Viral strains, virus isolation and DNA extraction
KHV-I originating from koi in Israel [10] was propagated on the koi fin cell line (KF-1) which supports the growth of KHV as described by Hedrick et al. [5]. Cells were incubated at 20°C. The channel catfish herpesvirus (CCV) strain CA80-5 served as the unrelated herpes-like virus control and was propagated on channel catfish ovary (CCO) cell line. The CCO cell line was grown in minimum essential media (MEM) supplemented with 7.5% fetal bovine serum (FBS), 50 IU penicillin/ml, 50 μg streptomycin/ml, and 2 mM L-glutamine (MEM-7.5). The FBS concentrations of the growth medium were reduced to 2 % (MEM-2) when CCO cells were infected with CCV. Cells were incubated at 25°C following inoculation with CCV.
The CHV DNA was a gift from Dr. T. Sano and Dr. H. Fukuda, Tokyo University of Fisheries [19]. Viral and cell DNAs were extracted according to Gilad et al. [10].
KHV was isolated from koi head kidney tissues of infected or diseased fish according to the method described by Hedrick et al. [5] with a slight modification. Replicate wells of a 12-well tissue culture plate containing monolayers of KF-1 cells were inoculated with 0.2 ml of a 1:10 dilution (volume /volume or weight/volume) of the original head kidney homogenate. After an adsorption period of 1 h, 2 ml of MEM-2 containing 0.015 M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 50 IU of penicillin/ml, 50 μg streptomycin/ml, and 2 mM L-glutamine were added to each well and the plates were incubated at 20°C. The plates were observed daily for evidence of cytopathic effects (CPE). After 7 days, if there was no CPE, the medium was used to infect new KF-1 cells and incubated as above. Three such blind passages were performed before a clinical sample was described as negative for virus isolation.
Cloning of the thymidine kinase
KHV genomic DNA fragments following digestion with Sau 3AI were separated on 1% agarose gels adjacent to a DNA ladder. DNA fragments in the 3–6 kb region were extracted from the gel, purified and ligated overnight at 4°C into a predigested Bam HI site of the multiple cloning site arms of the Lambda Zap Express Vector (Stratagene, La Jolla, CA, USA.). The Lambda Zap Express vector allows in vivo excision and recircularization of the cloned inserts within the lambda vector to form a phagemid containing the cloned insert. Packaging of the Lambda Zap Express vector and excision of the pBK-CMV phagemid with the inserts from the Lambda Zap-Express vector were done according to the manufacturer's instructions. E. coli XLOLR colonies containing the recombinant phagemids were isolated, grown overnight in LB -Kanamycin broth at 37°C and the plasmids were extracted using the Wizard plus SV miniprep kit (Promega, Madison WI, USA).
One hundred clones with an average 3 kb insert were isolated and purified. Sixty clones have been sequenced using primers T3 (5'-AATTAACCCTCACTACTAA GGG-3') and T7 (5'-TAATACGACTCACTATAGGG-3') found on both sides of the BamH I insertion site. Inserts were completely sequenced by walking using relevant primers. The sequences of 60 pBK-CMV inserts were compared with the complete virus protein and nucleotide database from the National Center for Biotechnology Information using the BLASTX and BLASTN programs [20] and the Mac Vector software (Oxford Molecular Ltd. San Diego CA. USA).
Characterization of the recombinant thymidine kinase
The hypothetical TK gene was amplified by PCR from purified genomic KHV DNA using the primers: IMPTK5 (5'-GTTGTTCATATGGCTATGCTGGAACTGGTG-3') and IMPTK3 (5'-GTTGTTTGCTCTTCCGCACAGGATAGATATGTTACAAGAA CG-3'). To prove that the sequences of the amplified TK originated from KHV DNA and not from a contaminating agent or fish cellular sequences, Southern blot analyses were performed. The TK gene was labeled with digoxigenin (DIG) (Roche Applied Science, Mannheim Germany) according to the manufacturer instructions. Purified TK DNA and Carp DNA (1–2 μg) were incubated with 10 U of BsaA1 or with Pvu II that cut only once within the TK sequence for 4 h at 37°C. DNA fragments were separated by electrophoresis on 1.0% agarose gels and observed after staining with 1% ethidium bromide. DNA was transferred to positively charged nylon membranes (Roche Applied Science, Mannheim Germany) and treated as recommended by the manufacturer (Roche Applied Science, Mannheim Germany). Prehybridization was performed in 10 ml DIG easy hyb solution (Roche Applied Science, Mannheim Germany) within a roller bottle at 65°C for 30 min. Hybridization was carried out in 6 ml of DIG easy hyb containing 250 ng of DIG labeled probe at 65°C overnight. Membranes were washed twice with low stringency wash buffer (2 × SSC+0.1% SDS) at room temperature followed by two washes with high stringency wash buffer (0.1 × SSC +0.1% SDS) at 60°C. The membrane was blocked for 30 minutes with 40 ml blocking solution and then incubated with a 1:10000 dilution of anti Dig AP conjugated antibody (Roche Applied Science, Mannheim Germany). After washing, the membrane was placed on a plastic film, evenly covered with 500 μl of ready-to-use chemiluminescent alkaline phosphatase substrate (CSPD) (Roche Applied Science, Mannheim Germany), and incubated for 5 min. at room temperature. The membranes were then exposed to super RX X-ray film (Fuji photo film, Tokyo, Japan).
