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WRAC TERMINATION REPORT, September 2010 Part I: Summary

PROJECT TITLE: Potential threat of VHS Virus in the Western United States

REPORT GIVEN IN YEAR 2010

PROJECT WORK PERIOD: 7/01/2008–8/31/2009; no-cost extension approved through 8/31/2010.

AUTHOR: Gael Kurath

PARTICIPANTS:

Gael Kurath (Work Group Chair) USGS Western Fisheries Research Center (WFRC), Seattle Jim Winton USGS-WFRC, Seattle Paul Hershberger USGS-WFRC, Marrowstone Marine Station Carolyn Friedman* University of Washington, Seattle Jerri Bartholomew* (outreach) Oregon State University Kenneth Cain, Technical Advisor: University of Idaho Scott E. LaPatra , Industry Advisor: Clear Springs Foods, Inc. Chang Hoon Moon, Postdoctoral Fellow, University of Washington Evi Emmenegger USGS-WFRC, Seattle REASON for TERMINATION: Project complete, funds expended

PROJECT OBJECTIVES: 1. Assemble and distribute biosecurity information currently available for dealing with VHSV

2. Develop diagnostic assays to differentiate Great Lakes VHSV IVb from endemic West coast VHSV IVa

3. Test susceptibility of yellow perch, rainbow trout, herring, and Chinook salmon to disease and mortality caused by Great Lakes VHSV IVb, West coast VHSV IVa and European VHSV I

4. Test ability of relevant host species to act as carriers and/or reservoirs of different VHSV genotypes

5. Develop outreach materials to communicate project results PRINCIPAL ACCOMPLISHMENTS: Funding for this project became available in July 2008. Dr. Chang Hoon Moon was hired as a post-doctoral fellow in August 2008, and worked full-time on the project in the Kurath laboratory at the USGS Western Fisheries Research Center (WFRC) until the funding was expended in November 2009. There have been no funded personnel on the project since November 2009.

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Objective 1. Assemble and distribute biosecurity information currently available for dealing with VHSV. Biosecurity information for the European VHSV type I and the Great Lakes VHSV type IVb strains was assembled in year 1 and a Powerpoint presentation focusing on biosecurity was prepared by the outreach coordinator, Jerri Bartholomew. This file was provided to the WRAC office early in year 2 for use on the website when available. The presentation has been given orally at a workshop for fish farmers and at a second workshop for extension agents, and is attached to this termination report. This information, together with results of the research project, have been assembled into a draft "WRAC FAQs about VHSV" document that is intended to answer frequently asked questions concerning VHSV, with a focus on issues pertinent to Western U.S. user groups: fish farmers, anglers and fish health diagnosticians. This FAQ sheet is currently in review by extension personnel and will be published as a USGS fact sheet available on both the WFRC and WRAC and websites. General information on VHSV from a national perspective has been assembled into fact sheets available from various other agencies including NRAC. Rather than duplicate this effort we have provided links to these resources on our WRAC FAQs sheet. During the project six oral presentations on VHSV that include information on biosecurity issues were given by project investigators.

Objective 2. Develop diagnostic assays to differentiate Great Lakes VHSV IVb from endemic West coast VHSV IVa. During this project we have used a general quantitative real-time polymerase chain reaction (qRT-PCR) developed by Dr. Kyle Garver of the Pacific Biological Station in Nanaimo, British Columbia. The development of genotype-specific qRT-PCR assays that specifically detect type IV a only, or type IVb only, was originally a priority for this project, but during the project period several factors arose that caused us to consider this a less urgent need. Results obtained in objectives 3 and 4, dynamic changes initiated at the national level for standardization of VHSV diagnostics, and availability of existing genetic typing methods that distinguish genotypes IVa and IVb, all combined to support a decision not to pursue development of genotype specific qRT-PCR assays at this time. Detailed explanation of the basis for this decision is provided in part II.

Objective 3. Test susceptibility of yellow perch, rainbow trout, herring, and Chinook salmon to disease and mortality caused by Great Lakes VHSV IVb, West coast VHSV IVa and European VHSV I. This objective has all been completed and is being prepared for publication in a peer-reviewed journal by the first author, Evi Emmenegger. Susceptibility of four fish species to disease and mortality due to Great Lakes VHSV type IVb (designated VHSV IVb-GL) has been determined in controlled wet laboratory challenge studies, where virus was delivered by intraperitoneal (IP) injection at two different viral doses referred to as "low", and "high". For comparisons of different VHSV genotypes, West coast type IVa, Great Lakes type IVb, and European type I strains were tested simultaneously. In addition, an Atlantic coast VHSV type IVb strain from New Brunswick (IVb-NB) was included in challenge studies when possible. All infection studies were conducted on juvenile fish (2-5g) at 12°C in the WFRC Biosafety Level 3 (BSL-3) wetlab. Mortality in mock-infected control groups was negligible.

Virulence trials by IP injection in yellow perch: Yellow perch are used in this project as a positive control host for the Great Lakes VHSV IVb, since epidemics have occurred recently in yellow perch in the Great Lakes. Challenges were conducted by IP injection with high and low doses of VHSV strains representing genotypes I, IVa, IVb-GL, and IVb-NB. In the high dose treatment groups, average final cumulative percent mortality (CPM) ranged from 84-100%, and low dose treatments groups ranged 30-93%. Relative virulence of the four VHSV strains, in order from highest to lowest, was IVa > IVb-GL > IVb-NB > I. In general, by the IP injection delivery route yellow perch were highly susceptible to all strains, but the low dose treatment indicated it is least susceptible to VHSV genotype I.

