Source of koi and sampling.
Six koi (designated K1 to K6) between 2 and 15 years old were from premises that had previous KHV infections or exposure. Three (K2, K4, and K5) were recently imported koi that had been housed in the same quarantine facility. Prior to donation, K4 and K6 tested positive for KHV by serum antibody ELISAs carried out at the Immunology and Virology Laboratory, Veterinary Medicine Teaching Hospital, University of California, Davis (29
). The remaining three koi (K1, K2, and K5) were survivors from a pond associated with a suspected KHV outbreak in 1998 and a confirmed KHV outbreak in 2003, based on a positive PCR test at the University of Georgia Infectious Disease Laboratory. To investigate whether KHV becomes latent in the peripheral leukocytes, 0.5- to 2.0-ml blood samples from fish K1 to K6 were collected and stored in EDTA tubes at 2 weeks, 1 month, and 2 months following arrival of the fish at the Oregon State University, Salmon Disease Research Lab (OSU-SDL). Three sets of blood samples from these six koi were collected to ensure the consistency of KHV genome detection as persistence of the genome over time is a characteristic of latency. The OSU-SDL is specifically designed for conducting in vivo
experiments with infectious diseases. The incoming water is from a deep well and is pretreated with UV irradiation and is thus not a source for KHV.
Five 2-year-old koi were obtained from facilities with no known history of KHV problems, and these were designated KI to KVI. An additional four 2-year old koi were obtained from a local pet store, and these were designated KVI to KIX. These four koi were home bred by a local pet owner, and there are no records of KHV infection in the breeding facility. Both groups of koi were kept segregated and maintained at 12°C in 4-ft-diameter tanks at OSU-SDL in accordance with the Animal Care and Use Committee regulations. All blood samples were collected via caudal vein puncture after the koi were anesthetized with MS-222 (100 ppm). To investigate whether KHV becomes latent in certain tissues, all koi in this study were euthanized via MS-222 (500 ppm) overdose. Tissues, including the brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas, were collected at necropsy.
CCB and KF-1 cell lines.
Both a common carp brain (CCB) cell line and koi fin cell line (KF-1) (gift of Ronald Hedrick, University of California, Davis) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma-Aldrich, Inc., St. Louis, MO) and incubated at 22°C. The strains of KHV from the United States and Israel (KHV-U and KHV-I, respectively) were gifts of Ronald Hedrick.
Detection of persistent infection by examining KHV in body secretions.
The presence of KHV DNA or infectious virus in body secretions is indicative of persistent infection. To determine whether persistent infection was present, we swabbed fish K1 to K6 every other day for 1 month to see if we could detect the presence of KHV or KHV DNA on gill surfaces and within the anal vent. These Dacron swabs were placed in 0.5 ml of sterile DMEM (Invitrogen, Carlsbad, CA) containing penicillin (200 U/ml) and streptomycin (200 μg/ml) (Sigma-Aldrich, Inc., St. Louis, MO). Five-microliter aliquots of swab solution were tested by real-time PCR for KHV DNA, and 0.2 ml of swab solution was inoculated to CCB cells seeded in 12-well plates for KHV virus detection.
Separation of peripheral WBC and total DNA extraction from WBC and plasma.
Blood collected from the caudal vein into a syringe previously coated with heparin (Sigma-Aldrich, Inc., St. Louis, MO) at 1,000 U/ml in phosphate-buffered saline (PBS) was transferred to an EDTA tube. Blood was centrifuged at 650 × g at 4°C for 10 min; the buffy coat was collected and exposed to 3 to 4 volumes of red blood cell lysis buffer (Tris-NH4Cl). The remaining white blood cells (WBC) were washed twice in sterile DMEM (Invitrogen) by centrifugation at 650 × g at 4°C for 10 min (Becman XJ). Then, WBC from each blood sample were subjected to a total DNA extraction (yielding WBC total DNA) using a High Pure PCR Template Preparation Kit (Roche Diagnostics, Indianapolis, IN). Total DNA was extracted similarly from the plasma pellet that was collected after ultracentrifugation of 250 to 500 μl of the plasma layer at 25,000 rpm for 1 h min at 4°C (Beckman model XL-70) in an SW28 rotor. Approximately, 0.1 to 0.5 μg/μl total DNA can be isolated from WBC obtained from each fish. The DNA concentration was adjusted to 0.1 μg/μl before use in PCR.
Virus culture and virus isolation.
