Methodology article

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Herve Bercovier¹ , Yolanta Fishman¹ , Ronen Nahary¹ , Sharon Sinai¹ , Amir Zlotkin⁴ , Marina Eyngor² , Oren Gilad³ , Avi Eldar² and Ronald P Hedrick³

¹  Institute of Microbiology, Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Ein Karen, Jerusalem, Israel

²  Department of Poultry and Fish Diseases, The Kimron Veterinary Institute, Beit Dagan, Israel

³  Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616, USA

⁴  Department of Infectious diseases, Sheba medical center, Tel Hashomer, 52621, Israel

author email corresponding author email

BMC Microbiology 2005, 5:13doi:10.1186/1471-2180-5-13

Published:	17 March 2005

Abstract

Background

Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.

Results

A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection.

Conclusion

The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.