruses KHV, CEV and SVCV. Both KHV and CEV were detected in common carp (healthy and dead), but not in any native species – this is the first report of these viruses in Minnesota. SVCV was not detected in any sample. KHV appears to be widespread in Minnesota, overlapping with high carp abundance. The discovery of CEV has gained international attention since this is only the second report of CEV in North America and we found it in six locations from Minnesota, Iowa, Wisconsin and Pennsylvania. In addition, the confirmation of dual infections of KHV/CEV is quite remarkable and is the first report in wild common carp. These findings have reinforced the decision to focus on KHV as a candidate virus for biocontrol. CEV is also an interesting candidate, but until a culture model can be developed its application is unlikely. Below is the map of carp sampling locations and diagnostic results: Figure. a) KHV and CEV detections from mortality events of wild common carp in Iowa, Minnesota, and Wisconsin during this study; b) All KHV and CEV detections from wild common carp during this study; c) All published KHV and CEV detections from wild common carp in North America from Cornwell et al. (2012), Xu et al. (2013), Thresher et al. (2018), and Lovy et al. (2018). Symbols denote: detection of KHV alone, filled black circles; detection of CEV alone, filled red circles; detection KHV and CEV, red circles with black bullseye; no detection of KHV or CEV from mortality events in this study, diamonds. Major watersheds (HUC 8) are outlined in light grey. Major rivers and lakes (1x10^6 scale) are denoted in light blue. State and province boundaries are denoted in dark grey.

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A peer-reviewed manuscript discussing these results has been accepted by the Journal of Fish Diseases. Padhi, S. K., I. E. Tolo, M. McEachran, A. Primus, S. K. Mor, N. B. D. Phelps. In press. Koi herpesvirus and carp edema virus: Infections and coinfections during mortality events of wild common carp in the United States. Journal of Fish Disease. ACTIVITY 2: Characterize virome – Conduct virus discovery by next generation sequencing (NSG) and culture potential pathogens. Description: Characterizing the virome of invasive carp and mussel species is an initial step towards risk assessment and evaluation of viruses as biological control agents. Phase I of this project was the first virus discovery effort in common carp and no similar survey has been done for zebra mussels. The methods developed and refined in Phase I proved the utility of next generation sequencing techniques, having identified 10+ novel viruses. There is little doubt important new viruses will be found in Phase II. A necropsy will be performed on all fish and mussels collected as part of Activity 1. Various tissues (i.e. kidney, spleen, gills, etc) will be collected by aseptic techniques for virus discovery. If the fish or mussels were collected from a fish kill (Objective 1b), a thorough diagnostic exam will be performed to determine the cause of death and, if present, understand the pathology of viral agents. All samples will be processed for NGS for the unbiased amplification of viral RNA and DNA viruses following our methods developed in Phase I. Samples will be pooled

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in groups of five for a total goal of 168 pools of invasive carps, 240 pools of zebra mussel and 80 pools from mortality events. We will multiplex (barcode) 50 samples in a single lane of HiSeq 250 paired end cycle run to reduce sequencing cost while maintaining critical sequence depth. Bioinformatics methods for virome analyses are already in place and in routine use by the Project Team and colleagues at the Minnesota Veterinary Diagnostic Laboratory (MVDL). The pipeline consists of two steps, sequence quality screening and sequence analysis. Quality sequence reads will be compared to the most closely related sequences available in GenBank. Specific primers will be designed for standardization of PCR for each virus detected by NGS. These specific assays will be used for screening of individual samples and future diagnostic needs.

If novel viruses are identified by NGS, the original sample will be subjected to virus isolation for further characterization. A variety of immortal fish cell lines (i.e. EPC, FHM, CHSE-214, etc) are currently in use by the Project Team and will be available for this project. No permanent cell lines derived from bivalves are available for isolation of viruses and the only permanent cell line from any mollusk is from the snail, Biomphalaria glabrata (Hansen 1976; American Type Culture Collection number CRL-1494). Ongoing work by the USGS and US FWS towards the development of a zebra mussel cell line looks promising and should be available for this project. However, the Project Team has experience developing primary cell lines and will pursue this approach if needed or use the CRL-1494 snail cell line as a last resort. To visualize viral particles, infected cell culture fluids will be freeze-thawed thrice followed by light centrifugation. The supernatant will be examined by negative contrast electron microscopy. Morphological classification of the viral isolate will support findings of NGS.

Summary Budget Information for Activity 2: ENRTF Budget: $272,448 Amount Spent: $272,448 Balance: $0

Activity Completion Date: Outcome Completion Date 1. Database and isolate archive of viruses of fish June 2019 2. Database and isolate archive of viruses of mussels June 2019

Activity Status as of July 31, 2017: Samples from six mortality events have been archived in preparation for Activity 2. These samples will be processed and analyzed in the coming weeks. Given the important finding of KHV, we aim to sequence the entire genome of this virus to determine the virus subtype (e.g., European vs. Asian strain). Knowing the strain is important for biocontrol considerations and identifying the introduction source. Activity Status as of January 29, 2018 All the collected tissue samples from common carp, silver carp and fish kills were homogenized in sterile phosphate buffer saline. These homogenized samples were centrifuged and supernatants were collected and stored at -80°C. Several experiments such as ultracentrifugation and filtration were conducted to figure out an optimal method for concentrating viral particle and removing host nucleic acid contamination for next generation sequencing (NGS). This has been important to increase our likelihood of finding viruses at low concentration (from healthy fish) and/or poor sample quality (from fish kills). Three NGS plates prepared to evaluate the different methods as described above and have been submitted to the University of Minnesota Genomics Center for sequencing. Results are expected in the coming months. From these data, an optimal method will be standardized and the remaining tissue samples will be processed. We had previously reported (Activity 1, 7/31/17 update) the important finding of koi herpes virus in Lake Elysian. From subsequent large-scale carp mortality events, we found an additional six locations to be KHV positive in the same region of Minnesota. This included: Lake Tetonka (Waterville County), Lake Washington, Lake Dora, Lake Sabre, and Gorman Lake (LeSeur County), and Cottonwood Lake (Lyon County). This is the

