NB: THIS DISEASE IS NO LONGER LISTED IN

CHAPTER 1.2.3 OF THE AQUATIC CODE

CHAPTER 2.1.10.

1
2
3

CHANNEL CATFISH VIRUS DISEASE

1.

Scope

5
6

For the purpose of this chapter Channel catfish virus disease (CCVD) is considered to be infection with Ictalurid herpesvirus 1. The commonly used
name is channel catfish virus (CCV).

2.

Disease information

2.1. Agent factors

2.1.1.

Aetiological agent, agent strains

10
11
12
13

On the basis of antigenic studies conducted with polyclonal rabbit antibodies, CCV isolates form a homogeneous group. However, the
use of monoclonal antibodies shows some variation in antigenic determinants between isolates (1). Some variation in the virulence of
CCV strains has been recorded during natural outbreaks of disease and has been demonstrated experimentally. Additionally,
molecular data indicate genetic variation within this species (5). The CCV genome was the first fish herpesvirus to be sequenced (7).

14

2.1.2.

15
16

Virions remain viable for 2 days in clean pond water at 25C. The virions readily adsorb to pond sediments (4) and interaction with
suspended clay particles in pond water may influence infectivity.

17

2.1.3.

18
19
20
21

Virions are rapidly inactivated by UV irradiation, heat (1 hour at 60C) (22), desiccation (24 hours on concrete, 48 hours on netting),
alcohols (70% ethanol or isopropanol), aldehydes, phenolics, strong acids and bases (muratic acid-hydrochloric acid, lye-sodium
hydroxide), and halide disinfectants (chlorine 540 mg/litre and iodine compounds 250 ppm) for 30 minutes (18). The infectivity of
virions in fish tissues is preserved by freezing (21).

22

2.1.4.

23
24

The life cycle involves fish to fish transmission, both horizontally and vertically. The virus establishes latency in exposed fish and
these fish act as the reservoir for CCV.

Survival outside the host

Stability of the agent (effective inactivation methods)

Life cycle

25

2.2. Host factors

26

2.2.1.

27
28

Channel catfish and the closely related blue catfish (Ictalurus furcatus) have been the only fish found to be infected with CCV, and
variations in susceptibility to CCV have been recorded depending on fish strain.

29

2.2.2.

30
31
32
33
34

The age of the fish is extremely important for overt infection. Although experimental data suggest that older fish are susceptible to
natural outbreaks of acute CCVD (12), the disease occurs almost exclusively in fish that are less than 1 year of age, and generally less
than 4 months of age. In experimental trials, very young fish (less than 2 weeks of age) are more resistant to immersion challenges
and they become more susceptible over the following 2 months as they age, and this resistance may be due to maternal antibodies
(10).

Susceptible host species

Susceptible stages of the host

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Chapter 2.1.10. Channel catfish virus disease

35

2.2.3.

Species or sub-population predilection (probability of detection)

36
37

Channel catfish are the most susceptible of ictalurid species. Molecular detection of latent pathogen appears to be most sensitive in
sac fry. These fish are infected by vertical transmission.

38

2.2.4.

39
40

CCV causes a generalised viraemia but the highest virus concentrations during replicative infection are usually found in the trunk
kidney tissue. Latent virus genome can be detected in various visceral organs and skin.

41

2.2.5.

42
43
44

CCV readily establishes long-term infection. The carrier state is latency with no infectious virus production. Methods that depend on
infectious virus for detection will not be useful for identifying carriers. Furthermore, overt viraemia is not a prerequisite for the
carrier state. Many fry obtain CCV infection by vertical transmission with no detectable virus replication (24).

45

2.2.6.

46

There are no known biotic vectors for CCVD.

47

2.2.7.

48
49

Wild and feral channel catfish may be reservoirs of CCVD. Many aquaculture populations are endemic for CCVD and these fish are
carriers for life and carriers transmit CCV to their offspring at a high rate (24). Therefore, feral fish are likely carriers.

Target organs and infected tissue

Persistent infection with lifelong carriers

Vectors

Known or suspected wild aquatic animal carriers

2.3. Disease pattern

50
51

2.3.1.

Transmission mechanisms

52
53
54
55
56
57
58
59
60

Infectious CCV can be detected in the water from tanks of experimentally infected fish, but the route of shedding has not been
determined. The sites where the virus is most abundant during the course of overt infection are posterior kidney, skin, gills, spleen
and intestine in decreasing magnitude, respectively (12, 13). The transmission of CCV is horizontal and vertical. Horizontal
transmission may be direct. The virus has been shown to readily adsorb to pond sediments (6) and interaction with suspended clay
particles in pond water may influence horizontal transmission. Experimental infection by immersion exposure causes peak viremia
and losses at 4 days post-infection (14). Horizontal transmission can occur without overt CCVD (24). Vertical transmission is common,
but the mechanism of vertical transmission is not known, as infectious virus has not been detected on the skin or in the sexual
products of spawning adults. When individual fry from CCV-positive parents were evaluated by polymerase chain reaction (PCR), the
detected rate of vertical transmission was 4075% (10).

61

2.3.2.

62
63
64

In a single years evaluation, sac fry of five commercial fingerling production facilities demonstrated a prevalence range of 11.7 to
26.7% and, when tracking three individual pond populations, the prevalence of all ponds increased from 12% to over 35% over the
summer without overt disease (24).

65

2.3.3.

66

CCVD has been documented in Channel catfish production areas in the USA and there was one isolated report in Honduras.

67

2.3.4.

68
69

Unapparent CCV transmission may be common but overt CCVD is characterised by rapidly increasing losses. These acute outbreaks
in heavily stocked ponds can result in over 90% mortality over a 2-week period.

