Our understanding of Cyprinid herpesvirus 3 (CvHV-3) is continually evolving. There are a number of forms of the virus, some of which are more virulent than others. New strains have resulted in new outbreaks of disease in ponds in the US and Europe. The CyHV-3 virus, like all known herpesvirus, has the ability to infect a host fish and live in the host in an "inactive" form for the rest of the koi's life.
Post infection, if the koi survives, the CyHV-3 (KHV) remains dormant (and usually undetectable) in a particular form of white blood cells (WBCs) similar to human plasma cells. These WBCs make IgM antibodies. Some of the research done to study the latency of KHV and identify where the dormant virus “hides” in the survivors was sponsored by hobbyist thru Project KHV. Funds were collected specifically for KHV research and the Project KHV committee contracted with Dr. Ling Jin’s team at Oregon State University to study the latency issue. Links to three of the pertinent papers follow.
The emergence of several different strains of the virus with different virulence suggest this disease will be an ongoing problem. Viruses mutate ensuring their long term survival, with changes in the sequence of the genetic material. A koi which has survived one strain of KHV and is subsequently exposed to another virulent strain of KHV may or may not have adequate immunological protection. If a koi infected with KHV is quarantined or introduced to an immunologically naive collection at less than permissive temperatures, the virus likely will not grow and multiply demonstrating clinical disease until the water warms to permissive temperatures. As water warms to the viruses' permissive temperatures, the number of copies of the virus in the fish increases unless the fish immune system can keep the virus from multiplying. As the koi viral load increases (increased numbers of copies of the virus), clinical disease may become apparent. If the viral load is suppressed, no disease may be apparent. There have been several new documented cases of KHV outbreaks in the US in 2017 and 2018.
The presence of the latent virus may be verified by collecting several milliliters of anti-coagulated blood. The blood is overnighted to the research lab and the live WBC are separated from other blood products. After other steps, the WBC DNA is extracted and virus is identified by nested-PCR technology. These special research techniques are not practical for general testing.
The serological studies discussed in these pages (ELISA) will usually be negative in the carrier state. Many fish have likely been exposed to the virus at an early age (first year? or during an outbreak) and are survivors. Some imported to the US that have been tested do harbor the virus in their WBCs (although they usually have a negative ELISA). Unless the PCR is done on the purified live WBCs, PCR tests are usually negative in asymptomatic koi (unless the fish viral load is high). The virus is widespread in waters of Japan and the US as well as other parts of the world. Those collections most likely to be infected are those which have been "isolated" and have had new additions, especially when water is cool. When the water reaches permissive temperatures, the virus may be shed by recently infected fish or by KHV survivors (carriers) which may shed the virus.
The emergence of several different strains of the virus with different virulence suggest this disease will be an ongoing problem. Viruses mutate ensuring their long-term survival, with changes in the sequence of the genetic material. A koi which has survived one strain of KHV and is subsequently exposed to another virulent strain of KHV may or may not have adequate immunological protection. If a koi infected with KHV is quarantined or introduced to an immunologically naive collection at less than permissive temperatures, the virus likely will not grow and multiply demonstrating clinical disease until the water warms to permissive temperatures (Quarantine in cold water does not allow the KHV to grow and show signs of infection.). As water warms to the viruses' permissive temperatures, the number of copies of the virus in the fish increases unless the fish immune system can keep the virus from multiplying. As the koi viral load increases (increased numbers of copies of the virus), clinical disease may become apparent. If the viral load is suppressed, no disease may be apparent. There have been several new documented cases of KHV outbreaks in the US, Europe and elsewhere in 2017 and 2018.
With the ongoing spectra of Koi Herpes Virus, the hobbyist is faced with a dilemma. If there are no problems with koi quarantined at permissive temperatures, how does the hobbyist know if an asymptomatic carrier (KHV survivor) is present amongst his newly quarantined koi. If the koi is a KHV survivor, it will carry the KHV virus in “B” white blood cells. The virus has the potential to “reactivate” and shed the virus to other fish. In the carrier state the virus count in the “B” WBCs is likely too low to be detected by conventional PCR. Special testing of concentrated WBCs for KHV is needed to determine if a asymptomatic koi is a carrier.
In 2010, from an isolated research pond, 5-10 cc whole blood samples on KHV survivors 8 years post outbreak were sent to Dr. Jin’s team in Oregon overnight for testing. They separated out the WBC for nestled PCR testing and identified the virus in all of the tested KHV survivor koi.
To the author's knowledge, testing for the carrier state by separating out the WBCs for nestled PCR study is not available to breeders in the countries from which most koi are imported. Unless testing is done for the carrier state, asymptomatic koi which have been infected with KHV will likely serve as a ongoing source for virulent strains of KHV with resultant outbreaks of KHV epidemics.