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largest reported outbreak of KHV in the United States (most other outbreaks happen in isolation). It is unclear at this time if we are documenting the emergence of KHV in Minnesota or our project has resulted in an increased reporting of an endemic disease – a critical question to know for long term applications for biocontrol. We are currently investigating this question through genetic analysis. Activity Status as of July 31, 2018: A total of 132 tissue samples collected from healthy fish, fishkills and zebra mussels have been processed through a newly optimized viral nucleic acid extraction method for RNA-NGS. All samples were submitted to the University of Minnesota genomics center for HiSeq sequencing. Out of 132 submitted samples, 130 passed the internal quality control check, which is better than normal suggesting our optimized processing methods were successful. The sequencing results are expected to be delivered in the month of August. Once we get the results of RNA-NGS, the search for the total viral population will be carried out through specific pipeline of bioinformatics tools. We have also submitted two KHV positive DNA samples to UMGC for DNA-NGS; however, while these samples passed initial quality control, they failed library preparation. We are modifying the methods to ensure better outcomes with DNA extraction optimization and all the downstream processes. Genotyping of samples positive for KHV have been carried out for five of the lakes in Minnesota, a reservoir in Pennsylvania, and two lakes in Wisconsin where mortality of wild common carp occurred in 2017. This analysis is currently in progress for two additional lakes were KHV appears to have caused mass mortalities of wild common carp as well as a mortality event of domestic koi. Genotyping analysis will be continued to describe the apparent outbreak of KHV in Minnesota. We have received two common carp cell lines, Common Carp Brain (CCB) and Koi Fin (KF-1) from Dr. Thomas Waltzek, University of Florida. These cell lines are recommended by OIE for isolation of KHV. A trial experiment is ongoing to culture KHV from KHV positive tissue homogenate. Activity Status as of January 31, 2019: All samples have been collected and are at various stages of processing, sequencing, analysis or final results for both RNA and DNA virus discovery. Briefly, 92 samples of healthy carp and fish kill have been submitted for RNA virus discovery. All samples passed quality control and are currently in queue for sequencing on the Hiseq platform. We are processing an additional 48 samples collected from healthy silver carp, fish kills, zebra mussels, and experimental study samples for submission to the sequencing lab the first week of February. For DNA virus discovery, 120 samples passed DNA quality control and the samples are in queue for sequencing by Hiseq, which will be completed by February 15. The samples that failed quality control and new remaining samples will be submitted by February 15 another Hiseq run. We have developed an improved bioinformatics pipeline for analysis and reporting of NGS data. The RNA Hiseq data of 212 samples have been received and the final analysis of 84 samples has been completed. The remaining 128 samples, including zebra mussel samples, are in the pipeline for analysis. The table below summarizes the results available at this point in the project. As analysis is completed, we are assembling the complete genome of these novel viruses and making phylogenetic comparisons between viruses identified during this study and all other known viruses available in GenBank. Using several new cell lines, we have continued to work on the culture of our KHV isolates. While this has remained a challenged, we continue to discuss with our international collaborators and optimize our protocols accordingly. We remain hopeful that we will be able to grow the virus in culture before the end of the project. Activity Status as of July 31, 2019:

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Over the last 6 months we have completed the bioinformatics analysis of all 266 samples that were sequenced using the Illumina HiSeq platform. This was a major accomplishment that resulted in the identification of many novel undescribed viruses, and known viruses of interest (see summary table below). We are currently in the process of making the sequence data available in either the National Center for Biotechnology Information (NCBI; cleaned and complete viral genomes) the University of Minnesota Data Repository (DRUM; partial viral genomes and raw sequencing data). Table 11. Virus discovery using Illumina HiSeq next generation sequencing

Species Sample type

Number of samples

NGS analysis

dsRNA viruses ssRNA viruses DNA viruses

Common carp

Healthy 17 Completed Totiviridae Reoviridae

Astrovirus Picornavirales Alphanodavirus Caliciviridae Nodaviridae

Anelloviridae Circoviridae Iridoviridae Parvoviridae Herpesvirales

Common carp

Fish kill 83 Completed Birnaviridae Reoviridae Arbovirus Totiviridae

Rhabdoviridae Astroviridae Hepeviridae Picornavirales Negarnaviricota

Herpesvirales Iridoviridae Circoviridae Cruciviridae Anelloviridae Parvoviridae Mimiviridae Poxviridae

Silver carp Healthy 16 Completed

-- Picornavirales Iridoviridae Circoviridae

Native fish Fish kill 99 Completed Reoviridae Rhabdoviridae Astroviridae Caliciviridae Hepeviridae Picornavirales Mononegavirales Ortervirales

Ambidensovirus Circoviridae Poxviridae (Unclassified)

Zebra mussel

Healthy 20 Completed Picobirnaviridae Picornavirales Nodaviridae Unclassified uncultured viruses Changjiang crawfish virus

Parvoviridae Circoviridae

Zebra mussel

Challenge study

29 Completed Astroviridae Picornavirales Unclassified uncultured virus Unclassified Riboviria sp.

Iridoviridae Parvoviridae Circoviridae Unclassified Mimiviridae Unclassified Pacmanvirus Unclassified Lavidaviridae

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Unclassified Marseilleviridae

In addition to the sequencing described above, we evaluated the ability of the MinION, a new nanopore unbiased sequencing tool, to provide longer sequencing reads of our samples within a few hours (instead of weeks) and at lower cost. This comparison with Illumina HiSeq had never been done for fish and mussel viromes, yet showed significant promise based on preliminary data by our project team. Due to a late budget amendment to make this possible, we have not yet completed the final analysis for all 198 samples; however, we have developed and optimized the pipeline for sample processing and analysis. Early results have reaffirmed the benefits of this approach. On a single run we were able to recover and assemble a ~276kb genome of the carp edema virus. We were also able to assemble the complete genome of koi herpes virus and perform the necessary phylogenetics to determine its European origin.

