Abstract

In 1998 our laboratory investigated epidemics characterized by high mortality occurring in koi (Cyprinus carpio) populations in both the USA and Israel. In addition, fish tissues from fish in Israel and the USA were obtained for examination by both light and electron microscopy. 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 koi and common carp (Hedrick et al. 2000).

The development of the koi fin cell line (KF-1) made possible the isolation of KHV as commonly used cell lines by fish virology laboratories were not susceptible to the new virus (Hedrick et al., 2000). The virus was isolated from numerous 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 20°C. 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 15-25°C with an optimum at 20°C (Gilad et al. 2003). Electron microscopy 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). Subsequently, the same agent was isolated 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.

Previous studies have demonstrated that the virus grown in KF-1 cells can 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), the agent known to cause carp pox (Sano et al. 1995), was suggested by differences in the diseases caused by both agents and because KHV fails to react with anti-CyHV-1 antibodies using immunofluorescence assays. Differences in the virion proteins and genomic sequences was additional proof the two viruses were different (Gilad et al. 2002).

The genome size of KHV, 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 phylogenetic position of KHV. Using degenerate primers developed by Dr. Larry Hanson of Mississippi State University, we amplified, by polymerase chain reaction (PCR), a 500 bp region of the DNA polymerase gene from KHV. Amplicons were visualized using a 1.5% agarose gel, and PCR purified and cloned following standard molecular protocols, and then sequenced using an ABI 377 automated sequencer. Sequences were aligned using the computer program MacDNASIS, version 3.7 and additional primers were designed specifically for KHV to determine the amount of intraspecific genetic variation in the DNA polymerase gene from European, Asian, and North American isolates. Phylogenetic relationships between KHV and other fish and amphibian herpes-like viruses were conducted using Paup 4.0 and phylogenetic reconstruction was performed using discrete and distance methodologies.

The objective of this project was to clarify relationships between KHV with other fish and amphibian herpes-like viruses currently placed in the family Herpesviridae. Understanding which viruses are most closely related to KHV may provide insight into the extreme virulence exhibited by KHV, answer questions about the mode of infection (i.e., persistent or latent), and lead to the development of appropriate therapeutics.

References

1.  Gilad O, S Yun, KB Andree, MA Adkison, A Zlotkin, H Bercovier, A Eldar, RP Hedrick. 2002. Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi. Dis Aquat Org 48:101-108.

2.  Gilad O, S Yun, KB Andree, MA Adkison, K Way, NH Willits, H Bercovier, RP Hedrick. 2003. Molecular comparison of isolates of an emerging fish pathogen, the koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi. J Gen Virol 84: 1-8.

3.  Hedrick RP, O Gilad, S Yun, JV Spangenberg, GD Marty, RW Nordhausen, MJ Kebus, H Bercovier, A Eldar. 2000. A herpesvirus associate with mass mortality of juvenile and adult koi, a strain of a common carp. J Aquat Anim Health 12:44-57.

4.  Ronen A, A Perelberg, J Abramowitz, M Hutoran, S Tinman, I Bejerano, M Steinitz, M Kotler. 2003. Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio. Vaccine 21:4677-4684.

5.  Sano T, H Fukuda, M Furukawa, H Hosoya, Y Moriya. 1985. A herpesvirus isolated from carp papilloma in Japan. Fish Shell Pathol 32:307-311.