Development of Monoclonal Antibodies Against Channel Catfish Virus, the Causative Agent of Channel Catfish Virus Disease
IAAAM 1991
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, 1980).

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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Speaker Information
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Kristen D. Arkush
University of California, Bodega Marine Laboratory
Bodega Bay, CA, USA

Ronald P. Hedrick, PhD
Department of Medicine and Epidemiology
School of Veterinary Medicine, University of California, Davis
Davis, CA, USA

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