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Virulence trials by IP injection in rainbow trout: Rainbow trout are used here due to their significance as a major western aquaculture species, and also as a positive control host for VHSV genotype I. In the high dose injection groups CPM was 3-86%, and in low dose groups CPM was 2-98%. Relative virulence of the four VHSV strains, in order from highest to lowest, was I >> IVa = IVb-NB > IVb-GL. In general, by IP injection rainbow trout were highly susceptible to genotype I, but not very susceptible to genotypes IVa, IVb-GL, or IVb-NB.

Virulence trials by IP injection in Chinook salmon: Chinook salmon were also tested as an important western aquaculture species. In the high dose treatment groups, CPM ranged 13-76%, and in low dose groups CPM was 5-47%. Relative virulence of the four VHSV strains, in order from highest to lowest, was I >> IVb-GL=IVb-NB > IVa . In general, the susceptibility pattern of Chinook salmon was similar to that of rainbow trout, but Chinook had slightly lower mortality with genotype I, and seemed nearly resistant to the new genotype IVb-GL.

Modified virulence trials by IP immersion in herring: Pacific herring were included in this project as a positive control host species for VHSV genotype IVa, which has caused large-scale marine epidemics of herring and pilchard off the west coast. A trial was conducted in herring using challenge by immersion rather than injection, and CPM ranged 43-80%. As expected, herring were very susceptible to genotype IVa, and only moderately susceptible to the other three VHSV genotypes.

Objective 4. Test ability of relevant host species to act as carriers and/or reservoirs of different VHSV genotypes. This objective involves comparing infection levels of individual VHSV strains in different fish hosts regardless of the occurrence of disease signs or mortality. Quantification of virus in fish that died during the injection challenges described above showed that all VHSV strains replicated in all fish species, but with slightly lower titers in Chinook. Among asymptomatic fish that were randomly sampled at 7 days post-challenge (dpc), yellow perch, rainbow trout, and Chinook salmon showed replication of all virus strains in all fish species. In herring sampled 14 dpc all virus strains persisted in most fish, but at 1-2 logs lower titers than in herring that died. In survivors samples at 30 dpc, yellow perch and Chinook salmon showed all four virus strains persisted in at least some fish, but in rainbow trout only genotypes I and IVa persisted, in a small number of fish.

To compare in vivo growth of VHSV genotypes I, IVa, and IVb-GL, time-course experiments were conducted measuring viral loads in rainbow trout or yellow perch challenged by immersion at 15°C. Virus replication peaked at day 3 for all 3 strains. In rainbow trout the viral load of VHSV genotype I was 1-2.5 logs higher than genotypes IVa and IVb. This variation in viral growth among the 3 VHSV strains correlated very well with the levels of mortality in the rainbow trout virulence trials. Host immune gene response was also measured, and mirrored viral loads, being most notable in fish infected with genotype I. A similar time-course in yellow perch at 12°C showed more rapid replication of VHSV type IVa and IVb, to higher viral load levels than type I, again correlating with the differences between VHSV types in virulence challenge studies.

Collectively, these data indicate that the four fish species tested differ in their susceptibility to disease and infection by the various VHSV types, and they can all act as carriers and reservoirs of Great Lakes VHSV.

Objective 5. Develop outreach materials to communicate project results. Four oral presentations and one poster focusing on data from this project have been presented by project investigators at regional and national fish health meetings. Data from the project was also included in 4 of the 6 general talks listed under objective 1. An invited presentation was made to the Idaho Aquaculture association, and separate workshops for fish farmers and extension

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personnel have been held. Educational materials that include project results include the Powerpoint presentation and "WRAC FAQs about VHSV" fact sheer created by Jerri Bartholomew. These also include biosecurity information and are detailed in objective 1. IMPACTS:

Relevance: Viral hemorrhagic septicemia virus (VHSV) was first isolated from fish in the Great Lakes in 2003, and was reported in 2005 as the causative agent of a large-scale die-off of freshwater drum in Lake Ontario. Since then numerous epidemics in multiple host species have occurred in the Great Lakes region, resulting in an extreme level of concern and severe restrictions on aquaculture activities. This project involved outreach and research objectives that address specific needs of fish farmers in the western region of the United States, and contribute to the national response to the emergence of VHSV in the Great Lakes.

Response: The Kurath lab conducted research to address concerns of western aquaculture producers about the threat of the Great Lakes strain of VHS virus for western aquaculture species. Four strains of VHSV representing genetic types from the Great Lakes, the Pacific Coast, the Atlantic Coast and Europe were tested for virulence and persistence in four fish species. The fish selected for study were relevant to western aquaculture, or to VHS epidemics in the Great Lakes and the Pacific Ocean. Funds of $100,000 supported a 15 month research and outreach project.

Results: Variations in virulence and persistence in different fish host were identified for different strains of VHS virus. No biological difference was demonstrated between VHSV strains from the Great Lakes and those currently endemic in Pacific marine fish species. Rainbow trout and Chinook salmon had low susceptibility to North American VHSV strains but high susceptibility to European VHSV. In contrast, yellow perch and Pacific herring were more susceptible to the Great Lakes strain than the European strain.