KHV was cultured in CCB or KF-1 cell lines that were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma-Aldrich, Inc., St. Louis, MO) at 22°C. KHV virus isolation from tissue samples was performed by homogenizing frozen tissue samples in DMEM (1:5 ratio [wt/vol]), centrifuging the tissue homogenate at 2,000 × g for 10 min, filtering the homogenate through a 0.45-μm-pore-size filter (Waterman), and inoculating supernatant of the tissue homogenate into 25-cm2 flasks seeded with CCB cells (0.5 ml of tissue preparation per 25-cm2 flask). To monitor virus shedding in the gill and feces, each gill or fecal swab medium (0.2 ml/swab) was inoculated onto CCB cells seeded in 12-well plates. Visible characteristic KHV cytopathogenic effect (CPE) was considered positive isolation. If initial inoculation failed to produce KHV CPE the first time, total cell lysate was reinoculated onto CCB cells seeded in 12-well plates and incubated at 22°C. Swabs or tissue preparations failing to produce KHV CPE in CCB cells the second time were considered KHV negative. Stock KHV cultures were inoculated onto one well of each plate as a positive control.
Purification of KHV DNA.
Viral DNA for positive controls was extracted from either virions or purified intracellular nucleocapsids as described previously (13
). Briefly, the purified virions or nucleocapsids were digested in 10 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1% N
-lauroyl sarcosine, and 200 μg/ml proteinase K overnight at 55°C. The viral DNA was extracted twice with an equal volume of phenol-chloroform (1:1 [vol/vol]) and then precipitated with two volumes of ethanol and 1/10 volume of sodium acetate. The precipitate was washed once in 70% ethanol and dissolved in TE buffer (10 mM Tris-HCl [pH 8.0]-1 mM EDTA).
KHV reactivation by temperature stress.
To determine whether KHV latency can be reactivated, the koi tank water temperature was increased from 12°C to 23°C at a rate of 1°C per day. The temperature was then held constant for 4 days at 23°C before being dropped back to 12°C at a rate of 1°C per day (see A). Anal vent and gill swabs were collected from all six koi every other day starting at day 2 post-temperature increase until day 16 post-temperature stress. Swabs were collected and transported in 0.5 ml of sterile PBS containing penicillin (200 U/ml) and streptomycin (200 μg/ml) (Sigma-Aldrich, Inc., St. Louis, MO). Five-microliter aliquots of swab solution were tested by real-time PCR for KHV DNA, and 0.2 ml of swab solution was inoculated onto CCB cells seeded in 12-well plates for KHV virus detection.
Fig. 3. Water temperature change and detection of KHV DNA in fecal and gill swabs. (A) Tank water temperature change. Vent and gill swabs were collected from all six koi every other day starting at day 2 post-temperature increase until day 16 post-temperature (more ...)
Two koi, designated K5 and K6, died on day 20 post-temperature stress (i.e., 20°C). These fish were necropsied, and tissue samples were collected, including the brain, spleen, gills, heart, eye, intestine, and kidney. One month after completion of the temperature stress regime, the remaining four koi were bled as described above and then euthanized to permit collection of tissues, including the brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas, via necropsy.
Total DNA extraction from tissue samples.
Tissue samples (approximately 100 to 200 mg) obtained from freshly euthanized or dead koi were stored at −80°C. Before DNA extraction, the frozen tissues were homogenized in 800 μl of 1× lysis buffer by 2.5-mm silica beads (Biospec Product) and digested overnight at 55°C in the presence of 100 μg of proteinase K. Genomic DNA was then extracted from the tissue lysates with a High Pure PCR Template Preparation Kit according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). Approximately 0.1 to 1 μg/μl total DNA could be extracted from each tissue. All the tissue DNA was adjusted to 0.1 μg/μl before being used in PCRs. For each sample, 5 μl of total DNA (about 0.5 μg) was used in real-time PCR or PCR.
Total RNA extraction from WBC and KHV-infected KF-1 cells.
To determine if persistent infection occurs in the WBC, viral mRNA expressed during lytic infection was examined in total white blood collected from four healthy koi obtained from a local pet store; these fish had tested positive for KHV DNA in WBC by PCR. Total RNA was extracted from the combined WBC from the four koi using TriZol (Invitrogen). As a positive control, total cellular RNA from KHV-infected KF-1 cells was harvested at 8 days postinfection and extracted by using TriZol in accordance with the manufacturer's instructions. Total RNA from uninfected KF-1 cells served as a negative control. The isolated total RNA was resuspended in RNase-free H2O and examined for KHV lytic infection by reverse transcription-PCR (RT-PCR). The extracted total RNA from WBC (WBC total RNA) and KF-1-infected or uninfected cells was adjusted to 0.5 μg/ml before use in RT-PCRs.
Primers and probes.
