Report on KHV Study by Oregon State University

[Steve E.]

Hi All,
I have copied an article appearing in the July/August 2011 KOI USA magazine. It was written by Raymond Jordan, chair of the Project KHV Committee of which I am a member. It is reproduced here as it appeared with the following changes: 2 pictures were removed and the last part of the side bar article was eliminated as it contained the OSU address for sending samples. I have in the past received permission from KOI USA for reprinting articles and discussed posting this here with Ray who had no objection. There is also additional information out there regarding the verticle transmission of the virus but not included in the article as it came to our knowledge after the article had been submitted for publicaton.

Also there are several additional studies that have been done in Europe and the USA:

Koi herpesvirus workshop, O. Haenen1* and R. Hedrick2
1 CIDC-Lelystad, NRL for Fish and Shellfish Diseases, 8203 AA Lelystad, The Netherlands; 2Dept. of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, 95616 USA.

Detection of koi herpes virus (CyHV-3) in goldfish, Carassius
auratus (L.), exposed to infected koi
J Sadler, E Marecaux and A E Goodwin
Aquaculture Fisheries Center, University of Arkansas at Pine Bluff, Pine Bluff, AR, USA

Sampling in the frame of an effective sample collection for KHV detection in latent infected fish, Sven M. Bergmann, Heike Schutze, Jolanta Kempter*, Meike Riechardt, Uwe Fischer und Dieter Fichtner. *Universitat of Szczecin, Dep. of Aquaculture. (must apologize here as I do not know how to show two small dots [umlat?] above the u in Heike Schutze's name)

I wanted to pass on this information as I know folks here may not be aware that the money donated to AKCA for KHV research has been well spent for the purpose it was collected.

Steve

Up Date on AKCA - Project KHV Funded Research at Oregon State University
By Ray Jordan
About two years ago AKCA - Project KHV entered into an agreement with Oregon State University to fund a KHV Latency Research Project under the direction and supervision of Dr. Ling Jin, Ph.D., DVM. as Principal Investigator. Dr. Jin is an Associate Professor in Virology in the Department of Biomedical Sciences and Section Head for Virology at the Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Oregon State University (OSU). Co-Investigators include: Dr. Michael Kent, Ph.D., Department of Biomedical Sciences, College of Veterinary Medicine, OSU; Dr. Jerry Heidel, D.V.M., Ph.D., DACVP, Department of Biomedical Sciences and Director of the Veterinary Diagnostic Laboratory, College of Veterinary Medicine, OSU; Dr. Tim Miller-Morgan, D.V.M., Ornamental Fish Health Program, Department of Biomedical Sciences, Section Head for Aquatics at the Veterinary Diagnostic Laboratory, College of Veterinary Medicine/Oregon Sea Grant Extension, OSU.
The OSU KHV latency research project began in August 2009 and is projected to end in September 2011. $79,940.00 is the amount of funding dedicated in the AKCA – Project KHV and Oregon State University agreement. I am very pleased to report that the OSU research project has made exciting positive progress in terms of results and also remains within budget.
Overview of the specific research project objectives and outcomes to date:
1) Identify which tissue(s) KHV becomes latent
Significant success has been made with this objective. A number of tissues have been identified that harbor latent KHV DNA but for our hobby the most significant finding may be the discovery that KHV can be detected in koi/carp white blood cells extracted from blood samples which means we no longer need to sacrifice the koi to be tested for a latent KHV infection. On going research is being conducted to reconfirm this exciting finding. You can read more about this finding later in this article.
2) Discover which viral genes remain active during latency
Progress is being made on mapping the KHV DNA during latency and the principal investigator feels it likely this objective will be completed by the conclusion of the study.
3) Study spontaneous reactivation from latency in KHV infected koi
This research has demonstrated that reactivation most often requires permissive temperatures (approximately 68F to 80F) and a stressor that lowers the immune system.
4) Investigate stressors that can cause reactivation of latent KHV
Numerous stressors were investigated with koi that have been previously exposed to KHV to determine if latent KHV virus infections could be reactivated. These stressors included overnight transportation/shipping, temperature increased into the KHV permissive range of (68F to 80F), and steroid injections to suppress the immune system in a similar manner to stressor like poor water quality.
Prior to the OSU research project, there was no proven method of detecting latent KHV infection in a koi without extracting key internal organs for testing. Obviously this method was useless to koi hobbyists wishing to determine if their koi were free of KHV. Regardless of whether the KHV testing was positive or negative for KHV the individual koi tested had to be sacrificed. The only certified and accepted test for KHV is the Polymerase Chain Reaction (PCR) testing method that enables researchers to produce millions of copies of a specific DNA sequence in a few hours to identify a specific virus. PCR testing is accurate when used to identify KHV in situations of active KHV disease because there are billions of KHV virions present, which can more easily be detected. However, in a latent KHV infection the KHV virus survives in relatively low amounts and “hiding” in areas where a standard PCR testing cannot accurately be utilized in non-fatal tissue samples such as a blood sample.
There is a different type of blood test available to screen for KHV “antibodies” , which is called an Enzyme-Linked Immunosorbent Assay (ELISA) test. This test is narrow in application because it can only detect KHV antibodies within a brief period after KHV exposure. It would be expected that koi exposed to KHV more than a year ago could test negative for KHV antibodies with ELISA. Also some koi after a KHV infection produce lower amounts of antibodies than can be easily detected by ELISA testing. The OSU research team has developed a modification of the PCR test that can detect KHV in white blood cells isolated from total blood. The OSU study has shown that they can detect KHV DNA with their modified PCR protocols on white blood cells. In fact in a side bar to this article you will find the specific directions necessary to send a blood sample to the OSU diagnostics lab for this special modified PCR test.
In early March 2011 the first scientific paper based on the OSU KHV research project titled “Results of total DNA measurement in Koi by tissue Koi Herpesvirus real-time PCR” was published in Journal of Virological Methods.
A second research article on KHV latency titled “Investigation of Koi Herpesvirus Latency in Koi” was published in May 2011 in the Journal of Virology, which is a top research virology journal in which experts in Virology review and recommend articles for publication. For example in general/internal medicine the New England Journal of Medicine is considered one of the top line journals and whenever new medical research is published in the NEJM it is known to have been screened by experts and considered to be novel and important information. In other words this study is viewed as important new credible research. The Journal of Virology is a top line journal for viral diseases and the publication is confirmation of the validity and value of this research. Here is the publication information:
J Virol. 2011 May; 85(10):4954-62. Epub 2011 Mar 9. Investigation of koi herpesvirus latency in koi. Eide KE, Miller-Morgan T, Heidel JR, Kent ML, Bildfell RJ, Lapatra S, Watson G, Jin L.
Link to the abstract of the study: http://www.ncbi.nlm.nih.gov/pubmed/21389134