Impact: Two workshops were held to communicate these results along with biosecurity information to fish farmers and western region extension agents. Biosecurity measures to prevent introduction of any VHSV into aquaculture facilities will reduce costs to producers, both in terms of disease impacts and regulatory compliance. Several general VHSV talks and talks on the project results were presented to various interest groups, and a document titled "WRAC FAQs about VHSV" has been prepared as a fact sheet.

RECOMMENDED FOLLOW-UP ACTIVITIES: The results obtained in this project have succeeded in answering the most urgent questions pertinent to salmonid aquaculture in the Western U.S. by finding low susceptibility, but carrier potential, in rainbow trout and Chinook salmon. Follow-up activities might include: - test virulence and carrier potential in Atlantic salmon, hybrid striped bass and/or sturgeon. - test virus delivered by immersion in fish species where mortality was seen by injection. - conduct susceptibility studies with wild/free-ranging fish species present in the West that is already on list of 28 host species in the Great Lakes. VHSV IVb has been isolated from these species, but many have not been tested for level of susceptibility to disease, which would assess the risk they pose as potential reservoir species if VHSV IVb is introduced in the west. - if VHSV IVb is introduced and is detected in western aquaculture facilities this would elevate the priority of developing genotype specific qRT-PCR assays for VHSV IVa and IVb.

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SUPPORT:

YEAR

WRAC-USDA

Funding

OTHER SUPPORT

Total Support University Industry Other

Federal Other** Total

2008 & 2009*

$72,097 $72,097

2010 $27,903 $27,903

TOTAL $100,000 $100,000

* The funds for this project were emergency funds awarded outside the standard timing of the WRAC funding cycle. Funds became available initially in July of 2008, so that in FY 2008 there were only 2 months of activity on the project. Therefore, FY2008 and FY2009 have been combined together in the table above as "Year 1" of the project, which was funded at $72,097. In FY 2010 the funds remaining for year 2 were $27,903, which supported approximately 3 months of work. **Substantial in-kind support provided throughout this project included donated time of G. Kurath, E. Emmenegger, P. Hershberger, and J. Winton at the USGS-WFRC lab, and donated pathogen-free fish stocks from P. Hershberger (USGS-WFRC Marrowstone Marine Station), S. LaPatra (Clear Springs Foods, Inc.), and F. Goetz and F. Binkowski (UWM, Great lakes WATER Institute). PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: See next page

September 14, 2010 SUBMITTED BY: Title: (Work Group Chair or PI) Date

APPROVED: September 14, 2010 Technical Advisor Date

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PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Publications in Print J. Bartholomew, Outreach product. Problems presented by emerging pathogens: Potential threat of Great Lakes VHS virus in the

Western United States. Powerpoint provided to WRAC for addition to WRAC website. Manuscripts in preparation Emmenegger EJ, Moon CH, and Kurath G. Variable Susceptibility of salmonid, cyprinid,

clupeid, and percid fish species to VHSV genotypes from freshwater, brackish water, and marine environments. In preparation for Diseases of Aquatic Organisms.

Bartholomew J, and Kurath G. WRAC FAQs about VHSV. In review for publication as a USGS

fact sheet. Papers Presented: General outreach information and project results Kurath G. VHSV: The history and basics of the virus and the disease. 33rd Eastern Fish Health

Workshop, VHSV Continuing Education Session, 4/4/08, Atlantic Beach, North Carolina. Emmenegger EJ*, Kurath G, Wargo A, Binkowski F, and Goetz R. Yellow perch (Perca

flavescens) susceptibility to viral hemorrhagic septicemia virus isolates from Europe and North America. Western Fish Disease Workshop, Ocean Shores, WA, June 2008.

Kurath G*, Emmenegger EJ, Wargo A, Binkowski F, and Goetz R. Susceptibility of yellow perch

to VHS virus strains from the Great Lakes, Pacific coast, and Europe. AFS-Fish Health Section Annual meeting, Charlottetown, Prince Edward Island, Canada, July 2008

Kurath G*, Emmenegger EJ, Moon CH. Susceptibility of Pacific salmonids, yellow perch, and

koi to viral hemorrhagic septicemia virus strains from the Great Lakes, Pacific and Atlantic coasts of Canada, and Europe. Aquaculture America, Invited talk for session on RAC research on VHSV. 2/17/09, Seattle, Washington.

Kurath G. How we got here: A review of VHS in the US . Invited talk for special "Producers

session" on VHSV at Aquaculture America, 2/17/09, Seattle, Washington. Kurath G. Fish RNA viruses: VHSV epidemiology and evolution. Invited guest lecture in Fish

Disease course at Oregon State University, 2/23/09, Corvallis, Oregon. Kurath G. History and current affairs of VHS virus. Invited talk in special VHSV session at the

International Conference on Aquatic Invasive Species, 4/23/09, Montreal, Canada. Emmenegger EJ*, Moon CH, and Kurath G. Susceptibility of western aquaculture species to

different strains of VHS virus, including Great Lakes VHSV. Idaho Aquaculture Association, 6/09, Twin Falls, Idaho.