Selection of primers for KHV sequence amplification was based on conserved DNA sequences of KHV (AF411803). Real-time PCR primers were selected as previously described (8
): KHV-86f (5′-GACGCCGGAGACCTTGTG-3′), KHV-163r (5′-CGGGTTCTTATTTTTGTCCTTGTT-3′), and TaqMan probe KHV-109p (5′-6FAM-CTTCCTCTGCTCGGCGAGCACG-TAM-3′, where FAM is 6-carboxyfluorescein and TAM is 6-carboxytetramethylrhodamine). Another real-time PCR primer set specific for a host gene encoding glucokinase was also used as an internal control to equalize the amount of input total DNA. All the real-time PCRs for KHV DNA were run with equal amounts of DNA estimated by real-time PCR of the glucokinase gene. The primer sequence and TaqMan probe were selected as reported previously (8
). The primers used for screening for the presence of viral DNA in tissue samples and tissue culture fluid were complementary to the KHV DNA polymerase and open reading frame 26 (ORF26) sequences: KHVDF-242 and KHVDR-242 for the KHV DNA polymerase gene; KHVF-447 and KHVR-447 for KHV ORF26 (). To further probe the amplified DNA sequence specific for KHV, another set of nested primers was selected: KHVNF242 and KHVNR242 as the DNA polymerase gene probe () and KHV263F and KHV263R as the ORF26 probe ().
Primer pairs used to detect KHV DNA and KHV gene transcription
First-strand cDNA was synthesized from 2.5 μg of total RNA by using 10 pmol of random primer and Superscript Reverse Transcriptase III (Invitrogen) according to the manufacturer's recommendations. PCR amplification with KHV-specific primers for detection of cDNA of KHV genes expressed during lytic infection was performed using a 25-μl reaction mixture consisting of 22.5 μl of amplification buffer (Platinum PCR Supermix; Invitrogen, Carlsbad, CA), a 1.25 μM concentration of each primer, and 2.5 μl of the completed RT reaction mixture. The mixture was subjected to 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s and then incubated at 72°C for 5 min after the final cycle.
PCR amplification with KHV-specific primers for detection of viral DNA in tissues or culture fluid was performed using a 25-μl solution consisting of 19 μl of amplification buffer (Platinum PCR Supermix; Invitrogen, Carlsbad, CA), a 0.4 μM concentration of each primer, and 5 μl of total DNA (~0.5 μg) or 5 μl of swab medium. The mixture was subjected to 94°C for 2 min, and 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a 10-min elongation reaction at 72°C after the final cycle. Real-time PCR was performed according to the manufacturer's instructions for quantitative PCR (qPCR) Supermix (Platinum qPCR Supermix-UDG with ROX; Invitrogen, Carlsbad, CA).
Plasmid preparation for KHV DNA real-time quantitation.
The PCR product amplified from KHV-U DNA with real-time PCR primers was cloned by a TOPO 2.1 PCR cloning vector (Invitrogen, Carlsbad, CA). The correct insert was screened by restriction digestion and then sequenced by the Center for Genome Research and Biocomputing (CGRB) at Oregon State University. This plasmid product was employed to set up the standard curve for measuring the viral DNA copy number in tissue samples or swab fluid.
PCR products (40% of total PCR with 0.5 μg of DNA template) generated using WBC or tissue DNA were electrophoresed through a 1.5% agarose gel, transferred to a nylon membrane (14
), and then UV cross-linked to the membrane. The DNA products were then probed with a digoxigenin (DIG)-labeled DNA probe. The probe was generated with nested PCR primers that are specific for the target genes. To make digoxigenin-labeled PCR products, digoxigenin-labeled deoxynucleoside triphosphates (Roche Diagnostics, Indianapolis, IN) were added to the PCR mixtures according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). The membrane was prehybridized with prehybridization buffer (Roche Diagnostics, Indianapolis, IN) at 68°C and then hybridized with the DIG-labeled DNA probes specific for the gene coding for DNA polymerase or major capsid protein at 68°C. After incubation with the probe, membranes were washed with 0.1% sodium dodecyl sulfate and 10% 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) before incubation with an antidigoxigenin antibody conjugated with peroxidase. The membrane was then developed by incubation with a chemiluminescent peroxidase substrate (Roche Diagnostics, Indianapolis, IN). The blots were exposed to film (Kodak) at room temperature for 30 min to 2 h. The molecular masses of the resulting bands were estimated by using a 1-kb DNA ladder (Invitrogen, Carlsbad, CA).
DNA sequencing and analysis.
The PCR products were cleaned with a ChargeSwitch PCR Clean-Up Kit (Invitrogen, Carlsbad, CA) before sequencing and were sequenced by the CGRB at Oregon State University. The nucleotide sequences were analyzed with the Geneiou software.