We have uncovered many novel and undescribed viruses; however, it is often difficult to interpret the results of exploratory surveys like this – ‘what does the detection of each novel virus mean for Minnesota?’ More research is clearly needed to better understand the implications, but some early reaction to viruses associated with mortality events or related to viruses of concern are below:

1. Detection of Rhabdoviridae family viruses in native fish kill and carp kill samples highlight the importance of our virome study. Further characterization of these viruses will help to understand the relationship to two related rhabdoviruses of concern: viral hemorrhagic septicemia virus (native species) and spring viremia of carp virus (common carp). In general, Rhabdoviruses are well known to cause moderate to severe mortality in different fish species.

2. A novel Seadornavirus, a member of Reoviridae family, was identified in both native fish and common carp mortality event samples.

3. Identification of crayfish-like virus in zebra mussels is another important finding. The crayfish virus is well known to cause high mortality and white spot syndrome disease in crayfish. This virus was recently detected in Louisiana. https://agfax.com/2019/04/30/louisiana-crawfish-white-spot-virus-in-ponds/

4. Viruses of Picornavirale order have been detected in all types of fish and mussel samples, indicating widespread prevalence. Picornaviruses are known to evolve rapidly and may become pathogenic for particular species. It is important to keep eye on such viruses.

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5. RNA viruses (such as birnavirus, arbovirus, totivirus) and DNA viruses (parvovirus, herpesvirus, iridovirus, mimivirus) are differential viruses detected only in common carp mortality events and not in native fish kills. In contrast, calicivirus is a differential virus detected only in native fish kill samples.

6. Picobirnavirus (dsRNA virus) and nodavirus (ssRNA virus) were detected in zebra mussels survey samples but not in experimental samples. Picornavirus and parvovirus were detected in both zebra mussels survey and experimental samples.

Final Report Summary: The ultimate goal of this activity was to identify potential viral biocontrol candidates that caused disease in invasive species and were not present in native species or did not cause them harm. This comprehensive diagnostic survey resulted in the identification of many novel viruses and known viruses of interest in all types of samples tested. We expected to find novel viruses, but the diversity of viruses was surprising even to the project team. The implications of these results are discussed above but remain largely unclear and warrant further study. Of particular note: The discovery of Herpesvirales (e.g. koi herpes virus) only in common carp and never in native fish kills is important and will be the subject of further research as a candidate biocontrol agent. During the course of this study, we demonstrated the value for both short read (Illumina HiSeq) and long read (MinION, Oxford Nanopore) methods for the detection and characterization of fish and zebra mussel viromes. The pipelines that we developed to process, sequence and analyze samples directly from fish tissue (often in poor post-mortem condition) is a major advancement for the field of aquatic animal health and have informed testing by the Minnesota Veterinary Diagnostic Laboratory. ACTIVITY 3: Disease challenge – Determine the disease-causing potential of two selected viruses, one for native and invasive fish and the other for native and invasive mussels. Description: The potential use of viral agents as a means of biological control for invasive carp and zebra mussels is dependent on the identification of a virus that is capable of negatively impacting the health of the invasive species while at the same time having little or no impact on native species. Two novel viral agents (one from fish and one from mussels) identified in Activity 2 will be selected based on the pathogenic potential to the target species (i.e. common carp and zebra mussels) and ability to propagate in cell culture. These viruses will be used to challenge the target species as well as a native fish species (fathead minnow) and a native mussel species (three ridge mussel). The fathead minnow was chosen as a representative native fish species because it is closely related to common carp and suitable for laboratory confinement. The three ridge mussel was chosen as a good representative native mussel species because of its accessibility and suitability for captive culture.

Fish and mussels will be held at the Minnesota Aquatic Invasive Species Research Center’s Containment Laboratory in in 30-gallon flow-through aquaria and monitored at least once daily throughout the experiment. Flow-through rates will be adjusted to maintain water quality, while minimizing the need for costly effluent treatment. Prior to initiating a viral challenge, animals will be acclimated for a period of at least 14 days and a subsample of each species involved will undergo a full diagnostic examination to ensure suitability for participation in the study. Each species will be challenged at two temperatures (10-15°C and 25-30°C) and two viral inoculum concentrations; each challenge will be conducted in duplicate, and one negative control per species per temperature will be used. Each species will therefore require the use of ten tanks. Each tank will contain thirty individuals. Animals will be assigned to challenge tanks (and therefore treatment groups) without any intentional bias.

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Fish will be challenged by intraperitoneal (IP) injection of viral inoculum. Briefly, fish will be anesthetized with buffered tricaine methanesulfonate (TMS). Once fish reach stage 3 anesthesia, each fish will be injected with 0.1ml of viral inoculum or cell culture growth medium (negative control). Two different viral concentrations will be used for the inoculum of challenged fish: ~ 108 pfu/ml and ~105 pfu/ml (which, when administered in a 0.1 ml dose will result in administration of ~107 and ~104 pfu per fish, respectively). Mussels will be challenged using a static bath challenge method whereby water flow is terminated and a standardized amount of viral inoculum/infectious material is added to the tank water; water flow may be turned back on after a fixed amount of time (e.g. 24 hours post inoculation). If the mussel virus that is used is amenable to cell culture, preparation of viral inoculum stock solution and quantification of virus in stock solution will follow the procedures outlined above for the fish virus; final concentrations of ~103 and ~101 pfu/ml would be ideal for bath challenge. If the mussel virus selected is not amenable to in vitro amplification, infected tissues will be used to create a stock solution; the stock solution will then be used for the higher concentration viral challenge treatment groups, and a 100-fold dilution will be used for the lower concentration viral challenge. In the later scenario, quantitative PCR would be used to quantify the dose. Animals will be closely monitored for 60 days to evaluate host response to the virus, at which time all surviving animals will be humanely euthanized. Clinically ill individuals will be euthanized prior to the end of the study if necessary. A complete diagnostic exam will be completed on all organisms in the study to determine health status, as well as persistence and replication of the virus within the individual. All fish studies will run simultaneously and will require 4 months’ continuous time in the infectious disease challenge room. All mussel studies will run simultaneously and will require an additional 4 months’ continuous time in the infectious disease challenge room.