Moon CH*, Emmenegger EJ, and Kurath G. Susceptibility of Pacific salmonids, yellow perch,

and koi to four strains of VHS virus, including Great Lakes VHSV. Poster presentation at AFS Fish Health Section Annual meeting, 6/10/09, Park City, Utah.

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Kurath G*. Epidemiology of fish rhabdoviruses part 2: VHSV. Invited guest lecture in Oregon State University Salmon Disease Workshop, 7/21/09, Corvallis, Oregon.

Bartholomew J. Problems presented by emerging pathogens: Potential threat of Great Lakes

VHS virus in the Western United States. Workshop for fish farmers at Hagerman Fish Culture Experiment Station, 8/14/09, Hagerman, Idaho.

Bartholomew J. Problems presented by emerging pathogens: Potential threat of Great Lakes

VHS virus in the Western United States. Workshop for extension agents, presented in conjunction with WRAC meeting 10/8/09, Spokane, Washington.

Kurath G. Viral hemorrhagic septicemia virus: and old virus with new tricks. Plenary talk, 6th

International Symposium on Aquatic Animal Health, 9/8/10, Tampa, Florida.

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Part II: Detail PROJECT TITLE: Potential threat of VHS Virus in the Western United States

REPORT GIVEN IN YEAR 2010

PROJECT WORK PERIOD: 7/01/2008–8/31/2009; no-cost extension approved through 8/31/2010.

AUTHOR: Gael Kurath

PARTICIPANTS:

Gael Kurath (Work Group Chair) USGS Western Fisheries Research Center (WFRC), Seattle Jim Winton USGS-WFRC, Seattle Paul Hershberger USGS-WFRC, Marrowstone Marine Station Carolyn Friedman* University of Washington, Seattle Jerri Bartholomew* (outreach) Oregon State University Kenneth Cain, Technical Advisor: University of Idaho Scott E. LaPatra , Industry Advisor: Clear Springs Foods, Inc. Chang Hoon Moon, Postdoctoral Fellow, University of Washington Evi Emmenegger USGS-WFRC, Seattle PROJECT OBJECTIVES: 1. Assemble and distribute biosecurity information currently available for dealing with VHSV

2. Develop diagnostic assays to differentiate Great Lakes VHSV IVb from endemic West coast VHSV IVa

3. Test susceptibility of yellow perch, rainbow trout, herring, and Chinook salmon to disease and mortality caused by Great Lakes VHSV IVb, West coast VHSV IVa and European VHSV I

4. Test ability of relevant host species to act as carriers and/or reservoirs of different VHSV genotypes

5. Develop outreach materials to communicate project results TECHNICAL SUMMARY AND ANALYSIS: Funding for this project became available in July 2008. Dr. Chang Hoon Moon was hired as a post-doctoral fellow in August 2008, and worked full-time on the project in the Kurath laboratory at the USGS Western Fisheries Research Center (WFRC) until funding was expended in November 2009. Evi Emmenegger, a USGS GS12 research microbiologist at WFRC

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contributed significantly to the project as the designated WFRC specialist in exotic and invasive pathogens. There have been no funded personnel on the project since November 2009. Objective 1. Assemble and distribute biosecurity information currently available for dealing with VHSV. Biosecurity information for the European VHSV type I and the Great Lakes VHSV type IVb strains was assembled in year 1 and a Powerpoint presentation focusing on biosecurity was prepared by the outreach coordinator, Jerri Bartholomew. This file was provided to the WRAC office early in year 2 for use on the website when available. The presentation has been given orally at a workshop for fish farmers and at a second workshop for extension agents, and is attached to this termination report. This information, together with results of the research project, have been assembled into a draft "WRAC FAQs about VHSV" document that is intended to answer frequently asked questions concerning VHSV, with a focus on issues pertinent to Western U.S. user groups: fish farmers, anglers and fish health diagnosticians. This FAQ sheet is currently in review by extension personnel and will be published as a USGS fact sheet available on both the WFRC and WRAC and websites. General information on VHSV from a national perspective has been assembled into fact sheets available from various other agencies including NRAC. Rather than duplicate this effort we have provided links to these resources on our WRAC FAQs sheet. During the project six oral presentations on VHSV that include information on biosecurity issues were given by project investigators.

Objective 2. Develop diagnostic assays to differentiate Great Lakes VHSV IVb from endemic West coast VHSV IVa. During this project we have used a general quantitative real-time polymerase chain reaction (qRT-PCR) developed by Dr. Kyle Garver of the Pacific Biological Station in Nanaimo, British Columbia. This assay detects both North American VHSV types IVa and IVb, and has been used in objectives 3 and 4 below for general detection of VHSV. The development of genotype-specific qRT-PCR assays that specifically detect type IV a only, or type IVb only, was originally a priority for this project, but during the project period several factors arose that caused us to consider this a less urgent need. First, results obtained in fish challenge studies in objectives 3 and 4 showed that the main western aquaculture species tested here, rainbow trout and Chinook salmon, were of low susceptibility to both VHSV types IVa and IVb. There was no discernible biological difference between VHSV IVa and IVb, but all fish species tested could be carriers, suggesting that western producers should be equally cautious about avoiding introduction of either virus type IVa or IVb into their facilities. In the event that VHSV is detected by general diagnostic methods in a western aquaculture facility, the isolate can identified as IVa or IVb within days using existing genetic typing methods developed in the Kurath laboratory. On the national level diagnostics for VHSV has been a very dynamic area, with changes to the AFS/USFWS Blue Book and initiation of development of a National Aquatic Animal Pathogen testing Network (NAAPTN) as part of the implementation of the National Aquatic Animal Health Plan. Toward this effort the PI (Kurath) has participated in the VHSV work group of the new NAAPTN, contributing specifically to the qRT-PCR assay sub-group. As these efforts are all in early stages, and the existing genetic typing methods can distinguish genotypes IVa and IVb, we decided not to pursue development of genotype specific qRT-PCR assays at this time, although they might be of greater interest in the future.