70

2.3.5.

71
72

The disease is restricted to high temperatures (over 25C with highest losses occurring above 27C). High stocking densities and
stress due to handling or low oxygen are predisposing factors.

Prevalence

Geographical distribution

Mortality and morbidity

Environmental factors (e.g. temperature, salinity, season, etc.)

2.4. Control and prevention

73
74

2.4.1.

75

Experimental evidence suggests that CCVD vaccines are possible (15, 2527) but none are commercially available.
2

Vaccination

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Chapter 2.1.10. Channel catfish virus disease

76

2.4.2.

77

There are no commercially available treatments for CCVD.

78

2.4.3.

79

Treatment of channel catfish fingerlings with Poly I:C makes them resistant to CCV (16) but this is no longer being used commercially.

80

2.4.4.

81
82

There are differences in the sensitivity of different strains of channel catfish to CCVD (20) but the use of genetics for controlling
CCVD has not gained widespread commercial application.

83

2.4.5.

84
85

Biological resistance to CCVD has been demonstrated in a similar species (blue catfish, I. furcatus, are more resistant than channel
catfish) (19, 23). However, the culture of this species has not gained widespread commercial application.

86

2.4.6.

87

None.

88

2.4.7.

89

Egg disinfection is a good practice when bringing egg masses from ponds, but it does not prevent vertical transmission of CCV.

90

2.4.8.

91
92
93
94
95
96

Management to reduce CCVD-associated losses is the best option when the pathogen becomes established on a facility or is endemic
in a region. This is done by avoiding crowding (stock at levels below 400,000/ha) and stressing events, especially in fry and young
fingerlings when the temperatures are above 25C. Also, maintaining quality nutrition and feeding levels of brood stock, fry and
fingerlings will help prevent losses. If fry experience CCVD in the hatchery before being stocked in the pond, the affected population
should be destroyed and the hatchery sterilised before bringing in new eggs. If possible, reducing water temperature in a pond will
reduce losses after a CCVD outbreak has started (17).

97
98

3.

Chemotherapy

Immunostimulation

Resistance breeding

Restocking with resistant species

Blocking agents

Disinfection of eggs and larvae

General husbandry practices

Sampling
3.1. Selection of individual specimens

99
100
101
102

Samples for inspections/surveillance must be representative of all groups present in a population that is derived from multiple independent
stockings. Sampling from ponds should include any moribund fish and fish that school behind aerators when oxygen levels are not stressful.
Sampling should not include the use of feed to attract fish or the use of baited hooks. Diseased fish often display spastic or uncoordinated
swimming and may occasionally be seen at the surface.

103

3.2. Preservation of samples for submission

104
105

Samples for CCV culture can be iced or refrigerated for up to 48 hours. For extended storage, isolated tissues can be frozen in serum-free
medium at 70C.

106

3.3. Pooling of samples

107

Samples of up to five fish can be pooled for virus isolation.

108

3.4. Best organs or tissues

109
110
111
112

Whole fry or small juveniles (body length 3 cm), viscera including kidney (3 cm body length 6 cm) or, for larger size fish, kidney, and
spleen are sampled for cell culture. When evaluating of tissues by immunohistochemistry samples must be processed for producing
cryostat sections. If using whole fry, the sections that are evaluated must include the kidney tissue. For PCR analysis of brood fish, a 6 mm
diameter biopsy of the caudal fin may be used.

113

3.5. Sample/tissues that are not suitable

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Chapter 2.1.10. Channel catfish virus disease

The use of decomposing fish for CCV diagnosis is not reliable.

114
115

4.

Diagnostic methods
4.1. Field diagnostic methods

116
117

4.1.1.

Clinical signs

118
119
120
121

Generally, outbreaks are seen in populations of young channel catfish (less than 4 months of age) that are in heavily stocked ponds
(over 400,000 fish/ha) at elevated temperatures (above 25C). Mixed infection with Flavobacterium columnare (columnaris disease)
and/or Edwardsiella ictaluri (enteric septicaemia of catfish) are common. However, many populations of channel catfish are endemic
for CCV with no signs of disease.

122
123
124
125

A CCVD-affected population may show rapidly progressing mortality with affected fish being lethargic in a head up position at the
pond surface. Some diseased fish will also demonstrate distressed uncoordinated swimming. As the outbreak worsens in the pond the
population will substantially reduce feeding activity. During acute outbreaks, diseased fish will often demonstrate protruding eyes,
abdominal distension and haemorrhages or reddening at the fin bases.

126

4.1.2.

127

Behavioural changes

4.2. Clinical methods

128
129

4.2.1.

Gross pathology

130
131

In addition to the external gross pathology listed above, the fish may display clear yellow ascites, enlarged and posterior kidney and
scattered haemorrhages in the musculature and viscera.

132

4.2.2.

133

Not evaluated.

134

4.2.3.

135
136

Extensive necrosis and inflammation of the interstitial tissue trunk kidney tissues is the most common histopathology associated with
acute infections.

137

4.2.4.

138

NA

139

4.2.5.

140

NA

141

4.2.6.

142

NA

143

4.2.7.

144
145
146
147
148

Replicating CCV are visible in infected cells as assembly centres in the nucleus with capsids budding from the nuclear membrane and
enveloped particles migrating through the cytoplasm, endoplasmic reticulum and cytoplasmic vacuoles. Virions can also be
concentrated from 0.45 m filtrate of tissue homogenates by centrifugation at 20,000 g. This concentrate can be evaluated by
negative staining. CCV virions are typical of herpesvirus particles 125 nm diameter icosahedral capsid with a distinct envelop. The
diameter of the enveloped virion is 175200 nm.