Summary Budget Information for Activity 3: ENRTF Budget: $94,600 Amount Spent: $82,919 Balance: $11,681

Activity Completion Date: Outcome Completion Date 1. Determine disease causing potential of selected fish virus December 2018 2. Determine disease causing potential of selected mussel virus June 2019

Activity Status as of July 31, 2017: Potential viral biocontrol candidates are still being considered. This Activity will begin in mid-2018. Activity Status as of January 29, 2018 Potential viral biocontrol candidates are still being considered. This Activity will begin in mid-2018. Activity Status as of July 31, 2018: We have decided to move forward with exposure trials for KHV to tests its potential biocontrol agent in Minnesota. We are currently in the process of culturing the virus to use for the trials. The experiment for viral infection to common carp and native species will begin in September 2018. Given the difficulty in collecting sick/dead mussels, we are considering an alternate approach for the mussel trial. Instead of isolating a disease-causing virus from wild populations and exposing captive mussels, we will stress mussels in the lab to induce disease. Details of this approach are being evaluated, but aim to increase our odds of effectively meeting our proposed outcomes and staying within budget. Activity Status as of January 31, 2019:

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Koi Herpes Virus (KHV) was selected as the virus to evaluate in fish for Activity 3 as it has been found in native common carp (Activity 1), it is known to be pathogenic to common carp, it is being evaluated as a biocontrol agent for common carp in Australia, and no other clearly more-suitable alternative was discovered in Activity 1. For the fish challenge experiment, which we planned to use to determine the disease-causing potential and species specificity of KHV to the fathead minnow (native cyprinid) and common carp (as a positive control), virus material for the challenge is typically grown in cell culture. Due to unforeseen challenges with culturing and amplifying this virus in cell culture, we were forced to try to amplify the virus in vivo (i.e. in live common carp) to produce enough suitable infectious material for the experimental challenge. This in vivo amplification procedure was attempted and samples are now being tested to evaluate the success of this approach. Alternative means of obtaining suitable challenge material (i.e. live KHV) are also be explored. As Activity 1 was unable to identify any suitable viruses for zebra mussel challenge, a new approach to identifying such a virus was employed here in Activity 3. This new approach involved collecting over 800 zebra mussels from a Minnesota lake, placing them in several different aquaria, and applying several different types of stressors – including elevated temperature, fluctuating daily temperatures, elevated salinity, and combination of elevated temperature and elevated salinity – to these mussels in the hope of inducing viral disease. Mussels were then observed daily and any sick or dead mussels were collected and saved for subsequent testing. Mussels were also collected at pre-determined time-points for subsequent testing. The experimental trial was completed in November 2018 and samples are currently being assessed for viruses by next generation sequencing (Activity 2). Activity Status as of July 31, 2019: Several additional efforts were made to grow KHV in cell culture. This involved obtaining fresh tissues from wild common carp positive for KHV, the use of multiple cell lines at different environmental conditions and consultation with other fish health laboratories. These efforts, however, were ultimately unsuccessful. In addition, the in vivo challenge of naïve common carp with homogenate derived from infectious tissues (conducted in attempt to amplify KHV in live fish) did not result in disease of detectable KHV in the challenged group. Finally, several wild common carp from lakes with a history of KHV infection were obtained and brought to the laboratory alive with the hope if infecting naïve common carp and/or naïve fathead minnows. These efforts were unable to demonstrate KHV transmission. Final Report Summary: Koi Herpes Virus (KHV) was selected as the virus to evaluate in fish for Activity 3 because 1) it has been found to cause high mortality in local common carp (Activity 1), 2) it is thought to be pathogenic only to common carp, 3) it is being evaluated as a biocontrol agent for common carp in Australia, and 4) no other clearly more-suitable alternative was discovered in Activity 1. For the fish challenge experiment, which we planned to use to determine the disease-causing potential and species specificity of KHV to the fathead minnow (native cyprinid) and common carp (as a positive control), virus material for the challenge is typically grown in cell culture. Several efforts were made to grow KHV in cell culture. This involved obtaining fresh tissues from wild common carp positive for KHV, the use of multiple cell lines at different environmental conditions and consultation with other fish health laboratories. These efforts, however, were ultimately unsuccessful. Due to the unforeseen challenges of culturing and amplifying this virus in cell culture, we were forced to try to amplify the virus in vivo (i.e. in live common carp) to produce enough suitable infectious material for the experimental challenge. The in vivo challenge of naïve common carp with homogenate derived from infectious tissues (conducted to attempt to amplify KHV in live fish) did not result in disease of detectable KHV in the challenged group. Finally, several wild common carp from lakes with a history of KHV infection were obtained and brought to the laboratory alive with the hope if infecting naïve common carp and/or naïve fathead minnows. These efforts were unable to demonstrate KHV transmission.

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In addition, as Activity 1 was unable to identify any suitable viruses for zebra mussel challenge, a new approach to identifying such a virus was employed here in Activity 3. This new approach involved collecting over 800 zebra mussels from a Minnesota lake, placing them in several different aquaria, and applying several different types of stressors – including elevated temperature, fluctuating daily temperatures, elevated salinity, and combination of elevated temperature and elevated salinity – to these mussels in the hope of inducing viral disease. Mussels were then observed daily and any sick or dead mussels were collected and saved for subsequent testing. Mussels were also collected at pre-determined time-points for subsequent testing. The experimental trial was completed in November 2018 and samples were assessed for viruses by next generation sequencing. Viruses of seven families (2 RNA and 5 DNA virus families) were detected. The large DNA viruses such as iridovirus, mimivirus and pacmanvirus may be associated with mortality or stress of zebra mussels alone or in combination with small RNA and DNA viruses. ACTIVITY 4: Communication – Communicate findings to scientific, management, and public stakeholders Description: Disseminating research findings to relevant stakeholders and framing the future conversation on the use of pathogens for biocontrol of invasive species is an important part of this project. In so doing, we will provide stakeholders with the information to fill key knowledge gaps, guide future research and inform long-term approaches to manage AIS. A diverse communication approach will be used to ensure appropriate delivery and detail to each relevant stakeholder group. The data generated as part of this project will result in the publication of three peer-reviewed articles in high-impact journals. Efforts will be made to provide open access availability to all published articles. The results will be presented at the American Fisheries Society – Fish Health Section annual meeting, or similar meeting each year. In addition, all research findings will be disseminated through the MAISRC newsletter, social media, and annual Research Showcase. Publication and presentation topics will be determined based on the project progress and findings. Through active and ongoing collaborations with MN DNR fish health and AIS specialists, important project findings will be shared directly. Given the acknowledgement that the use of pathogens for biocontrol is a high-risk, high-reward research endeavor, we must look forward to the potential implementation of such a strategy. We will prepare a thorough review paper for journal submission to frame the conversation on the use of this strategy for aquatic invasive species control in inland waters of United States.