The modified KHV PCR testing techniques, that are able to detect latent KHV virons in a specially prepared blood samples, has already been shared in a variety of ways with veterinary diagnostic labs across the U.S. by Dr. Jin’s research team.
The AKCA Project KHV committee will be publishing the final report about this KHV latency research project in KOI USA as it becomes available after the conclusion of the study in a few months. Special thanks to all our AKCA - Project KHV donors. Without your generous donations we would not have been able to fund this significant research project. We also want to thank the AKCA - Project KHV Steering committee and the AKCA Board of Directors for their assistance with the research overview process.
--------------------------------------------------------------------------------------------
SIDE BAR ARTICLE:
by Ray Jordan
I have been asked, what are some of the key accomplishments from the hobbyist’s point of view regarding the KHV latency research done so far at OSU? Here are the top results in my opinion:
1) KHV latency is a significant threat to our koi collections that can remain undetected for many years until a combination of permissive water temperatures and stress turns on KHV genes and permits them to multiply into a full blown KHV infection and population disease outbreak.

2) ELISA testing is somewhat limited in utility because koi only produce levels of KHV antibodies that are detectable for a fairly short period of time post infection and at a narrow range of detection level. Also it has been recognized that some koi post KHV infection do not produce sufficient antibodies to be detected by ELISA testing.

3) The discovery by Dr. Jin’s team that KHV can be detected by a modified PCR test on white blood cells extracted from blood samples means we no longer need to sacrifice a koi to test for latent KHV infection.

4) Koi with latent KHV infections are likely a much greater percentage of the general population than previously believed.

So what does all this mean for the average koi hobbyist today?
This research data reinforces the importance of quarantining all new koi for a period of at least three weeks at permissive temperatures. Proper quarantine remains the best defense against introducing KHV into our personal koi collections. The OSU data demonstrates the ability to reactivate KHV outbreaks in koi that had been exposed to KHV many years prior. The combination of overnight shipping stress followed by permissive temperatures for at least ten days permitted the re-emergence of KHV disease in koi previously exposed to KHV.
This data also provides a “new” methodology for a non-lethal KHV test using the modified PCR techniques developed by Dr. Jin and her research team.
PCR detection of KHV DNA in white blood cells of Koi
We are including some additional information regarding KHV testing. The OSU Veterinary Diagnostic Lab is now able to test koi blood samples for KHV latent infections. As this article is being submitted for publication this new modified PCR testing technique information is being made available to Veterinary Diagnostic Labs across the United States.
I am listing the information needed to have PCR testing and PCR detection of KHV latency in koi blood samples at the OSU Veterinary Diagnostic Lab. It is possible that you could have this modified PCR test done at a regional veterinary diagnostic lab in your area and you will need to contact them for their specific instructions.
Prior to sending blood samples for PCR detection of KHV call the OSU Veterinary Diagnostic Lab to confirm shipping information and notify them of your shipment. Plan for samples to arrive early in the week.
Sampling:
1) Submit 0.5-1.0 ml whole blood in EDTA or heparin coated tube.
2) Anti-coagulated tubes should be thoroughly mixed immediately after specimen collection
3) Sample should be evaluated within 48-72 hours.
Packing Guidelines
• Samples must be in a leak proof primary container. This container must be labeled with patient/owner ID and sample contents, test name. (John Hobbyist, Koi blood, PCR detection of KHV in WBC)
• Print and complete sample submission form from this link
• http://oregonstate.edu/vetmed/diagnostic/forms
• Request PCR testing of Koi blood sample with follow up testing for KHV latency if initial KHV PCR testing is negative.
• Acceptable containers include:
o Leak proof: blood tubes (vacutainer and transfer tubes), plastic centrifuge tubes, screw top plastic jars with a ridge on the inside of the jar.

[Manky Sanke]

Steve,

Thanks for that information, I also followed the link to the NCBI site and found enough information on there to occupy my reading time for the next few weeks.

A quick point about the umlaut that you didn't know how to type, there is a list of "extended ascii codes". These are characters that you might want to type into a document but for which you don't have a key on your keyboard.

This is a link to a typical site with a list and a simple explanation of how they work.
http://balch.org/asp/ascii.asp

alt 0220 does this Ü
alt 0252 is the lower case ü

Easy peasy - hope that helps.

[Steve E.]

Manky,

Thanks for the link and the information.

Steve