Clinical chemistry

Microscopic pathology

Wet mounts

Smears

Fixed sections

Electron microscopy/cytopathology

4.3. Agent detection and identification methods

149

4.3.1.

150

Direct detection methods

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Chapter 2.1.10. Channel catfish virus disease

151

4.3.1.1.

152

4.3.1.1.1. Wet mounts

153
154

Microscopic methods

NA

4.3.1.1.2. Smears

155

Tissue imprints from the posterior kidney may be evaluated by antibody-based detection methods.

156
157
158
159
160
161
162
163

The tissue sample is blotted with a laboratory wipe to remove blood, and then imprinted on an alcohol-cleaned slide. The imprint
is then allowed to dry. It is immersed briefly three times with cold acetone (stored at 20C) to fix the tissues, and then stained
using enzyme-linked immunostaining or the fluorescent antibody technique using a CCV-specific monoclonal antibody (MAb) (1), as
described below. Negative controls using a MAb that is not specific for CCV should be run simultaneously. Positive staining of
CCV-infected cells while negative staining of a match negative control is a confirmatory diagnosis. Cryostat sections from a
known CCV-infected fish can be used as a positive control. This method is used primarily as a rapid confirmatory diagnostic test
for CCV. It is not routinely used for inspections because CCV is easily cultured on cell culture and culture protocols can be more
sensitive.

164
165
166
167

4.3.1.1.3. Fixed sections

Formalin-fixed, paraffin-embedded sections can often be stained using immunohistochemical methods; however the reported
MAbs do not work on histologically processed tissues. Cryostat sections can be used for immunohistochemistry, as described
above.

168

4.3.1.2.

Agent isolation and identification

169

4.3.1.2.1. Cell culture/artificial media

170
171
172

Sampling and isolation of the agent: tissues indicated in Section 3.4 can be processed using routine virus extraction procedures

173
174

Inoculation of cell monolayers: the cell line of choice is channel catfish ovary (CCO). The brown bullhead cell line (BB) is also

175
176
177

i)

Make an additional tenfold dilution of the 1/10 organ homogenate supernatants and transfer an appropriate volume of each
of the two dilutions on actively growing cultures of the CCO cell line that are approximately 80% confluent. Inoculate at
least 2 cm2 of the cell monolayer with 100 l of each dilution.

178
179
180

ii)

Allow virus to adsorb for 0.51 hour at 2530C. Then (without withdrawing the inoculum), add the cell culture medium
buffered at pH 7.6 and supplemented with 10% foetal calf serum (FCS) (1 ml/well for 24-well drained cell culture plates),
and incubate at 2530C.

181

Monitoring incubation

182
183
184
185
186

i)

Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination at
40100 total magnification for 10 days. The use of a phase-contrast microscope is recommended. CPE is extensive and
rapidly progressing in cultures from overtly diseased individuals. CPE consists of cell fusion (syncytium) formation and
contraction leaving cytoplasmic spindles irradiating from the syncytium to points on the flask surface where the cells
were originally attached.

187

ii)

Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation.

188
189

iii)

If CPE appears in cell cultures inoculated with the dilutions of the tested homogenate supernatants, identification
procedures must be undertaken immediately (see below).

as presented in Chapter 2.1 (the use of neutralising antibodies to inactivate enzootic birnaviruses is not needed because these
viruses are not found in CCV-susceptible species).
acceptable, though cytopathic effect (CPE) development may be somewhat slower.

If a fish health surveillance/control programme is in place specifically for CCVD, approved health status of the production
unit from which the virus-positive sample originated will need to be suspended until confirmatory tests are run.

190
191
192
193
194

iv)

If no CPE develops in the inoculated cultures (despite normal progression of CPE in the virus controls), the inoculated
cultures should be subcultured for a further 7 days. Should the sample inoculated with the virus control fail to develop CPE,
the process should be repeated with fresh susceptible cells and new batches of samples.

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Chapter 2.1.10. Channel catfish virus disease

195
196
197
198
199
200
201
202
203

Expectations: catfish with acute CCVD generally have very high levels of virus. Extensive CPE is generally seen within 48 hours at

204
205
206
207

Alternatives: for disease diagnoses, cells can be infected at the time of seeding. To use this protocol a 1/2 split of a heavy

208
209

The use of the BF2 and/or EPC cell line in parallel with the CCO cell line is a useful addition during the initial culture protocol to
distinguish IcHV2 and catfish iridovirus.

30C. Latent carriers rarely produce culturable virus and, consequently, this method is not adequate for identifying latent
carrier fish. Chronic or subclinical detection may require longer incubation and blind passage. CCV is very host specific in
culture. It grows well in ictalurid and clarid cell lines and but not at all in BF2 or EPC cells. Other virus that may be found in
catfish include the black bullhead herpesvirus (Ictalurid herpesvirus 2 [IcHV2]), which produces similar CPE but replicates in BF2
and EPC cells, and catfish iridovirus, which causes a distinct CPE (rounding of cells and cytoplasmic inclusion bodies) and
replicates in BF2 and EPC cells. Another virus that has been occasionally isolated from channel catfish is the catfish reovirus.
This agent causes syncytia in CCO cells but the progression of single plaque growth is slower. Confirmation requires serology or
molecular-based methods.
monolayer of cells can be placed into control and test wells, and control samples can be added to the wells (a 25 cm2 flask can be
used to seed a 24-well plate). Since this method lacks the initial incubation with the more concentrated sample the sensitivity is
reduced, but during active CCVD the fish have very high levels of virus.