Summary Budget Information for Activity 4: ENRTF Budget: $21,000 Amount Spent: $14,020 Balance: $6,980

Activity Completion Date: Outcome Completion Date 1. Three peer-reviewed manuscripts submitted June 2019 2. Three scientific conference presentations June 2019 3. Dissemination of research findings via MAISRC communications June 2019

Activity Status as of July 31, 2017: We learned from Phase I of this project (MAISRC SubProject 7-1) that an increased communication effort was needed to generate collaboration on sample collection. To that end, the Project Manager (Dr. Nick Phelps) and Co-Investigator (Dr. Sunil Kumar Mor) attended the joint meetings of the American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop and the Great Lakes Fish Health Committee to present on this project. The presentations were titled: “Investigating fish kills: Looking back, looking deep and looking forward” and “Understanding the virome of invasive carp: What it could mean for biocontrol”. These presentations

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resulted in an active discussion on the potential use of viruses for biocontrol, interest to submit samples for the project and potential collaborations for future research efforts related to this project. Funding was only provided to Dr. Mor for attendance, Dr. Phelps used other funds. To discuss the applications of viral biocontrol, we have invited a world leader, Dr. Ken McColl (Commonwealth Scientific and Industrial Research Organization, Australia), to present at the 2018 iCOMOS meeting to be held at the University of Minnesota, more information: http://icomos.umn.edu. We expect that as part of Dr. McColl’s visit, we will host meetings with members of state and federal agencies to socialize this approach and generate ideas for future research needs. Dr. Nick Phelps is on the iCOMOS organizing committee and has arranged all costs to be covered from other sources. Activity Status as of January 29, 2018 Dr. Sunil Mor and Isaiah Tolo provided a presentation at the MAISRC showcase meeting in September 2017, entitled “Hitchhikers on the hitchhikers: Novel viruses to control invasive fish populations”. Dr. Nick Phelps attended the International Conference on Aquatic Invasive Species in October 2017, entitled “Understanding the carp virome: What it could mean for the control of invasive carp”. Lastly, Dr. Soumesh Padhi provided a presentation for the Minnesota Veterinary Diagnostic Laboratory in December 2017, entitled “Understanding the carp virome: What it could mean for the control of invasive carp”. Dr. Sunil Mor and Isaiah Tolo will be attending the Aquatic Invaders Summit III scheduled for Feb 28th/March 1st 2018 at the Earle Brown Heritage Center in Spring Lake Park. Activity Status as of July 31, 2018: Dr. Sunil Mor provided a presentation at 43rd Eastern Fish Health Workshop in April 2018, entitled “Carp Killing Virus Discovered In Minnesota: What Could It Mean For Biocontrol?”. This presentation resulted in a request from the European Association of Fish Pathologists to publish a paper on the topic in their journal. It also sparked ongoing conversations with researchers and managers on the potential of this approach for controlling aquatic invasive species. Dr. Sunil Mor also presented at the Aquatic Invaders Summit III in Spring Lake Park, MN. In addition, Dr. Nick Phelps presented at a special meeting of the Freshwater Mollusk Conservation Society focused on the health of native and invasive mussels. This was a good opportunity to network with freshwater mussel research, discuss the goals of the project and share diagnostic approaches that have been developed. A research manuscript detailing about current trends of KHV and CEV outbreak in state Minnesota is nearing completion and will be submitted soon. Dr. Ken McColl (Commonwealth Scientific and Industrial Research Organization, Australia) presented on the Australian progress towards the use of viral biocontrol for common carp at the 2018 International Conference on One Medicine One Science held at the University of Minnesota. He also provided a more technical research seminar on campus and had meetings with our research team, the US Fish and Wildlife service, MN Department of Natural Resources, and other interested researchers. This visit proved to be a useful opportunity to socialize this approach and generate ideas for future research needs. His visit was at no-cost to this project. Activity Status as of January 31, 2019: Dr. Soumesh Padhi provided a presentation at the International Symposium on Aquatic Animal Health in September 2018, entitled “Viruses as biocontrol agents for invasive common carp in Minnesota”. As expected, this presentation sparked active discussion during and after the meeting on the project – with interest for and against. Multiple media interviews were provided during the summer of 2018 related to large scale mortality events of common carp and the concept of using viruses as a biocontrol strategy. In addition, two videos were created to highlight the purpose of the project and current outcomes – they are available here: https://www.maisrc.umn.edu/native-pathogens. In December 2018, we met with the MN DNR Koi Herpes Virus Working Group to discuss the 2017-2018 KHV and

