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General Discussion of the virus and its pathogenicity:
When an outbreak occurs, surviving positive fish should be moved to a quarantine facility and held for further observation of the progression of the disease.
Positive fish that have been moved to “better” environment, such as a reduced density have upon subsequent testing shown to be free of the virus, or at least not at current detection limits.
Diagnostics continue to be questioned until we better understand the pathogenesis of the virus, and what quantitative levela of virus and stress is necessary to cause disease.
We need to understand the mode of transmission of the virus and the triggers for expression of the disease.
Caution needs to be exercised when working with multiple species as virus may cross infect.
A large scale challenge should be conducted with fish from 0.1g to 5g in size. It is interesting to note that larger fish (>10g) injected with the virus do not come down with the disease.
Encourage the National Marine Fisheries Service to sample wild fish for nodavirus as part of their surveys. What is the incidence in the wild? Could it contribute to stock decline?
  
Funding possibilities:
Most immediate, use existing dollars that researchers now have to get started.
Other:
NRAC,  pre-proposals due 12/31
UNH/OOA, uncertain amount of dollars available
SBIR, proposals due Jan 19^th
AquaNet, potential matching program
ARCDP
USDA
  
Broodstock management and testing:
We should also be sampling different organs in addition to brain, particularly ovarian fluid, sperm, blood and mucus.
Develop non-lethal test, i.e. virus has been detected in blood in symptomatic fish at MBL
            Is it detectable in non-symptomatic “carrier” brood?
Asian hatcheries only use early spawned eggs to reduce risk of vertical nodavirus transmission
Conduct paired matings, and evaluate eggs from a female(s) over the continuum of a spawning cycle
Is vaccination of brood practical?
Continue feeding  broodstock during spawning season to reduce mortality
 
Disinfection of water and eggs
U.V. light concentrations of >200KWsec/sq.cm. are reportedly strong enough to disinfect water.
Dosages of ozone (concentration x time) of 4 to 10 appear to be sufficient to disinfect halibut and haddock eggs experimentally infected with a low titre of virus.
No disinfection studies yet conducted on naturally infected eggs from infected cod broodstock. Such brood and eggs are currently be studied as part of a NRAC project involving Stewart Johnson from the NRC Canada, and Scott Lindell at the MBL in Woods Hole, MA.
 
Larvae and juvenile management and testing:
Mortality events appear to strike fish at 90 dph or later.  Fish at this stage are fully metamorphosed, weaned, and potentially able to be exposed to an immersion vaccine.
Periodically take and preserve samples for future testing so that if there is an outbreak, we can track back initial infection to a stage or incident.
Adjust sampling size to 1-2% (note: as larvae this will be a very large number)
Sample around grading (stressor) events, before and after
Best to preserve samples at –80C if available, and if not, then in RNA-later
Live feed inputs should also be tested as they may represent a vector of introduction
   
Future Research / Proposed Action
Purposely infect larval/juvenile fish and monitor the progression and detectability of the disease using the most sensitive methods. Ideally, we’d like to compare with naturally infected fish as well.
Develop vaccination and challenge trials
Produce a recombinant protein vaccine using native isolates for experimental trials – evaluate immersion form of vaccine
            Talk to Aquahealth about nodavirus vaccines
            May only need short-term protection to get through fragile juvenile phase
            Other natural  immuno-stimulants may be successful
Use LD50 from Norway as a starting point
Places where infection and vaccination trials could be conducted: URI, UNH, Microtechnologies (Maine), DFO (St. Andrews & Bedford)
Send blood and mucous to MBL for non-lethal testing
Are survivors of nodavirus outbreaks potentially disease-resistant breeding stock? Is there a place to hold them to find out? Canadian researchers are examining the correlation between genes and immune system function.
Develop a reproducible ELISA test for Cod Nodavirus. Compare sensitivity of cell culture with molecular methods.
 