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Chapter 2.1.10. Channel catfish virus disease

210

4.3.1.2.2. Antibody-based antigen detection methods

211

4.3.1.2.2.1. Neutralisation test

212
213
214

(Note: when developing antisera for CCV, most researchers have found a weak neutralising antibody response in rabbits, and
cross-reaction with cellular components also often occurs). The use of neutralising MAb produced by Arkush et al. 1992 (1)
provides the specificity to distinguish IcHV1 from IcHV2 (13).

215
216

i)

Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at 2000 g for 15 minutes at 4C to remove
cell debris.

217

ii)

Dilute virus-containing medium from 102 to 104 in 200 l aliquots.

218
219

iii)

Mix 100 l aliquots of each virus dilution with equal volumes of an antibody solution specific for CCV, and similarly treat 100
l aliquots of each virus dilution with cell culture medium.

220

iv)

In parallel, other neutralisation tests must be performed against:

221

A homologous virus (positive neutralisation test)

222

A heterologous virus (negative neutralisation test).

223

v)

Incubate all the mixtures at 25C for 1 hour.

224
225
226

vi)

Transfer aliquots of each of the above mixtures onto cell monolayers (inoculate two cell cultures per dilution) and allow
adsorption to occur for 0.51 hour at 25C; 24-well cell culture plates are suitable for this purpose, using a 50 l inoculum
per well.

227
228

vii)

When adsorption is complete add the cell culture medium, pH 7.37.6, supplemented with 10% FCS into each well and
incubate at 2530C.

229

viii) Check all cell cultures daily for CPE by microscopic examination and note the dilution and time of first detectable CPE.

230
231
232
233

ix)

234

4.3.1.2.2.2. Indirect fluorescent antibody test

235
236
237
238

i)

Seed at least six wells of a 24-well plastic cell culture plate or cover-slips with CCO cells to produce 80% confluency
within 1224 hours for each virus isolate to be identified. Four wells will be used for two dilutions of test virus with positive
serum and negative control serum (non-relevant antibody similar in structure to the test antibody), and two wells will be
for control virus with positive serum and negative control serum.

239
240

ii)

When the cell monolayers are ready for infection, inoculate them with 100 l of 100-fold and 1000-fold dilutions of the virus
suspensions directly in the cell culture wells.

241
242

iii)

Dilute the control virus suspension of CCV to obtain a virus titre of about 500010,000 plaque-forming units (PFU)/ml in
the cell culture medium and inoculate two wells with 100 l per well.

243

iv)

Incubate at 2530C and observe for early CPE.

244

v)

Remove the cell culture medium, rinse once with 0.01 M phosphate-buffered saline (PBS), pH 7.5, then fix in 100% methanol.

245

vi)

Let the fixative act for 10 minutes. A volume of 0.5 ml is adequate for 2 cm2 of cell monolayer.

246

vii)

Allow the cell monolayers to air-dry and process immediately or freeze at 20C.

247
248
249

viii) Prepare a solution of purified antibody or serum to CCV in 0.01 M PBS, pH 7.2, containing 0.05% Tween 80 (PBST), at the
appropriate dilution (which has been established previously; there is no commercial source for CCV-specific antiserum) or
use 1/2 dilution of cell culture supernatant if using an unconcentrated cell culture source of a MAb.

250

ix)

Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and remove this buffer completely.

251

x)

Cover the cell monolayers with the antibody solution for 1 hour at 37C in a humid chamber.

252

xi)

Rinse four times with PBST as above.

The tested virus is identified as CCV when CPE is eliminated or substantially delayed in the cell cultures that had received
the virus suspension treated with the CCV-specific antibody, when compared with all other cell cultures. In the absence of
neutralisation by neutralising antibody to CCV, conduct an indirect fluorescent antibody test (IFAT) with the suspect
sample, or use a CCV-specific nucleic-acid-based assay.

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Chapter 2.1.10. Channel catfish virus disease

253
254

xii)

Treat the cell monolayers for 1 hour at 37C with a solution of fluorescein isothiocyanate (FITC)-conjugated antibody to the
immunoglobulin used in the first layer and prepared according to the instructions of the supplier.

255

xiii) Rinse four times with PBST.

256
257

xiv) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using glycerol mounting
medium buffered to pH 8.5 prior to microscopic observation.

258
259

xv)

260

4.3.1.2.2.3. Enzyme-linked immunostaining

261
262
263

Samples are prepared the same as for IFAT the first steps are similar, but the secondary antibody is conjugated to horseradish
peroxidase. A third incubation step with a substrate (DMOB, Sigma) is performed for 10 minutes, and after washing and drying,
the wells or cover-slips are mounted in buffered glycerine and observed microscopically under normal transillumination.

Examine under an epifluorescent microscope. Positive and negative controls must be found to give the expected results
and are used for comparison for evaluating the tested sample.

4.3.1.2.3. Molecular techniques

264
265
266

Nucleic-acid-based techniques include specific PCR (2, 3, 9) and degenerate PCR followed by sequencing of a fragment of the
DNA polymerase gene (11).

267

4.3.1.2.3.1. Polymerase chain reaction

268
269
270
271

There are several published PCR assays for CCV. PCR is very susceptible to false-positive and false-negative results. Therefore
each assay and tissue extraction should include a negative control to rule out contamination. PCR reaction set-up should be done
in a separate physical location from where the PCR products are evaluated. PCR is a useful method for identifying the virus after
isolation in culture. PCR can also be used to evaluate latent CCV in fry (24) and in brood fish.