25

CEV outbreaks in Minnesota, DNR management response, and project progress. It was an informative meeting that will be helpful to guide future research direction. One discussion point that remained unanswered is ‘how can research satisfy concern for environmental safety?’ The project team has placed environmental safety at the top of the priority list and is working to address related questions, such as species specificity. However, defining risk tolerance and thresholds would be very helpful as this research moves forward. Activity Status as of July 31, 2019: Dr. Nick Phelps provided a presentation at the Eastern Fish Health Workshop in April 2019, entitled “Importance of next generation sequencing for the detection and characterization of the carp virome”. The presentation focused on the methods that had been developed during the course of this project and was very well received. We were also provided feedback and suggestions to improve our process. The project was featured in the New York Times Science section on June 4, 2019. The article can be found here: https://www.nytimes.com/2019/06/04/science/carp-whooshh-cannon.html Final Report Summary: We had learned during Phase 1 of this project (MAISRC Sub Project 7.1) that communication, outreach and transparency were very important for this type of project. To that end, the project team has spent considerable time engaging with managers, scientists, and the public in multiple formats. This has included formal in person meetings, local-national-international scientific conferences, peer-review publication, networking with internationally-renowned experts, video production, and print, radio and TV media. A summary of this is listed below: Formal in-person meetings: Great Lakes Fish Health Committee, MN DNR Koi Herpes Virus Working Group. Scientific conferences: American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop, MAISRC showcase (x3), International Conference on Aquatic Invasive Species, Minnesota Veterinary Diagnostic Laboratory, Aquatic Invaders Summit III, Freshwater Mollusk Conservation Society, International Symposium on Aquatic Animal Health. NOTE: Most of these conferences were supported by non-LCCMR funding. Peer-review publication: Padhi, S. K., I. E. Tolo, M. McEachran, A. Primus, S. K. Mor, N. B. D. Phelps. In press. Koi herpesvirus and carp edema virus: Infections and coinfections during mortality events of wild common carp in the United States. Journal of Fish Disease. Several other publications are in progress. Networking with experts: Dr. Ken McColl, Dr. Tom Waltzek, Dr. Mikolaj Ademek, and others. Video production: Video 1 (viewed 822 times), Video 2 (viewed 96 times). Media: New York Times, KSTP 5, KARE 11, Star Tribune, Minnesota Daily, MN DNR Press release, MAISRC newsletters. V. DISSEMINATION: Description: Dissemination of this project is critical to advancing the broader scientific understanding, achieving our goals, and successful implementation of the findings. To that end, communications have been explicitly included as Activity 4 to account for the associated budget and identify outcomes. Status as of July 31, 2017: We learned from Phase I of this project (MAISRC SubProject 7-1) that an increased effort was needed to communicate the need to collaborate on sample collection. To that end, the Project Manager (Dr. Nick Phelps)

26

and Co-Investigator (Dr. Sunil Kumar Mor) attended the joint meetings of the American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop and the Great Lakes Fish Health Committee to present on this project. The presentations were titled: “Investigating fish kills: Looking back, looking deep and looking forward” and “Understanding the virome of invasive carp: What it could mean for biocontrol”. These presentations resulted in an active discussion on the potential use of viruses for biocontrol, interest to submit samples for the project and potential collaborations for future research efforts related to this project. Funding was only provided to Dr. Mor for attendance, Dr. Phelps used other funds. To discuss the applications of viral biocontrol, we have invited a world leader, Dr. Ken McColl (Commonwealth Scientific and Industrial Research Organization, Australia), to present at the 2018 iCOMOS meeting to be held at the University of Minnesota, more information: http://icomos.umn.edu. We expect that as part of Dr. McColl’s visit, we will host meetings with members of state and federal agencies to socialize this approach and generate ideas for future research needs. Dr. Nick Phelps is on the iCOMOS organizing committee and has arranged all costs to be covered from other sources. Status as of January 29, 2018 Dr. Sunil Mor and Isaiah Tolo provided a presentation at the MAISRC showcase meeting in September 2017, entitled “Hitchhikers on the hitchhikers: Novel viruses to control invasive fish populations”. Dr. Nick Phelps attended the International Conference on Aquatic Invasive Species in October 2017, entitled “Understanding the carp virome: What it could mean for the control of invasive carp”. Lastly, Dr. Soumesh Padhi provided a presentation for the Minnesota Veterinary Diagnostic Laboratory in December 2017, entitled “Understanding the carp virome: What it could mean for the control of invasive carp”. Dr. Sunil Mor and Isaiah Tolo will be attending the Aquatic Invaders Summit III scheduled for Feb 28th/March 1st 2018 at the Earle Brown Heritage Center in Spring Lake Park. Status as of July 31, 2018: Dr. Sunil Mor provided a presentation at 43rd Eastern Fish Health Workshop in April 2018, entitled “Carp Killing Virus Discovered In Minnesota: What Could It Mean For Biocontrol?”. This presentation resulted in a request from the European Association of Fish Pathologists to publish a paper on the topic in their journal. It also sparked ongoing conversations with researchers and managers on the potential of this approach for controlling aquatic invasive species. Dr. Sunil Mor also presented at the Aquatic Invaders Summit III in Spring Lake Park, MN. A research manuscript detailing about current trends of KHV and CEV outbreak in state Minnesota is nearing completion and will be submitted soon. Status as of January 31, 2019: Dr. Soumesh Padhi provided a presentation at the International Symposium on Aquatic Animal Health in September 2018, entitled “Viruses as biocontrol agents for invasive common carp in Minnesota”. As expected, this presentation sparked active discussion during and after the meeting on the project – with interest for and against. Multiple media interviews were provided during the summer of 2018 related to large scale mortality events of common carp and the concept of using viruses as a biocontrol strategy. In addition, two videos were created to highlight the purpose of the project and current outcomes – they are available here: https://www.maisrc.umn.edu/native-pathogens. In December 2018, we met with the MN DNR Koi Herpes Virus Working Group to discuss the 2017-2018 KHV and CEV outbreaks in Minnesota, DNR management response, and project progress. It was an informative meeting that will be helpful to guide future research direction. One discussion point that remained unanswered is ‘how can research satisfy concern for environmental safety?’ The project team has placed environmental safety at the top of the priority list and is working to address related questions, such as species specificity. However, defining risk tolerance and thresholds would be very helpful as this research moves forward.