Scott Lindell at MBL will serve as coordinator for further discussion and research as needed (email : slindell@mbl.edu )

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Comments from Thomas D. Kocher
Professor and Co-Director Hubbard Center for Genome Studies

1. Take a ‘nodavirus-eye’ view.
As I listened to the natural history data on this virus, I began to understand its ecological/evolutionary strategy. It seems well-adapted to transmission in marine life-cycles. My guess is that the ovarian fluids infect young larvae. To spread among these larvae at the low densities found in natural habitats the virus must be highly infectious. The spread to neural tissue is probably an adaptation of the virus to improve transmission (whirling larvae attract the attention of other larvae/predators). At older stages, the virus becomes latent, in order to survive until the next reproductive cycle. So, both vertical and horizontal tranmission are important, but at different stages in the life cycle. The virus itself is a tough opponent. It is extremely simple, and has a high rate of sequence evolution. It also has a very broad host-range. All of these factors will have an impact on strategies to control it.

2. Learn about the pathogenesis of infection to improve your diagnostic testing.
The molecular diagnostic tools that are available are quite adequate to detect very low concentrations of the virus. The problem is they are being applied blind, without an understanding of where the virus lives in the fish at various points in the life cycle. Use these tools to learn more about the nodavirus life cycle. I think this will be a relatively simple task, because I suspect this virus travels very much like similar (e.g. salmonid) pathogens.

3. Change a few key management practices.
Given the prevalence of nodavirus-infected adults, a proportion of the broodstock brought in from the wild will carry the virus, and will shed them in eggs/sperm. So, you are playing roulette. I don’t know if it is practical, but it seems to me each batch of gametes, from individual fish, should be tested for viral status. I think this means abandoning your strategy of natural tank spawning. Then I think you should try a flow-through system for egg/larval culture, in order to keep the viral titres low through the early stages. Essentially you are in a race to get the fish to the size at which the virus becomes latent. Frankly, I think this whole strategy is an unstable one. We don’t know enough about the parameters of transmission to know if you will be successful, but given the economic realities, I suspect you will try anyway.

4. Begin a selection program now!
The observation that the Norwegians no longer have problems with nodavirus in halibut is key. Long-term, the only way you will defeat this problem is to select nodavirus-resistant (or at least compatible) fish. This requires saving the survivors from disease outbreaks, and using them as broodstock for future production cycles. It should only take 1-2 rounds of selection to create cod which have much better natural defenses against the virus. They may not be virus-free, but they will not be significantly affected by the virus. Because this selection will likely be for general viral defense mechanisms, rather than for specific immunity, these selected broodstock will likely be more resistant to a variety of future pathogens, including new versions of the nodavirus as they evolve. It’s a real shame that you had to throw away the survivors of the last outbreak. It deprived us of the opportunity to see which parental combinations of your current broodstock gave the most disease-resistant fish, and delays the start of this important program of selection.

-Tom

*******************************************
Thomas D. Kocher, Professor and Co-Director
Hubbard Center for Genome Studies
University of New Hampshire
Suite 400, Gregg Hall
35 Colovos Road, Durham, NH 03824
603-862-2115; FAX 862-2940
Tom.Kocher@unh.edu
http://hcgs.unh.edu

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Comments from Luisa Villamil
University of Rhode Island
 
I think that the ideas that every body proposed yesterday are very useful since all of them are focused in the prevention.
The necessity of establish the origin of the nodavirus is very basic to
combat the problem.
In my opinion it is very important to know:
1. If the nodavirus has always being in the larvae but just in a very low prevalence, making the detection by qPCR very difficult. I agree with every body that you may just need to adjust the sampling strategy. But I also think that it is important to try other detection methods, there is not perfect diagnostic method and it is always useful to compare them. Maybe peroxidase in cell culture could be a good alternative.
2. If the nodavirus is incorporated in the system by the water, live food or pellet. Incorporation of pathogenic bacteria via rotifer and Artemia has being documented in several studies. Maybe it has never found virus because anybody had looked for them there. You can't discard the artificial food because the nodavirus is very resistant. Incorporation of pathogens in other cultured species could be common and one of the worst examples are the prions and of course the mad cows.
I also think that if in the hatchery are other species that are simultaneously cultured, is better to make sure that they are negative to the nodavirus test.

The vaccination is an other important strategy; this may take longer in the terms of science. We have in the laboratory the technology to produce recombinant proteins in yeast that may be useful in the creation of the vaccine. The new aquarium facility that is in the bay campus could be a good place to do the experimental infection but unfortunately there have being some problems with the water pumps that need to be solved.

Luisa Villamil
University of Rhode Island
Dept. of Fisheries, Animal & Veter
20A Woodward Hall
Kingston, RI 02881 USA

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