272
273
274
275

The following is a modification of the method of Boyle & Blackwell (3) and uses a modified target as an internal control (14). The
53 sequence of the upper primer is TCA-TCC-GAA-TCC-GAC-AAC-TGA and that of the lower primer is CCA-AGA-TCG-CGG-AGAAAC. To minimise the potential for contamination, all sample preparation and reaction set-up should be done with aerosolpreventing pipette tips.

276

Sample preparation for cell cultures:

277

i)

Collect 0.51ml of cell culture supernatant from affected wells into a 1.5 ml microcentrifuge tube.

278

ii)

Centrifuge at maximum speed in a microfuge (18,00020,000 g) for 30 minutes.

279
280

iii)

Discard the supernatant and resuspend the pellet in 10 l proteinase K buffer (50 mM KCl, 15 mM Tris/HCl, pH 8.3, and
0.5% Nonidet P-40) containing 0.5 mg/ml proteinase K and incubate at 55C for 1 hour.

281
282

iv)

Heat inactivate the proteinase K at 95C for 10 minutes. Centrifuge at maximum speed for 10 seconds. Use 3 l in the PCR
reaction.

283

Other standard DNA extraction methods may be used.

284
285
286
287
288
289

Sample processing of fish tissues: DNA from trunk kidney or residual tissue from tissue homogenates for cell culture

290

291
292

The mastermix should be made-up in a separate location from areas where diagnostic samples are prepared and PCR product
are analysed.

293
294

i)

preparations can be isolated using a commercial kit such as DNeasy Tissue Kit (Qiagen, Maryland, Delaware USA). To evaluate
latent carriers, a biopsy can be taken from the caudal fin using a 6-mm diameter biopsy punch and the DNA can be extracted
using a commercial kit as stated above. When evaluating for latency the DNA should be relatively pure (providing a 260nm/280
nm OD ratio of 1.62.0), PCR should be run on 1 g of total DNA and at least five reactions should be run per fish. If the DNA
extraction is not of sufficient purity it can be re-extracted.
PCR set-up

Make-up the mastermix. Prepare at least one extra aliquot for each of the10 reactions planned. Include a positive and a
negative (water blank treated the same as the cell culture extract) control for every 10 samples.
Per sample:
distilled water

295
296
8

33.6 l
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Chapter 2.1.10. Channel catfish virus disease

10 buffer
Upper primer (50 pmole/l)
Lower primer (50 pmole/l
dNTPs (2.5 mM each)
25 mM MgSO4
Internal control (0.025 pg/l)
Taq polymerase (5 U/l)

297
298
299
300
301
302
303

5.0 l
0.4 l
0.4 l
2.0 l
5.0 l
0.3 l
0.3 l

304

ii)

Add 47 l per reaction tube.

305

iii)

Add 3 l of sample or negative control.

306

v)

Run the reaction. Use 30 cycles of 93C for 30 seconds, 60C for 30 seconds, and 72C for 30 seconds.

307
308

vi)

Electrophorese 10 l of the product on a 10% polyacrylamide gel with a 100 bp size ladder. Stain the gel with ethidium
bromide. Observe and photograph under UV transillumination (17).

309
310
311
312
313
314
315
316
317
318

Evaluate the results: the internal control produces a 149 bp product. CCV produces a 136 bp product. Expect a 149 bp band within
the negative control and on negative samples. Expect a 136 bp product and a 149 bp product on CCV-positive samples. If CCV is
present at a high level, the 136 bp band will be very strong and the 149 bp band may be missing. If no bands are present, the PCR
reaction did not work indicating a failure of one of the PCR components or an inhibitor in the sample. If the negative control
shows a 136 bp product, then there is a contaminant in the PCR set-up and the assay must be carried out again. If an aberrant
size band is produced, or to confirm that the PCR product is from CCV, the PCR can be undertaken again using no internal
standard and the product can be directly sequenced. The sequence can then be evaluated using BLAST on the National Center for
Biological Information internet site (http://www.ncbi.nlm.nih.gov/BLAST/) to identify sequences with high homology. The PCR
amplifies the region from 107827 to 107962 of the CCV genome (GenBank accession M75136) representing a portion of open
reading frame 73 (7). Expect at least 95% identity to this sequence.

319
320

TCA-TCC-GAA-TCC-GAC-AAC-TGA-CGC-GTC-GGT-AGC-CCG-ACC-GAT-CCG-TAT-GTT-ACG-GGTGCG-GGG-GTC-GAC-ACC-GTG-CTC-GCC-GCG-ATG-AGG-CTG-ACC-GCG-GAC-ACG-GGG-GGT-CCC-CCT-CGT-TTC-TCC-GCG-ATC-TTG-G

321

Figure 1. Sequence of CCV PCR product. Italic underlined portions indicate primer sequences.

322

4.3.1.2.3.2. Real-time PCR

323
324

A CCV-specific real-time PCR has been developed. This assay can be applied to cell culture isolated virus or for CCV diagnosis on
tissues of a suspect case.

325
326
327
328

DNA from trunk kidney or residual tissue from tissue homogenates for cell culture preparations can be isolated using a
commercial kit such as DNeasy Tissue Kit (Qiagen- Maryland, Delaware USA). DNA from virus produced in cell culture can be
prepared as stated above. To evaluate latent carriers, a biopsy can be taken from the caudal fin using a 6-mm diameter biopsy
punch and DNA extracted as stated above. When evaluating for latency at least five reactions should be run per fish.