27

Status as of July 31, 2019: Final Report Summary: We had learned during Phase 1 of this project (MAISRC Sub Project 7.1) that communication, outreach and transparency were very important for this type of project. To that end, the project team has spent considerable time engaging with managers, scientists, and the public in multiple formats. This has included formal in person meetings, local-national-international scientific conferences, peer-review publication, networking with internationally-renowned experts, video production, and print, radio and TV media. A summary of this is listed below: Formal in-person meetings: Great Lakes Fish Health Committee, MN DNR Koi Herpes Virus Working Group. Scientific conferences: American Fisheries Society – Fish Health Section, Eastern Fish Health Workshop, MAISRC showcase (x3), International Conference on Aquatic Invasive Species, Minnesota Veterinary Diagnostic Laboratory, Aquatic Invaders Summit III, Freshwater Mollusk Conservation Society, International Symposium on Aquatic Animal Health. NOTE: Most of these conferences were supported by non-LCCMR funding. Peer-review publication: Padhi, S. K., I. E. Tolo, M. McEachran, A. Primus, S. K. Mor, N. B. D. Phelps. In press. Koi herpesvirus and carp edema virus: Infections and coinfections during mortality events of wild common carp in the United States. Journal of Fish Disease. Several other publications are in progress. Networking with experts: Dr. Ken McColl, Dr. Tom Waltzek, Dr. Mikolaj Ademek, and others. Video production: Video 1 (viewed 822 times), Video 2 (viewed 96 times). Media: New York Times, KSTP 5, KARE 11, Star Tribune, Minnesota Daily, MN DNR Press release, MAISRC newsletters. VI. SUB-PROJECT BUDGET SUMMARY: A. Preliminary ENRTF Budget Overview: *This section represents an overview of the preliminary budget at the start of the project. It will be reconciled with actual expenditures at the time of the final report. See the Sub-Project Budget document for an up-to-date project budget, including any changes resulting from amendments.

Budget Category $ Amount Explanation Personnel: $229,831 Dr. Sunil Kumar Mor (1.0FTE total), Dr. Alex

Primus (0.25 FTE total), TBD Graduate student (1.0FTE total), and student support (0.5FTE total). All positions will be supported for a total of two years.

Professional/Technical Services and Contracts:

$110,607 Diagnostic services (i.e. bacteriology, electron microscopy, histology, virology) will be provided by the Minnesota Veterinary Diagnostic Laboratory (MVDL) on a fee-for-service basis for a total budget of $27,350. Sequencing services (Ilumina MiSeq) will be provided by the University of Minnesota Genomics Center (UMGC) on a fee-for-service bases for a total of

28

$77,370. Service contracts with the MVDL and UMGC are assuming sample numbers and conditions are as proposed. Disease challenge experiments will be performed in the MAISRC Containment Laboratory for eight months for a total of $15,000. Publication costs for three manuscripts for a total of $6,000.

Equipment/Tools/Supplies: $67,300 A variety of consumable supplies will be purchased for this project, including molecular kits, diagnostic supplies, anesthesia, fish and food, disinfectant, etc for a total of $54,840. In addition, one laptop ($2,000) will be purchased for data management by the graduate student.

Capital Expenditures over $5,000: $7,718 One biosafety cabinet will be purchased to prepare samples for diagnostic testing at a cost of $8,000.

Travel: $9,490 Travel within Minnesota to collect samples and meet in-state stakeholders for a total of $3,000. Domestic travel to collect samples from river systems where invasive carp are present and travel for an investigator to attend three scientific conferences and present findings for a total of $13,000.

Other: $0 TOTAL ENRTF BUDGET: $424,946

Explanation of Use of Classified Staff: Explanation of Capital Expenditures Greater Than $5,000: One biosafety cabinet will be purchased to prepare samples for diagnostic testing at a cost of $8,000. This cabinet was budgeted in Phase I of this project, but was not purchased at that time because the project team had access to a shared biosafety cabinet, which is no longer available. Number of Full-time Equivalents (FTE) Directly Funded with this ENRTF Appropriation: 2.75 FTE Number of Full-time Equivalents (FTE) Estimated to Be Funded through Contracts with this ENRTF Appropriation: 0 FTE B. Other Funds:

Source of Funds $ Amount Proposed

$ Amount Spent Use of Other Funds

Non-state $ $ State $ $

TOTAL OTHER FUNDS: $ $ VII. SUB-PROJECT STRATEGY:

29

A. Sub-Project Team/Partners: The project team for this project has a track record of collaborative and successful research efforts. The Project Manager, Dr. Nicholas Phelps, will provide overall guideance for the project. Dr. Sunil Kumar Mor’s effort is critical to the success of the project. He will contribute to the sample collection and processing, diagnostics, sequence analysis, report preparation, and manuscript publication. Dr. Alex Primus will contribute diagnostic support and lead the disease challenges. One graduate student will be supported on this project and contribute to all activiites. Local, national, and international stakeholders will be encouraged to participate in the project to help guide implementation and maximize impact.

B. Sub-Project Impact and Long-term Strategy: There will be a variety of potential benefits from this research. Most importantly, the results will help to inform science-based risk assessments to pursue the use viruses for invasive species control. In addition, the results will benefit fish health management decisions that impact the short and long-term sustainability of native and invasive fish populations in Minnesota by identifying emerging threats. This is the second phase of a long-term effort to evaluate the use of viruses as biological control agents for aquatic invasive species. Understanding the virome is a critical first step, followed by infection trials, environmental and risk assessments, development of release strategies, and implementation. The current Project Team is well suited for the initial stages and aims to build collaborations for future work in the areas of risk assessment and release strategies. These conversations have already begun with researchers at the Commonwealth Scientific and Industrial Research Organization in Australia and the United States Department of Agriculture, among others. Meanwhile, it is critical to engage stakeholders at all levels to evaluate/ensure a viable path forward should the research prove promising.