Objective 3. Test susceptibility of yellow perch, rainbow trout, herring, and Chinook salmon to disease and mortality caused by Great Lakes VHSV IVb, West coast VHSV IVa and European VHSV I. The research in support of this objective has all been completed and is being written for publication in a peer-reviewed journal by the first author, Evi Emmenegger. Susceptibility of four fish species to disease and mortality due to Great Lakes VHSV type IVb (designated VHSV IVb-GL) has been determined in controlled wet laboratory challenge studies, where virus was delivered by intraperitoneal (IP) injection at two different viral doses. The challenge doses were 103 and 106 plaque forming units (PFU) per fish, and are referred to as

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"low", and "high" doses, respectively. For comparisons of different VHSV genotypes, West coast VHSV type IVa and European VHSV type I strains were tested simultaneously. In addition, an Atlantic coast VHSV type IVb strain from New Brunswick (IVb-NB) was included in these challenge studies when possible. All infection studies were conducted on juvenile fish (2-5g) at 12°C in the WFRC Biosafety Level 3 (BSL-3) wetlab for containment of the VHSV types I and IVb that are not endemic to the Western region.

Virulence trials by IP injection in yellow perch: See Figure 1.

Yellow perch are used in this project as a positive control host for the Great Lakes VHSV IVb, since epidemics have occurred recently in yellow perch in the Great Lakes . During year 1 of the project we conducted two pilot studies in yellow perch that confirmed our ability to re-create the disease process seen in the field, and provided data on optimal challenge doses. During year 2 the full-scale virulence trial was conducted using triplicate groups of yellow perch challenged by IP injection with high and low doses of VHSV strains representing genotypes I, IVa, IVb Great Lakes, and IVb New Brunswick. Mock groups of fish were injected with buffer only, and groups were observed for disease signs and mortality for 30 days. Overall the challenge experiment went well, with no mortality in mock-injected control groups, and good reproducibility among replicate tanks. In the high dose treatment groups, average final cumulative percent mortality (CPM) for groups challenges with VHSV genotypes I, IVa, IVb-GL, and IVb-NB was 86%, 100% 100%, and 84%, respectively. For low dose treatments groups average final CPM was 30%, 93%, 76%, and 70% respectively. Relative virulence of the four VHSV strains, in order from highest to lowest, was IVa > IVb-GL > IVb-NB > I. In general, by the IP injection delivery route yellow perch were highly susceptible to all strains, but the low dose treatment indicated it is least susceptible to VHSV genotype I.

Figure 1. Yellow perch injection challenges: Cumulative mortality and diseases signs in yellow perch challenged with VHSV types I (Europe, blue), IVa (Pacific coast, yellow), IVb-GL (Great Lakes, red), and IVb-NB (New Brunswick, green). Mock challenged groups (black) had no mortality.

Virulence trials by IP injection in rainbow trout: See Figure 2.

Rainbow trout are used here due to their significance as a major western aquaculture species, and also as a positive control host for VHSV genotype I, which has caused severe epidemics in European Rainbow trout farms over the last 60 years. The Clear Springs stock of rainbow trout

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were tested as described above for yellow perch. Again the challenge experiment went well, with no mortality in mock-injected groups, and good reproducibility among replicates. In the high dose injection groups, average final CPM for groups challenged with VHSV types I, IVa, IVb-GL, and IVb-NB was 86%, 38% 3%, and 28%, respectively. For low dose injection groups average final CPM was 98%, 8%, 2%, and 15% respectively. Relative virulence of the VHSV strains was I >> IVa = IVb-NB > IVb-GL. In general, by IP injection rainbow trout were highly susceptible to type I, and not very susceptible to types IVa, IVb-GL, or IVb-NB.

Figure 2. Rainbow trout injection challenges: CPM and disease signs as in Figure 1.

Virulence trials by IP injection in Chinook salmon: See Figure 3.

Chinook salmon were also tested as an important western aquaculture species, using the same protocol described above. The challenge experiment went well, with no mortality in mock-injected control groups, and good reproducibility among replicate tanks. In the high dose treatment groups, average final CPM for groups challenges with VHSV types I, IVa, IVb-GL, and IVb-NB was 76%, 13% 28%, and 18%, respectively. For low dose treatments groups average final CPM was 47%, 5%, 17%, and 22% respectively. Relative virulence of the four VHSV strains, in order from highest to lowest, was the same as for rainbow trout: I >> IVb-GL=IVb-NB > IVa . In general, by IP injection the susceptibility pattern of Chinook was similar to rainbow trout, but less susceptible to type I overall, and nearly resistant to type IVb-GL.