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330
331
332
333
334
335
336
337
338

Real-time PCR can be performed in 25 l reactions containing 2 l DNA (1 g of high purity DNA for latency evaluation or 0.1 g for
diseased fish or cell culture extracts), 12.5 l of Platimum Quantitative PCR Super Mix-UDG (Invitrogen, Carlsbad, California USA),
200 nM of each primer and 200 nM of probe (5 FAM flourophore and 3 Black Hole Quencher from Biosearch Technologies,
Novato California, USA). Reactions are run for 2 minutes at 50C, 3 minutes at 95C followed by 40 cycles of 95C for 15 seconds,
64C for 1 minute, and 72C for 15 seconds in a iCycler iQ PCR Detection System (Bio-Rad, Hercules, CA). Positive results for cell
culture samples or diseased fish are indicated by a threshold cycle of less than that of 0.01 pg of purified CCV DNA. Positive
results when analysing for latent carriers are indicated by a threshold cycle of less than 38. All reactions should include negative
controls consisting of water for virus samples or negative tissue that was extracted at the same time for tissue samples. If an
unusual curve is produced, the product can be evaluated by electrophoresis on a 10% polyacrylamide gel and stained with
ethidium bromide. The CCV product will be a distinct 113 bp band.
Table 4.1. Primers and probe for CCV specific real-time PCR
Forward primer

CTC-CGA-GCG-ATG-ACA-CCA-C

Reverse

TGT-GTT-CAG-AGG-AGC-GTC-G

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Chapter 2.1.10. Channel catfish virus disease

Probe

CCC-ATC-CCT-TCC-CTC-CTC-CCT-G

339

4.3.1.2.3.3. Degenerate PCR and sequencing

340
341
342
343
344
345
346

The use of degenerate PCR followed by sequencing of a fragment of the DNA polymerase gene (11) is useful for evaluating virus
isolates that show unexpected host range, culture characteristics, antigen profile or PCR results. The concept is based on the
use of two regions in the DNA polymerase gene that are conserved. The upstream primer is 5-cgg-aat-tct-aGA-YTT-YGC-NWSNYT-NTA-YCC-3 with the upper case being specific for the DNA pol target (Y= C or T, N= ACG or T, W = C or G) and the lower
primer being 5-ccc-gaa-ttc-aga-tcT-CNG-TRT-CNC-CRT-A-3 (R = A or G). Furthermore, the use of DNase to remove host
sequences from virion pellets can be used before DNA purification to increase the effectiveness of the degenerate PCR. This
method is most effective on virus from cell cultures but it can also be used on productively infected tissues.

347

Method

348
349

i)

Disrupt the cells/tissue to release the virions. Virus from infected cell cultures in 25 cm2 flasks are released from the
cells by three serial freezethaw cycles or by sonication.

350

ii)

Pellet the cellular debris by centrifugation at 1000 g for 5 minutes.

351

iii)

Filter the supernatant through a 0.45 m filter.

352

iv)

Concentrate the virus from the filtrate by centrifugation at 21,000 g for 30 minutes.

353
354
355

v)

Resuspend the pellet in 80 l of water, add 20 l of 10 buffer and 100 l of RQ1 DNAse (Promega) and incubate at 37C
for 2 hours. This is followed by the addition of 20 l stop buffer (20 mM EGTA [ethylene glycol tetraacetic acid], pH 8.0)
and a 10-minute incubation step at 65C to inactivate the DNAse.

356

vi)

Extract the DNA using DNeasy Tissue Kit or Purgene genomic DNA isolation system (Qiagen- Maryland, Delaware USA).

357

vii)

Run degenerate PCR.

358
359
360
361
362
363

PCR consists of approximately 100 ng of template DNA, 20 pmole of each primer, 4 l 10 mM dNTP, 5 l 10 buffer, 2.5 U
Taq polymerase mix (Fisher Scientific) in 50 l reactions. The reaction conditions are: 93C, 1 minute for one cycle
followed by 93C, 30 seconds; 45C, 2 minutes; 72C 3 minutes for 35 cycles followed by a single cycle at 72C for 4
minutes. The product is evaluated by electrophoresis on 1.5% agarose gels followed by staining with ethidium bromide or a
similar stain and UV transillumination. Bands of interest are then excised from the gel and the DNA can be recovered using
GenElute Agarose Spin Columns (Supelco, Bellefonte, PA).

364
365
366

viii) Clone predominant bands of the appropriate sizes (450800 bp) using the TOPO TA cloning kit (Invitrogen) or a similar
rapid cloning system. Selected candidate clones are evaluated for a DNA insert of the appropriate size using colony PCR.
Then plasmid is purified for sequencing from 1 ml cultures using the QIAQUIK plasmid purification kit (Qiagen).

367
368

ix)

369
370
371

Variations that are helpful are (a) to use larger amounts of cells or tissues in samples that are suspected of having low numbers
of virions and (b) to run a negative control of non-infected tissue when multiple weak bands are produced to distinguish cellular
products from virus product candidates

Sequence the inserts of 34 plasmids for each band of interest and use BLASTx to compare the translated sequence with
deduced amino acid sequences in GenBank.

4.3.1.2.4. Agent purification

372

CCV produces high numbers of progeny in cell culture and the virions are easily purified for use as antigen. CCV virions can be
purified from infected cell cultures using sucrose (1) or ficoll gradient centrifugation (8). Basically, CCO cells in 1020 150 cm2
flasks are infected with 1 PFU/cell. When CPE involves all cells, the cells are dislodged from the flasks. The cells are pelleted by
centrifugation at 100 g for 5 minutes. The cell-free virus can be concentrated by centrifugation at 20,000 g for 1 hour onto a
40% sucrose cushion underlay. The cytoplasmic-associated virus can be obtained by suspending the cells in 3 ml of TNE (0.5 M
NaCl, 20 mM Tris/HCl pH 7.5, 2 mM EDTA [ethylene diamine tetra-acetic acid]), with 1% triton X-100 and briefly sonicating the
cells on ice using a probe sonicator. The cellular debris is then pelleted out by centrifugation at 5000 g for 10 minutes. The
supernatant can then be combined with the extracellular fraction and overlayed on to a 515% ficoll gradient in serum-free,
phenol-red-free medium in a thin-walled tube and centrifuged at 70,000 g for 1 hour. The band can be seen by overhead
illumination and harvested using a syringe. This band can then be diluted with three volumes of TNE and pelleted by centrifugation
at 20,000 g for 1 hour, or placed in dialysis tubing and dialysed in 1 litre of TNE.