C. Spending History: Funding Source M.L. 2008

or FY09

M.L. 2009 or

FY10

M.L. 2010 or

FY11

M.L. 2011 or

FY12-13

M.L. 2013 or

FY14

(add or remove rows and columns as needed) VIII. ACQUISITION/RESTORATION LIST: N/A IX. VISUAL ELEMENT or MAP(S): N/A X. ACQUISITION/RESTORATION REQUIREMENTS WORKSHEET: N/A XI. RESEARCH PROPOSAL: See attached research proposal. XII. REPORTING REQUIREMENTS: Periodic work plan status update reports will be submitted no later than July 31, 2017, January 31, 2018, July 31, 2018, January 31, 2019, and July 31, 2019. A final report and associated products will be submitted within two months of the anticipated sub-project completion of June 30, 2019.

Environment and Natural Resources Trust FundM.L. 2013 Sub-Project Budget of M.L. 2013-06a: Aquatic Invasive Species Research Center

Project Title: Aquatic Invasive Species Research Center Sub-Project #7: Developing erradication tools for invasive speciesLegal Citation: M.L. 2013, Chp. 52, Sec. 2, Subd. 06aProject Manager: Dr. Nicholas PhelpsOrganization: University of MinnesotaSub-Project Budget: $445,210Sub-Project Phase 2 Length and Completion Date: 2.5 years, June 30, 2019Project Length and Completion Date: 6 Years, June 30, 2019Date of Report: August 16, 2019

ENVIRONMENT AND NATURAL RESOURCES TRUST FUND BUDGETBUDGET ITEM Activity 1

Budget Amount SpentActivity 1Balance

Activity 2 Budget Amount Spent

Activity 2Balance

Activity 3 Budget Amount Spent

Activity 3Balance

Activity 4 Budget Amount Spent

Activity 4Balance

Personnel (Wages and Benefits) - Total $45,880 $45,880 $0 $140,740 $140,740 $0 $46,030 $37,211 $8,819 $6,000 $6,000 $0 $238,650 $229,831 $8,819Dr. Alex Primus, Assistant Professor: $20,000 Total salary, $6,740 Total benefits (33.7% fringe rate), 0.2 Total FTE for 2 years Dr. Sunil Kumar Mor, Assistant Professor: $21,000 Total salary, $7,077 Total benefits (33.7% fringe rate), 0.2 Total FTE for 2 years Soumesh Kumar Padhi - Post Doctoral Fellow: $96,000 salary, $20,544 benefits (21.4% fringe rate); 2.0 total FTETodd Knutson - Researcher 6: $16,816 salary, $3,600 benefits (21.4% fringe rate); 0.34 total FTEIsaiah Tolo, Graduate Student: $22,201 salary, $18,912 (37% tuition, 9% fringe rate), 0.5 Total FTE for 1 yearTBD, Professional/Undergraduate Student: $12/hr, 240hrs per year; (0% fringe rate) 0.25 Total FTE for 2 years

Professional/Technical Services and Contracts - Total $282 $36 $246 $75,781 $75,781 $0 $32,350 $32,350 $0 $6,000 $2,440 $3,560 $114,413 $110,607 $3,806Services- lab & medical (Diagnostic services at the Minnesota Veterianry Diagnostic Laboratory)

$0 $0 $0 $10,677 $10,677 $0 $17,350 $17,350 $0 $0 $0 $0 $28,027 $28,027 $0

Services- lab & medical (Sequencing services at the University of Minnesota Genomics Center)

$0 $0 $0 $65,104 $65,104 $0 $0 $0 $0 $0 $0 $0 $65,104 $65,104 $0

Services- lab & medical- (MAISRC Containment Lab) $0 $0 $0 $0 $0 $0 $15,000 $15,000 $0 $0 $0 $0 $15,000 $15,000 $0Publication costs (n=3) $0 $0 $0 $0 $0 $0 $0 $0 $0 $6,000 $2,440 $3,560 $6,000 $2,440 $3,560Shipping $282 $36 $246 $0 $0 $0 $0 $0 $0 $0 $0 $282 $36 $246

Equipment/Tools/Supplies - Total $6,000 $5,733 $267 $48,209 $48,209 $0 $16,220 $13,358 $2,862 $0 $0 $0 $70,429 $67,300 $3,129Supplies- lab & field (anesthesia, fish, fish food, molecular kits, other misc laboratory supplies)

$4,000 $3,733 $267 $48,209 $48,209 $0 $16,220 $13,358 $2,862 $0 $0 $0 $68,429 $65,300 $3,129

management by the graduate student. The computer will remain at MAISRC after the project is completed and could be used by other employees or students of MAISRC.)

$2,000 $2,000 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $2,000 $2,000 $0

Capital Expenditures Over $5,000 - Total $0 $0 $0 $7,718 $7,718 $0 $0 $0 $0 $0 $0 $0 $7,718 $7,718 $0Cap expenditures over $5,000: (Biosafety cabinet, $8,000) $0 $0 $0 $7,718 $7,718 $0 $0 $0 $0 $0 $0 $0 $7,718 $7,718 $0

Travel - Total $5,000 $3,910 $1,090 $0 $0 $0 $0 $0 $0 $9,000 $5,580 $3,420 $14,000 $9,490 $4,510Travel - MN (Milage to collect samples and meet in-state stakeholders) $2,500 $2,500 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $2,500 $2,500 $0Travel - Domestic (Milage to collect samples from river systems where invasive carp are present, travel for an investigator to attend three scientific conferences and present findings.

$2,500 $1,410 $1,090 $0 $0 $0 $0 $0 $0 $9,000 $5,580 $3,420 $11,500 $6,990 $4,510

Other - Total $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0 $0$0 $0 $0 $0 $0 $0 $0 $0 $0 $0$0 $0 $0 $0 $0 $0 $0 $0 $0 $0

COLUMN TOTAL $57,162 $55,559 $1,603 $272,448 $272,448 $0 $94,600 $82,919 $11,681 $21,000 $14,020 $6,980 $445,210 $424,946 $20,264

TOTALSPENT

Activity 4: Communication

TOTALBALANCE

Activity 1: Collect samples Activity 2: Characterize virome Activity 3: Determine disease causing potential

TOTAL BUDGET