Development of Monoclonal Antibodies Against Channel Catfish Virus, the Causative Agent of Channel Catfish Virus Disease
Kristen D. Arkush; Ronald P. Hedrick, PhD
Aquaculture and Fisheries Program, Department of Medicine, School of
Veterinary Medicine, University of California, Davis, CA
Channel catfish virus (CCV), a herpesvirus, is the causative agent of an
acute, hemorrhagic, and lethal infection occurring in populations of young channel catfish,
Ictalurus punctatus, (Fijan et al., 1970). The disease was first described by Fijan (1968), and
epizootics have been reported principally in the southeastern USA (Plumb, 1977) but also in
California (Amend and McDowell, 1983).
The virus can be recovered from fish showing signs of channel catfish virus
disease (CCVD) and replicates optimally in cell lines derived from ictalurid fish (Wolf and
Darlington, 1971; Plumb, 1971). Shortly after fish recover from natural and experimental
infections, the virus is rarely isolated from the tissues (Plumb and Gaines, 1975; Plumb,
1977). In one report, however, CCV was isolated from adult channel catfish broodfish which
appeared robust and healthy (Bowser et al., 1985). Demonstrations of prior exposure to CCV or
potential latent infections have been attempted by demonstrating the presence of serum anti-CCV
antibodies or by detection of viral antigens in fish tissues. Plumb (1973) first identified
anti-neutralizing activity in the sera of fish previously infected with CCV, and passive
transfer of sera with this activity was shown to protect juvenile channel catfish from lethal
challenges of CCV (Hedrick and McDowell, 1987). Various investigators have attempted to detect
viral antigen both in actively and potentially latently infected fish. Using the fluorescent
antibody technique, Plumb et al. (1981) reported detection of viral antigens in the ovary of
adult catfish. Wise and Boyle (1985) constructed a nucleic acid probe for CCV using recombinant
DNA techniques and reported detection of viral genome in normal appearing but experimentally
infected yearling catfish.
The use of hybridoma cultures to produce Mabs as reagents in the detection
of various pathogens has become a desirable technique since they have the advantage of being
available in an unlimited homogeneous supply (Woodhead et al., 1988). In particular, Mabs have
been developed against many fish viruses, such as infectious hematopoietic necrosis virus
(Schultz et al., 1985; Winton et al., 1988), infectious pancreatic necrosis virus (Dominguez et
al., 1990), and Egtved virus, the causative agent of viral hemorrhagic septicemia (Lorenzen et
al., 1988). This presentation describes the development of the first Mabs against CCV and some
of their preliminary applications.
CCV isolated from juvenile channel catfish suffering an epizootic of CCVD
in the Coachella Valley, California was propagated in the channel catfish ovary (CCO) cell
line, purified and used to immunize Balb/c mice. Hybridoma cultures were produced by a
modification of the methods described by Mishell and Shiigi (1980). Both polyclonal mouse sera
(prior to the fusion) and hybridoma culture supernatants were screened for the presence of
anti-CCV specific antibody. Serum from non-immunized mice and phosphate buffered saline (PBS),
pH 8.0, respectively, served as negative controls for polyclonal sera and hybridoma supernatant
screening. An indirect fluorescent antibody test (IFAT) incorporating fixed, CCV-infected CCO
cells was used to detect anti-viral antibody, and micro-neutralization assays were performed to
detect neutralizing antibody. Seven clones were selected which produced anti-CCV antibody, and
4 of these were able to neutralize virus. The selected clones were expanded and later injected
into mice for ascites production.
These Mabs were used to detect differences between the homologous CCV
isolate (from California) and CCV isolated from channel catfish in Alabama, Louisiana, and
Oklahoma by IFAT and neutralization tests. By IFAT, all Mabs reacted with the 4 CCV isolates
tested. In each case, anti-viral staining was punctate and cytoplasmic to perinuclear. None of
the Mabs reacted with uninfected CCO cells or with another cell line, rainbow trout gonad
(RTG-2), infected with two other herpesviruses, Oncorhynchus masou virus (OMV), or
Herpesvirus salmonis. This specific staining suggests that the Mabs could be directed to
CCV-coded antigens (e.g. glycoproteins) that accumulate on the host membranes and are later
incorporated into the viral envelope as particles bud through the nuclear envelope.
All 4 neutralizing Mabs prevented the onset of cytopathic effect for the 4
CCV isolates tested. Neutralization indices (NI), defined as the difference in log10
(TCID50 titer) between virus incubated with tissue culture media alone and those
incubated with ascites containing Mab, were similar for the CCV isolates from California,
Louisiana, and Oklahoma. Two of the 4 neutralizing Mabs were less effective at reducing the
titer of the Alabama CCV isolate, suggesting that this isolate may possess type-specific
antigenic determinants not recognized by the neutralizing Mabs. Similar findings have been
reported in neutralization tests comparing strains of herpes simplex virus (HSV) types 1 and 2
(Norrild, 1980). Antisera prepared to glycoproteins gC, gD, gE, and the mixture of gA and gB
were capable of neutralizing the infectivity of homologous virus, but the specificities of the
sera as determined by their reactivity with heterologous virus varied (Powell et al., 1974).
Results suggest that, for HSV, type-specific antigenic determinant sites are intermixed with
type-common sites in the glycoproteins projected at the surface of the virion (Norrild,
Those Mabs which demonstrated binding affinity for the CCV isolates tested
yet were unable to neutralize virus may be directed against glycoproteins which are not
responsible for virus infectivity, as is the case with the gC protein of HSV (Spear, 1984).
Alternatively, these binding Mabs may require the presence of complement to neutralize virus.
Further tests including complement in the neutralization assays will be performed in our
laboratory, as well as experiments to identify the epitopes recognized by these anti-CCV Mabs.
Clearly, though, the development of such Mabs makes possible the use of unique detection
reagents which can be produced in uniform and virtually unlimited quantities.
Current standard methods for the isolation and identification of fish
viruses require the use of established fish cell lines and serum neutralization tests (Amos,
1985). Sources of antibody specific for a particular virus other than hybridoma cultures are
host (fish) and non-host (rabbit, goat) derived polyclonal sera, which often have low
neutralization titers and relatively high cytotoxicity (Winton et al., 1988). All of the 7 Mabs
developed in our laboratory show good binding affinity for CCV, and thus would be appropriate
reagents for detection and quantification tests such as ELISA and IFAT techniques.
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