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Diseases of Aquatic Organisms

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DAO 60:179-187 (2004)  -  doi:10.3354/dao060179

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally-infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

Oren Gilad1, Susan Yun1, Francisco J. Zagmutt-Vergara1, Christian M. Leutenegger1,2,Herve Bercovier2, Ronald P. Hedrick1,*

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616 USA
2Lucy Whittier Molecular and Diagnostic Core Facility, University of California, Davis, California 95616, USA
3Institute of Microbiology, Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School,Ein Karen, Jerusalem, Israel
*Corresponding author. Email:

ABSTRACT: The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally-infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 101 to 107 molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28°C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28°C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 108 to 109 per 106 host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 × 102 per 106 host cells) as present in gill, kidney or brain tissues.

KEY WORDS: TaqMan PCR · Herpes-like viruses · Koi herpesvirus · KHV · Cyprinus carpio koi

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