Figure 3. Chinook salmon injection challenges: CPM and disease signs as in Figure 1.

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Modified virulence trials by immersion in herring: See Figure 4.

Pacific herring were included in this project as a positive control host species for VHSV genotype IVa, which has caused large-scale marine epidemics of herring and pilchard off the west coast. During our first year pathogen-free herring brought to the Seattle WFRC lab from the WFRC Marrowstone marine station showed poor survival after transport and during holding in the BSL-3, likely due to difficulties in maintaining cold temperatures while providing static seawater. In addition, our co-PI Paul Hershberger correctly advised that herring would not survive injection challenge or holding in the 1 foot diameter tanks in the BL3 wetlab. Therefore during year 2 we conducted another trial in herring with modified conditions including challenge by immersion rather than injection, single larger tanks for each treatment group, and holding for only 14 days. This challenge was successful, with only 10% CPM in the mock control group. In the immersion challenges the final cumulative percent mortality (CPM) for groups challenges with VHSV genotypes I, IVa, IVb-GL, and IVb-NB was 43%, 80% 52%, and 49%, respectively. Thus, as expected, herring were very susceptible to genotype IVa, and only moderately susceptible to the other 3 genotypes.

Figure 4. Pacific herring immersion challenges: CPM and disease signs as in Figure 1

Objective 4. Test ability of relevant host species to act as carriers and/or reservoirs of different VHSV genotypes: This objective involves comparing infection levels of individual VHSV strains in different fish hosts regardless of their clinical status, to determine the potential of these fish species to act as carriers of reservoirs of VHSV.

4a) Quantification of virus in fish that died during injection challenges. See Figure 5.

Determination of virus titers in fish that died during the injection challenges described above showed that all VHSV strains replicated in all fish species. In both high and low dose groups, virus titers in yellow perch, rainbow trout, and herring mortalities were 106-107 plaque-forming units per gram (PFU/g),and titers in Chinook salmon were slightly lower, at 104-105 PFU/g.

4b) Quantification of virus in living fish sampled during injection challenges. See Figures 5 & 6.

Concomitant with the virulence experiments above, 20 additional fish were injected with the high dose of each virus as a group to be sampled for viral infection levels. Fish from these "sampling

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tanks" were collected and euthanized at 7 and 28 days post-challenge. For the herring challenge a separate sampling group was not possible, but survivors were assayed at the end of the challenge 14 dpc. Fish from the yellow perch, rainbow trout, and Chinook salmon challenges that were randomly sampled at 7 days post-challenge (dpc) showed replication of all virus strains in all fish species, with virus levels 105-108 PFU/g. In herring tested 14 dpc all virus strains persisted in most fish, but at 1-2 logs lower titers than in herring that died. Yellow perch and Chinook salmon survivors sampled 30 dpc showed all four virus strains persisted in at least some fish, but at reduced titers and lower prevalence, indicating viral clearance by some fish. In rainbow trout at 30 dpc only genotypes I and IVa persisted, in a small number of fish.

Figure 5. Virus titers in fish from virulence trials in 4 fish species. Numbers in each bar indicate # positive for virus out of # tested. Dead fish were from various times, survivors were sampled at the end of challenge (14 d for herring, 30d for all other fish).

Figure 6. Virus titers in fish sampled 7 days after high dose injection challenge.

Collectively, the data collected in objective 4a and 4b indicate that all VHSV strains tested can infect and grow in all four fish species. Virus titers in fish that died during challenge indicate VHS as the cause of death, and titers in live fish confirm these species can be carriers of VHSV.

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4c) Preliminary detection of virus shed into water during injection challenges (data not shown)

Water samples from the tanks were collected at 7 and 28 days post-challenge. Water samples 7d post-challenge were analyzed by the general VHSV qRT-PCR assay and only extremely low levels of virus near the detection threshold were found in a small number of the samples. Water samples 28d post-challenge were tested by both qRT-PCR and plaque assay and no virus was detected in any samples.

4d) Comparative time-course studies to compare the in vivo growth of VHSV genotypes I, IVa, and IVb-GL in different host species

A series of studies were conducted to compare the growth rates and viral titers during infection with three different VHSV strains: European type I, Pacific coast type IVa, and Great Lakes Type IVb-GL. Due to results of the virulence trials in objective 3 we chose to focus viral growth time-courses on yellow perch and rainbow trout, with experiments in rainbow trout at both 12°C and 15°C due to the higher temperature used in the majority of trout aquaculture in Idaho.

In each infection time-course experiment groups of fish were challenged by the more natural immersion route for 12 hours, and then held in individual beakers during the 10 day sampling period, to avoid cross-infection. Sub-groups of 7 fish were harvested from each treatment at the start of the 12 hour immersion (Time -12), and at 0, 1, 2, 3, 5, 7, and 10 days post-infection. Viral loads were quantified in each individual fish by RNA extraction and qRT-PCR using the Garver general VHSV assay that detects all VHSV genotypes. The VHS viral loads in individual fish were determined by quantitative PCR and average viral loads of all positive fish are shown in graphs 7-9 below, along with the prevalence in terms of number of fish with positive titers out of the number of fish sampled.

i) Viral growth in yellow perch at 12°C See Figure 7.