Additionally there is another virus affecting koi collections which has many of the same symptoms of CyHV-3 infection. Carp Edema Virus (CEV) was first recognized in the '70s in Japan and the '90s in the US and Europe. CEV may cause mass mortality. This virus infection also causes gill necrosis and clinical infection may be indistinguishable from KHV infection. Koi sleepy disease is caused by CEV. Treatment is refractory and 0.5% NaCl solution is utilized for supportive care. It is caused by a different class of virus (not a Herpesvirus). Super-infection with bacterial agents is common with debilitating viral infections.
Investigation of koi herpesvirus (KHV) latency in koi
Analysis of koi herpesvirus latency in wild common carp and ornamental koi in Oregon, USA
Identification of B Cells as a Major Site for Cyprinid Herpesvirus 3 Latency
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Koi Herpesvirus (KHV) represents a third cyprinid herpesvirus formally designated Cyprinid herpesvirus 3(CyHV-3). Other nomenclature (Carp Interstitial Nephritis and Gill Necrosis Virus (CNGV)) has been suggested. For purposes of this web site the causative agent of Koi Herpesvirus disease will be be abbreviated KHV and the semantics left to others.
We hope to provide enough practical information regarding KHV such that the reader will be able to make informed choices regarding the acquisition of koi and their subsequent quarantine and testing. Some of the material is admittedly oversimplified for clarity. A partial listing of references is included.
There are now approximately one hundred known herpesviruses isolated, with at least one herpesvirus for each species studied. There are eight known human herpesviruses, of which most of us have been infected by six. One of the characteristics of herpesvirus infection is the ability to cause recurrent infection by one of several mechanisms including reactivation of latent virus or persistent infections (chronic low grade virus multiplication). As a whole, the herpesviruses studied have evolved a number of mechanisms for "hiding" from the "immune system" during their latent or persistent infectious states. These places of hiding vary between herpesviruses but have been found to include nucleated nerve cells, lymphoid cells, salivary glands, kidney, etc. The ability of herpesviruses to avoid the immune response and remain latent as an intracellular virus makes it very difficult for the host animal to clear the virus. Once infected, many animals have the virus for life and may intermittently shed virus without showing signs of disease. The site of latency of KHV is unknown.
There are direct and indirect tests to demonstrate viral infections. Direct tests detect the presence of the virus or of a portion of a virus. Growing the virus in cell culture or a PCR test to a portion of the viral DNA would be examples of direct tests. Generally these are most useful when the virus is present in large numbers such as in a KHV disease outbreak. Indirect tests measure antibodies to a particular virus. They are most useful in detecting previous infection or the stage of infection.
Diagnosis of KHV disease outbreaks is usually made based on epidemiology, characteristic histopathology, and the presence of the virus in internal organs demonstrated by cell culture or PCR assay. Periodic KHV surveillance may be done in koi growing countries by periodically sampling fish from ponds and doing PCR testing. Some health certificates and certifications will state "KHV specific amplification was not detected when genomic DNA from specimens were used as template". Others may just say they are free of KHV. PCR technology has proven very useful for confirming diagnosis in acute disease outbreaks. Unfortunately, PCR has not been found sensitive enough to detect those KHV survivors which may carry the virus in a latent or persistent infective state (carriers). It currently takes measurement of anti KHV antibodies to determine if a koi has previously been infected with KHV and has the potential of periodic virus shedding, infecting other fish.
No KHV quarantine or testing procedure offers 100% assurance a koi previously infected with KHV will be detected. Through quarantine and serological testing we can greatly reduce the odds of infecting other koi. . Serological technology is available to measure antibodies to the KHV virus. Both Enzyme Linked Immunosorbant Assay (ELISA) and the Virus Neutralization Antibody Assay (VN) serologies are available at modest cost, the latter being the most sensitive. The KHV disease outbreak illustrated in the photos should have been prevented by testing the fish for the carrier state while in quarantine. Serology done on survivors strongly suggests some imports had survived a previous KHV infection as they carried low antibody titers. It is unclear how long a KHV survivor will maintain detectable antibodies but studies are in progress. The CEFAS group has demonstrated detectable antibodies a year or more following infection. Using serology on fish in quarantine is another tool we have available for protecting our collections. If a fish has detectable levels of antibodies to KHV, it has been previously infected and must be considered a carrier. Carriers have the potential to shed the virus at a later time, infecting other fish. Materials and procedures we find useful for drawing blood for anti KHV antibody testing are outlined.
PCR testing is useful when fish are sick and dying but is usually not of benefit when fish appear healthy. PCR testing is extremely helpful in confirming histopathology when fish are dying. Again, PCR testing is extremely unlikely to detect disease in healthy appearing fish. All PCR techniques are not equally sensitive. Current PCR technology is inadequate for detecting the latent or carrier state of Koi herpesvirus. .