The amplified TK gene was cloned into vector pYB1 (Impact system, New England Biolabs, Beverly MA. USA) with an intein tag that allows the purification of the recombinant protein in a non-denaturated form without any extra residues. The TK protein was expressed in E. coli ER2566 and purified according to the manufacturer's instructions (New England Biolabs). After affinity purification on a chitin column, SDS-PAGE analysis was performed to characterize the recombinant TK and the enzymatic activity of the recombinant TK was tested.
Thymidine kinase activity was determined by the DE-81 filter paper assay using tritium labeled deoxy thymidine and deoxy cytidine as substrates. [14]. A typical reaction mixture contained in 100 μl of Tris-Cl (140.0 mM) MgCl2 (2.0 mM), DTT (1.7 mM), NaF (8.0 mM), PMSF (5.0 mM), ATP (10.0 mM), KHV TK enzyme, and substrates methyl-3H thymidine (dThd) (25 ci/mM) or deoxy(5-3H)cytidine (dCyd) (22 ci/mM) (Amersham Biosciences, UK). Substrates were used at concentrations between 5–30 mM. Reactions were carried out at 37°C. At 15 seconds intervals, 20 μl samples were removed, and spotted onto the filters. The filters were washed 4 times in 5.0 mM ammonium formate followed by a 15 min wash in Ethanol 95 %. Amount of radioactivity was determined in a scintillation counter (LS 6000TA, Beckman Instruments Fullerton CA. USA).
To demonstrate that the KHV TK is expressed in an experimentally infected cell line, total RNA was extracted using the SV total RNA isolation system (Promega, Madison WI. USA) from KF-1 cells infected with KHV. Uninfected KF-1 cells served as a control. RT-PCR (Access RT-PCR system, Promega, Madison WI. USA) was performed using internal primers of the TK gene (Upstream primer TK-RTf: (5'-ATGGCTATGCTGGAAC-3'); downstream primer TK-RTrl: (5'-TGGAGCGGCT GACACG-3') on experimentally infected KF-1 cells at different time points post infection. PCR products were analyzed on 1% agarose gel. The identity of the amplified products was confirmed by Southern blot hybridization with the labeled KHV TK gene as described above.
PCR assays
Head kidneys were removed from koi carp infected naturally or intentionally by KHV in order to obtain survivors in Israeli farms [18]. Template DNA from head kidney of koi carp was prepared using High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim Germany) according to the manufacturer's recommendations. A TK based PCR assay to detect KHV DNA was developed using specific primers, KHV-TKf (5'-GGGTTACCTGTACGAG-3') and KHV-TKr (5'-CACCCAGTAGA TTATGC-3'), derived from for the thymidine kinase gene (AJ535112) that produced a 409 bp amplicon. The TK based PCR was done using the above primers for 35 cycles of amplification under standard conditions (95°C 5 min, then 35 cycles: 95°C 30 sec, 52°C 30 sec, 72°C 1 min and finally 72°C 10 min).
Two other PCR assays were performed in parallel according to their original description and are denominated Gilad PCR (400 bp product obtained with Primers (5'-GACGACGCCGGAGACCTTGTG-3') and (5'-CACAAGTTCAGTCTGTTC CTCAAC-3') [10] and Gray PCR (290 bp product obtained with primers (5'-GACACCACATCTCAAGGG-3') and (5'-GAC ACATGTTACAATGGTGGC-3') [11] (Table 1).
The specificity of the PCR assays (discrimination of KHV from other viruses) was assessed as described by Gilad et al. [10]. A total of 50 ng of DNA obtained from purified KHV-I, CHV, CCV, or uninfected KF-1 cells were used as templates for the PCR assays. The sensitivity of the PCR assays was determined using 10 fold serial dilutions from 10 ng to 0.001 pg of KHV-I DNA and 10 fold serial dilutions of head kidneys (boiled in distilled water for 5 min) from diseased koi carp with proven virus isolation or the extracted DNA. Whole tissue or DNA extracted from negative head kidney samples from uninfected koi carp were run as controls. The resulting amplified products were analyzed by electrophoresis on 1% agarose gels after staining with ethidium bromide.