The infection time-course experiment conducted in yellow perch challenged by immersion at 12°C compared in vivo growth of VHSV genotypes I, IVa, and IVb-GL. VHSV types IVa and IVb had nearly identical growth patterns, growing slowly over days 0-7 days to reach final titers of 103-4 copies per gram of fish. The European genotype I exhibited lower prevalence and lower titers overall, with a peak of 102.5 copies per gram at 3 days post-infection. The differences in growth correlated well with the virulence differences seen in yellow perch in objective 3.

Figure 7. Viral growth in yellow perch at 12C shows VHSV type IVa = IVb, > I.

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ii) Viral growth in rainbow trout at 12°C See Figure 8.

The infection time-course experiment conducted in rainbow trout challenged by immersion at 12°C compared in vivo growth of VHSV genotypes I, IVa, and IVb-GL. VHSV type I grew notably faster and to higher viral loads, peaking 2d post-infection at over 106 copies per gram of fish. The Pacific type IVa exhibited lower prevalence and lower titers overall, with a peak of 104.2 copies per gram at 2 days post-infection. Great Lakes type IVb grew even more poorly, with low prevalences and a peak of 104 copies per gram at 3 days post-infection, representing a single fish (1/7). Again the differences in growth correlated with the virulence differences seen in objective 3, and therefore contrasted with the results seen in yellow perch.

Figure 8. Viral growth in rainbow trout at 12C shows VHSV type I > IVa > IVb.

iii) Viral growth in rainbow trout at 15°C See Figure 9.

To assess the potential effect of temperature, a viral growth study was conducted in rainbow trout at 15°C, for comparison with the results at 12°C described above. The 15°C temperature was chosen because it is characteristic of the Hagerman Valley trout growing region in Idaho.

Figure 9. Viral growth in rainbow trout at 15°C shows VHSV type I > IVa > IVb.

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In these condition virus replication peaked at day 3 for all 3 strains, but the viruses grew to different general viral load levels. The viral load of VHSV genotype I was 1-2.5 logs higher than genotype IVa at all time-points from 0-10 days. VHSV IVb-GL was similar to genotype IVa at days 1-3, but thereafter it was cleared below detectable levels from day 5-10. This variation in viral growth among the 3 VHSV strains correlated very well with the levels of mortality in the virulence trials. Overall, viral loads were higher than at 12°C, but the relative order of growth among the three virus types was consistent, with genotype I > IVa > IVb at both temperatures.

iv) Host innate immune response during viral growth in rainbow trout at 15°C See Figure 10.

Host immune gene response (Mx1 and IFN genes) was also measured and mirrored viral loads, being most notable in fish infected with genotype I.

Figure 10. Time-course showing VHSV genotype I (blue squares) grows to higher titers than genotypes IVa or IVb in rainbow trout at 15°C. Host immune response, indicated by Mx and IFN genes, is also greatest in fish infected with VHSV genotype I

Collectively, the data collected in objective 4d showed that the growth of different VHSV strains correlates well with the virulence differences identified in objective 3, and that host species, viral type, and temperature all profoundly influence VHS virus growth. The variation in viral load of the different genotypes of VHSV was consistently relevant to the risk each strain poses to rainbow trout, and host innate immune responses followed viral load as observed previously in other studies.

Objective 5. Develop outreach materials to communicate project results. Outreach for Fish Farmers: As described in Objective 1, a Biosecurity Workshop at the

Hagerman Fish Culture Experiment Station in Idaho (August, 2009) included a presentation

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on VHSV and the potential for risks to western aquaculture. This workshop was organized by Gary Fornshell and Ken Cain and was attended by approximately 40 members of the trout farming industry.

Outreach for Fish Health Professionals: Four oral presentations and one poster on data from

this project have been presented by project investigators at regional and national fish health meetings. In addition, Gael Kurath participated in a VHSV Continuing Education Session held in conjunction with the 33rd Eastern Fish Health Workshop (April, 2008).

Outreach for Extension Specialists: In addition to the workshop for fish farmers (see Objective

1), a 3 hour VSHV project workshop was organized for extension specialists and followed the WRAC IAC/TC meeting in Spokane, October 2009. Presentations included a biosecurity presentation by Jerri Bartholomew and an overview of the history and biology of VHSV along with current project results by Jim Winton.

Powerpoint presentation on biosecurity for the website. The powerpoint presentation "Problems presented by emerging pathogens: Potential threat of Great Lakes VHS virus in the Western United States" has been provided to the WRAC website administrator so that it is freely available on-line.

Fact sheet: A draft fact sheet (WRAC FAQs) that answers basic questions about VHS virus types IVa and IVb relevant to fish farmers, anglers and fish health professionals in the Western Region has been prepared and will be available on the website after review (Gary Fornshell and Ken Cain) and formatting (USGS).

IMPACTS: Outreach materials and presentation of project results at various venues have enhanced awareness and understanding of the emergence of VHSV in the Great Lakes among different interested parties including academic fish health specialists and aquaculture groups.

IMPACTS:

Relevance: Viral hemorrhagic septicemia virus (VHSV) was first isolated from fish in the Great Lakes in 2003, and was reported in 2005 as the causative agent of a large-scale die-off of freshwater drum in Lake Ontario. Since then numerous epidemics in multiple host species have occurred in the Great Lakes region, resulting in an extreme level of concern and severe restrictions on aquaculture activities. This project involved outreach and research objectives that address specific needs of fish farmers in the western region of the United States, and contribute to the national response to the emergence of VHSV in the Great Lakes.