373
374
375
376
377
378
379
380
381
382
383

4.3.2.

384
10

Serological methods
Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.1.10. Channel catfish virus disease

ELISA methods have been developed to detect catfish antibodies to CCV (6). The method uses whole purified virions at 10 g of virus
protein/ml in carbonated buffer (0.1 M Na2CO3, pH 9.6) at 100 l/well to coat polystyrene flat-bottomed ELISA plates. The plates are
incubated at room temperature over night in a humid chamber, washed five times with PBST and then blocked by the addition of 200
l of 10% non-fat dry milk in PBST for 1 hour. The plates are then washed with PBST, incubated for 1 hour with dilutions of the sera to
be tested, washed, and incubated with anti-fish-species immunoglobulin serum (MAb 9E1 can be used for channel catfish), washed, and
incubated with an antibody conjugate (either horseradish peroxidase or alkaline phosphatase) specific to the secondary antibody.
Then the plate is washed, the chromogenic enzyme substrate is added, the colour is allowed to develop and the plate is read. Each
assay must have a positive (serum from a fish with known antibodies to CCV) and a negative control (serum from a CCV nave fish).
Samples with a mean optical density that is 2 standard deviations above the mean of the negative control sample are considered
positive. Optimal working concentrations must first be determined for each reagent used in the test. This technique may be applicable
for screening populations of fish for previous exposure. However, the delay after exposure in the immune response, and seasonal and
genetic variability in immune responses makes these methods unreliable for inspection purposes. In CCV-endemic populations CCVspecific antibodies appear to be highest in the fall.

385
386
387
388
389
390
391
392
393
394
395
396
397
398

5.

Rating of tests against purpose of use

399
400
401
402
403
404

The methods currently available for targeted surveillance and diagnosis of CCVD are listed in Table 5.1. The designations used in the Table indicate:
a = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity; b = the method is a
standard method with good diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or other
factors severely limits its application; and d = the method is presently not recommended for this purpose. These are somewhat subjective as
suitability involves issues of reliability, sensitivity, specificity and utility. Not all of the tests listed as category a or b have undergone formal
standardisation and validation.

405

Table 5.1. Methods for targeted surveillance and diagnosis

Method

406
407

Targeted surveillance

Presumptive
diagnosis

Confirmatory
diagnosis

Clinical CCVD

Latent CCV

Gross signs

Histopathology

IFAT or immunostaining

Culture

PCR

Degenerate PCR sequencing

Real-time PCR*

Antibody-specific ELISA

IFAT = indirect fluorescent antibody test; *= relatively new test with limited testing for routine diagnostic work; PCR = polymerase chain reaction,
ELISA = enzyme-linked immunosorbent assay.

408

6.

409
410
411
412

Brood fish should be caudal fin biopsied and evaluated by PCR using at least five PCR reactions per fish. Offspring should be subsampled before 8
days of age and evaluated by PCR. This should be combined with diagnostic analysis that includes CCO cell culture of posterior kidney homogenate
at 2830C any time moribund fish are identified. Negative results for 2 consecutive years would indicate a free status and annual evaluation of
fry can be used to monitor this declaration.

413

7.

414

Test(s) recommended for targeted surveillance to declare freedom from channel catfish virus disease

Corroborative diagnostic criteria

7.1. Definition of suspect case

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11

Chapter 2.1.10. Channel catfish virus disease

415

CCVD will be suspected if any of the following occurs:

416

a.

Channel catfish are found displaying clinical signs of CCV at temperatures above 25C.

417

b.

Any population that has been diagnosed with CCVD in the past even if no clinical signs are present.

418

c.

Any population with detectable antibodies to CCV.

419

d.

Any population that produces CPE typical of CCV on CCO cells but not on BF2 or FHM cells.

420

7.2. Definition of confirmed case

421

CCVD will be confirmed if any of the following are found:

422
423

a.

Fish show clinical signs of disease and tissue smears contain IFAT or immunostaining positive staining of cells using CCV specific
antibodies or tissues test positive for CCV using CCV specific PCR or Real-time PCR.

424

b.

Virus is isolated by culture and it tests positive for CCV by antibody based detection or molecular methods.

425

8.

426
427

1.

428
429

2.

430

3.

BOYLE J. & BLACKWELL J. (1991). Use of polymerase chain reaction to detect latent channel catfish virus. Am. J. Vet. Res., 52, 19651968.

431
432

4.

BRADY Y.J. & ELLENDER R.D. (1982). The role of sediment in transmission of channel catfish virus disease. 1982, Mississippi Sea Grant
Consortium.

433
434

5.

COLYER T.E., BOWSER P.R., DOYLE J. & BOYLE J.A. (1986). Channel catfish virus: Use of nucleic acids in studying viral relationships. Am. J. Vet. Res.,
47, 20072011.

435
436

6.

CRAWFORD S.A., GARDNER I.A. & HEDRICK R.P. (1999). An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to channel catfish
virus (CCV) in channel catfish. J. Aquat. Anim. Health, 11, 148153.

