Initial Isolation and Characterization of a Herpes-like Virus (KHV)
from Koi and Common Carp

R.P. Hedrick, O. Gilad, S.C. Yun, T.S. McDowell, T.B. Waltzek, G.O. Kelley, and M.A. Adkison

Department of Medicine and Epidemiology, School of Veterinary Medicine
University of California, Davis, California 95616, USA

ABSTRACT

In September of 1998 our laboratory was asked to investigate epidemics characterized by high mortality occurring in koi populations in both the USA and Israel. Either live fish or frozen tissues arrived from both locations in September and November of 1998. In addition, tissues fixed for both light and electron microscopy from fish in Israel and the USA were obtained for examination. The virus observed and then isolated in 1998 from these initial samples has become the focus of a worldwide concern for the health and welfare of both captive and wild populations of koi and common carp (Hedrick et al., 2000).

The development of the koi fin (KF-1) cell in our laboratory in 1997 made possible the isolation of KHV as commonly used cell lines by fish virology laboratories were not susceptible to the new virus. The virus was isolated from numerous tissues of fish with signs of the disease, including the gill, kidney, spleen, gut and liver. The virus induced cell fusion and intense cytoplasmic vacuolation in KF-1 cells within 5 days after inoculation at 20⁰C. More complete cytopathic effects (CPE) were evident at 7-10 days and progressed to involve all the cells after 14 days. The virus grew in the KF-1 cell line at temperatures from 15-25⁰C with an optimum at 20⁰C (Gilad et al., 2003). Electron microscope of the virus present in and released from infected KF-1 cells was shown to possess a morphology and size consistent with viruses in the family Herpesviridae. The virions were composed of an inner capsid with icosadeltahedron symmetry of approximately 100-110 nm in diameter. Mature virions contained a loosely applied envelope giving the virion an overall diameter of 170-230 nm. Identical virus particles were observed in tissues from koi from fish involved in the epidemics in Israel and the USA. Based on the morphology and size of the virus and the sequential development in the host cell nucleus, the new virus was initially designated KHV for koi herpesvirus (Hedrick et al., 2000). The same agent was isolated subsequently from koi and common carp by Ronen et al. (2003) although they preferred the designation of carp nephritis and gill necrosis virus (CNGV) for the agent.

The KHV was the cause of the disease epidemics established by experimental demonstrations that the virus grown in KF-1 cells could reproduce the disease among koi exposed by either bath or intraperitoneal injections in the laboratory (Hedrick et al., 2000). That the virus differed from Cyprinid herpesvirus 1 (CyHV-1) or the agent known to cause carp pox (Sano et al., 1995) was suggested by differences in the diseases caused by both agents and when anti-CyHV-1 antibodies failed to react with KHV in immunofluorescence assays. Differences in the virion proteins and genomic sequences was additional proof the two viruses were different (Gilad et al., 2002).

The large size of the genome of KHV or CNGV, which is estimated at 277 kbp, exceeds that of 250 kbp known for members of the family Herpesviridae (Ronen et al., 2003). While genome size is not currently considered a criterion for placement or exclusion of viruses in the family, the differences in sequences so far obtained for KHV have shown little similarities to those known from herpesviruses from birds or mammals. This has led to a reasonable debate on taxonomy of KHV that remains unresolved. Certain proposals however, are developing that may involved considerable taxonomic changes in the family Herpesviridae which in turn may allow more specific assignments to viruses such as KHV.

Since the initial isolation of KHV in 1998, a number of new and improved methods have been developed to detect the virus. That isolation of the virus was difficult was shown early and thus other detection methods have been rigorously pursued. The polymerase chain reaction (PCR) method has proven to be an effective means to detect viral DNA in a number of fish tissues from animals during the acute disease and following recovery. As many as 7 different PCR tests for KHV are currently in use in different laboratories throughout the world (Gilad et al., 2002; Gray et al., 2002; H. Bercovier, K. Way, M. El-Matbouli, pers. comm.). These tests have greatly improve the ability to detect evidence for the virus but leave unresolved whether carriers with persistent or latent infections occur following acute disease. The detection of anti-KHV antibodies in the serum of koi and common carp is currently being used as both a diagnostic and research tool to demonstrate prior exposure of fish to the virus.

Recent comparisons in our laboratory of a range of herpes-like viruses found in carp, goldfish, eel, sturgeon, trout and frogs indicate they form a group of unique viruses quite distant from other members of the Family Herpesviridae and other viruses in general. Sequences obtained from the DNA polymerase and other genes of KHV and 11 representatives of the dish and amphibian herpes-like viruses shows that the viruses separate generally according to host species. KHV was found most closely related to Cyprinid herpesvirus 1 and 2 as found in carp and goldfish, respectively. Thus KHV should be considered as Cyprinid herpesvirus 3 (CyHV-3) in the family Herpesviridae. It is recognized however, that changes to the Family Herpesviridae to accommodate the distant relationships between the fish and amphibian herpes-like viruses and the herpesviruses from mammals, birds and reptiles are needed.