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

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.

Figure 1

Amino acids sequence alignment of the KHV TK gene. Multiple sequence alignment of KHV TK with representative TK sequences from Poxviridae, Herpesviridae and Trypanosomatidae families, performed by the Clastall W algorithm.

Figure 2

Southern blot hybridization of Carp and KHV DNA with the TK gene. KHV (A) and Carp (B) DNA were digested with BsaAI (I) or PvuII (II) and hybridized with a labeled TK probe. BCG (Bacillus Calmette Guerin) DNA (C) was used as a negative control.

Figure 3

Analysis of KHV TK mRNA in KF-1 cell line infected with KHV. KF-1 cells were infected with KHV. At 0 to 7 days post infection total mRNA was prepared from the infected cells and was used in an RT-PCR reaction using primers TK-Rtforward- TK and RTr long (A). 0 time control mRNA was prepared immediately after infection. lanes: M- size markers, 1–8- mRNA of days 0–7 post infection, 9- direct PCR of KHV DNA, 10- uninfected KF-1 cells 11- RNase treated mRNA of day 5 post infection. To confirm the identity of the RT-PCR products, Southern blot hybridization (B) was performed using the cloned TK gene as probe and mRNA of days 0–7 lanes 1–8 consecutively, control KHV – lane 9, and uninfected KF-1 cells as negative control – lane10. The arrow marks the position of the 621 bp product.

Figure 4

Sensitivity of three PCR assays for the diagnosis of KHV infection. A TK based PCR was used to detect KHV DNA and was compared to two other PCR protocols: the Gilad et al. (2002) and Gray et al. (2002). Details of primers, products and protocols are described in table 1. To compare the sensitivities of the assays, 10 fold serial dilutions of purified KHV DNA were prepared starting from 10 ng DNA per PCR reaction. PCR products were analyzed on a 1% agarose gel and visualized by ethidium bromide. For each protocol the highest dilutions that still give products are shown. The arrows denote the 200 and 500 bp. size markers.