Response: The Kurath lab conducted research to address concerns of western aquaculture producers about the threat of the Great Lakes strain of VHS virus for western aquaculture species. Four strains of VHSV representing genetic types from the Great Lakes, the Pacific Coast, the Atlantic Coast and Europe were tested for virulence and persistence in four fish species. The fish selected for study were relevant to western aquaculture, or to VHS epidemics in the Great Lakes and the Pacific Ocean. Funds of $100,000 supported a 15 month research and outreach project.

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Results: Variations in virulence and persistence in different fish host were identified for different strains of VHS virus. No biological difference was demonstrated between VHSV strains from the Great Lakes and those currently endemic in Pacific marine fish species. Rainbow trout and Chinook salmon had low susceptibility to North American VHSV strains but high susceptibility to European VHSV. In contrast, yellow perch and Pacific herring were more susceptible to the Great Lakes strain than the European strain.

Impact: Two workshops were held to communicate these results along with biosecurity information to fish farmers and western region extension agents. Biosecurity measures to prevent introduction of any VHSV into aquaculture facilities will reduce costs to producers, both in terms of disease impacts and regulatory compliance. Several general VHSV talks and talks on the project results were presented to various interest groups, and a document titled "WRAC FAQs about VHSV" has been prepared as a fact sheet.

PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED:

See next page

September 14, 2010 SUBMITTED BY: Title: (Work Group Chair or PI) Date

September 14, 2010 APPROVED: Technical Advisor Date

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PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Publications in Print J. Bartholomew, Outreach product. Problems presented by emerging pathogens: Potential threat of Great Lakes VHS virus in the

Western United States. Powerpoint provided to WRAC for addition to WRAC website. Manuscripts in preparation Emmenegger EJ, Moon CH, and Kurath G. Variable Susceptibility of salmonid, cyprinid,

clupeid, and percid fish species to VHSV genotypes from freshwater, brackish water, and marine environments. In preparation for Diseases of Aquatic Organisms.

Bartholomew J, and Kurath G. WRAC FAQs about VHSV. In review for publication as a USGS

fact sheet. Papers Presented: General outreach information and project results Kurath G. VHSV: The history and basics of the virus and the disease. 33rd Eastern Fish Health

Workshop, VHSV Continuing Education Session, 4/4/08, Atlantic Beach, North Carolina. Emmenegger EJ*, Kurath G, Wargo A, Binkowski F, and Goetz R. Yellow perch (Perca

flavescens) susceptibility to viral hemorrhagic septicemia virus isolates from Europe and North America. Western Fish Disease Workshop, Ocean Shores, WA, June 2008.

Kurath G*, Emmenegger EJ, Wargo A, Binkowski F, and Goetz R. Susceptibility of yellow perch

to VHS virus strains from the Great Lakes, Pacific coast, and Europe. AFS-Fish Health Section Annual meeting, Charlottetown, Prince Edward Island, Canada, July 2008

Kurath G*, Emmenegger EJ, Moon CH. Susceptibility of Pacific salmonids, yellow perch, and

koi to viral hemorrhagic septicemia virus strains from the Great Lakes, Pacific and Atlantic coasts of Canada, and Europe. Aquaculture America, Invited talk for session on RAC research on VHSV. 2/17/09, Seattle, Washington.

Kurath G. How we got here: A review of VHS in the US . Invited talk for special "Producers

session" on VHSV at Aquaculture America, 2/17/09, Seattle, Washington. Kurath G. Fish RNA viruses: VHSV epidemiology and evolution. Invited guest lecture in Fish

Disease course at Oregon State University, 2/23/09, Corvallis, Oregon. Kurath G. History and current affairs of VHS virus. Invited talk in special VHSV session at the

International Conference on Aquatic Invasive Species, 4/23/09, Montreal, Canada. Emmenegger EJ*, Moon CH, and Kurath G. Susceptibility of western aquaculture species to

different strains of VHS virus, including Great Lakes VHSV. Idaho Aquaculture Association, 6/09, Twin Falls, Idaho.

Moon CH*, Emmenegger EJ, and Kurath G. Susceptibility of Pacific salmonids, yellow perch,

and koi to four strains of VHS virus, including Great Lakes VHSV. Poster presentation at AFS Fish Health Section Annual meeting, 6/10/09, Park City, Utah.

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Kurath G*. Epidemiology of fish rhabdoviruses part 2: VHSV. Invited guest lecture in Oregon State University Salmon Disease Workshop, 7/21/09, Corvallis, Oregon.

Bartholomew J. Problems presented by emerging pathogens: Potential threat of Great Lakes

VHS virus in the Western United States. Workshop for fish farmers at Hagerman Fish Culture Experiment Station, 8/14/09, Hagerman, Idaho.

Bartholomew J. Problems presented by emerging pathogens: Potential threat of Great Lakes

VHS virus in the Western United States. Workshop for extension agents, presented in conjunction with WRAC meeting 10/8/09, Spokane, Washington.

Kurath G. Viral hemorrhagic septicemia virus: and old virus with new tricks. Plenary talk, 6th

International Symposium on Aquatic Animal Health, 9/8/10, Tampa, Florida.