437

7.

DAVISON A.J. (1992). Channel catfish virus: A new type of herpesvirus. Virology, 186, 914.

438
439

8.

DAVISON A.J. & DAVISON M.D. (1995). Identification of structural proteins of channel catfish virus by mass spectrometry. Virology, 206, 1035
1043.

440
441

9.

GRAY W.L., WILLIAMS R.J., JORDAN R.L. & GRIFFIN B.R. (1999). Detection of channel catfish virus DNA in latently infected catfish. J Gen Virol, 80,
18171822.

442
443

10.

HANSON L.A., RUDIS M.R. & PETRIE-HANSON L. (2004). Susceptibility of channel catfish fry to channel catfish virus (CCV) challenge increases with
age. Dis. Aquat. Org., 62, 2734.

444
445

11.

HANSON L.A., RUDIS M.R., VASQUEZ-LEE M. & MONTGOMERY R.D. (2006). A broadly applicable method to characterize large DNA viruses and
adenoviruses based on the DNA polymerase gene. Virol. J., 3, 28.

446
447

12.

448
449

13.

HEDRICK R.P., MCDOWELLL T.S., GILAD O., ADKISON M. & BOVO G. (2003). Systemic herpes-like virus in catfish Ictalurus melas (Italy) differs from
Ictalurid herpesvirus 1 (North America). Dis. Aquat. Org., 55, 8592.

450
451

14.

KANCHARLA S.R. & HANSON L.A. (1996). Production and shedding of channel catfish virus (CCV) and thymidine kinase negative CCV in immersion
exposed channel catfish fingerlings. Dis. Aquat. Org., 27, 2534.

12

References
ARKUSH K.D., MCNEILL C. & HEDRICK R.P. (1992). Production and characterization of monoclonal antibodies against channel catfish virus. J. Aquat.

Anim. Health, 4, 8189.

BAEK Y.-S. & BOYLE J.A. (1996). Detection of channel catfish virus in adult channel catfish by use of nested polymerase chain reaction. J. Aquat.

Anim. Health, 8, 97103.

HEDRICK R.P., GROFF J.M. & MCDOWELL T. (1987). Response of adult channel catfish to waterborne exposures of channel catfish virus. Prog. Fish

Cult., 49, 181187.

Manual of Diagnostic Tests for Aquatic Animals 2009

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NUSBAUM K.E., SMITH B.F., DEINNOCENTES P. & BIRD R.C. (2002). Protective immunity induced by DNA vaccination of channel catfish with early and
late transcripts of the channel catfish herpesvirus (IHV-1). Vet. Immunol. Immunopathol., 84, 151168.

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PLANT K.P., HARBOTTLE H. & THUNE R.L. (2005). Poly I:C induces an antiviral state against Ictalurid Herpesvirus 1 and Mx1 transcription in the
channel catfish (Ictalurus punctatus). Dev Comp Immunol, 29, 627635.

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17.

PLUMB J.A. (1973). Effects of temperature on mortality of fingerling channel catfish (Ictalurus punctatus) Experimentally infected with channel
catfish virus. J. Fish. Res. Bd Can., 30, 568570.

458

18.

PLUMB J.A. (1978). Epizootiology of channel catfish virus disease. Marine Fish. Rev., 3, 2629.

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460

19.

PLUMB J.A. & CHAPPELL J. (1978). Susceptibility of blue catfish to channel catfish virus. Proc. Ann. Conf. S.E. Assoc. Fish & WIldl. Agencies, 32,
680685.

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20.

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21.

PLUMB J.A., WRIGHT L.D. & JONES V.L. (1973). Survival of channel catfish virus in chilled, frozen, and decomposing channel catfish. Prog. Fish Cult.,
33, 170172.

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22.

ROBIN J. & RODRIGUE A. (1980). Resistance of herpes channel catfish virus (HCCV) to temperature, pH, salinity and ultraviolet irradiation. Rev.
Can. Biol., 39, 153156.

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23. SILVERSTEIN P.S., BOSWORTH B.G. & GAUNT P.S. (2008). Differential susceptibility of blue catfish, Ictalurus furcatus (Valenciennes), channel catfish,
I. punctatus (Rafinesque), and blue x channel catfish hybrids to channel catfish virus. J. Fish Dis., 31, 7779.

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THOMPSON D.J., KHOO L.H., WISE D.J. & HANSON L.A. (2005). Evaluation of channel catfish virus latency on fingerling production farms in
Mississippi. J. Aquat. Anim. Health, 17, 211215.

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VANDERHEIJDEN N., MARTIAL J.A. & HANSON L.A., (2001). Channel Catfish Virus Vaccine. United States Patent US 6,322,79312, p. 12.

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26.

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PLUMB J.A., GREEN O.L., SMITHERMAN R.O. & PARDUE G.B. (1975). Channel catfish virus experiments with different strains of channel catfish. Trans.

Am. Fish. Soc., 104, 140143.

WALCZAK E.M., NOGA E.J. & HARTMANN J.X. (1981). Properties of a vaccine for channel catfish virus disease and a method of administration.

Develop. Biol. Standard., 49, 419429.

ZHANG H.G. & HANSON L.A. (1995). Deletion of thymidine kinase gene attenuates channel catfish herpesvirus while maintaining infectivity.

Virology, 209, 658663.

476
477

*
* *

478
479

NB: There is an OIE Reference Laboratory for Channel catfish virus disease (see Table at the end of this Aquatic Manual or consult the OIE Web site
for the most up-to-date list: www.oie.int).

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13