MFR PAPER 1339

Phosphonoacetic Acid Inhibition ofChannel Catfish Virus Replication

Roger W. Koment is with the De­partment of Dermatology, Universityof Miami School of Medicine, Miami,FL 33152.

ROGER W. KOMENT

The virus responsible for epizooticoutbreaks of channel catfish diseasewas first isolated by Fijan et al. (1970)in cell cultures derived from ictaluridfish. Detailed characterization of thisvirus by Wolf and Darlington (197 [)clearly indicated the assignment ofchannel catfish virus (CCY) to the her­pesvirus group of DNA viruses. Mem­bers of this diverse family of viruses arefound throughout the phylogeny of thespecies, although presently only threerepresentatives have been associatedwith teleost fish (Wo[f, 1973; Wolf etaJ., 1976). The extent of parasitismshown by the herpesviruses ranges frompersistent subclinical infections to la­tent virus infections (Rapp and Jerkof­sky, 1973) to neoplastic changes result­ing in malignancy (Biggs et al., 1972).Although several herpesviruses arecapable of these multiple modes ofvirus-host interaction, the herpesvirusof channel catfish disease is knownprimarily for its direct, devastating ef­fects upon young populations of aqua­cultured catfish. The destruction of in­fected stock and decontamination of allfacilities is currently the only meansavailable to ensure complete eradi­cation of channel catfish disease out­breaks (Plumb, 1972).

Few viruses to date, regardless oftheir taxonomic classification or that oftheir host, will respond to conventionalchemotherapy. The herpesviruses areno exception. Recently, however, achemical derivative of the simplemolecule acetic acid has been shown to

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be effective in selectively inhibiting thereplication in tissue culture of represen­tative herpesviruses (Barahona et aJ.,1977; Duff and Overby, 1975; Huang,1975; Lee et aI., 1976; May et aI.,1977; Overby et aI., 1974; Summersand Klein, [976; Yajima et aI., [976).This compound, termed phosphonoace­tic acid (PAA), functions specificallyby inhibiting the herpesvirus codedDNA dependent DNA polymerase in itsprocess of replicating virus DNA (Maoand Robishaw, [975). In tissue culturesystems, PAA will not significantly in­hibit cell polymerase acti vity, represen­tatives of single-stranded RNA virusgroups, or other double-stranded DNAvirus groups (Duff and Overby, 1975;Leinbach et al., 1976; Mao and Robi­shaw, 1975). Vaccinia virus, a memberof the poxvirus group, was apparentlyan exception to this rule (Duff andOverby, 1975). In all cases, the effec­tive tissue culture dose of PAA whichinhibited herpesvirus replication hasbeen 100 j.tg PAA/ml or less.

On the basis of this information, itwas of interest to determine if CCYwould show similar sensitivity to PAA.Increasing concentrations of PAA wereprepared in overlay medium, and thesewere added to brown bullhead catfish(BB) cell cultures infected with aknown quantity of CCY. In all experi­ments, the results indicated that greaterthan 95 percent inhibition of CCY rep­lication was obtained in the presence of1,000 J-tg PANml. Viable cell countsas determined by trypan blue dye exclu-

sion at the beginning and end (72 hourspostinoculation) of each experiment in­dicated there was no cell death due todrug toxicity.

Dose dependency experiments indi­cated a direct relationship between thenumber of infectious virus particles in­fecting one cell and the amount of PAArequired to inhibit virus replication.This concentration ranged from greaterthan 500 J-tg PAA/ml for cultures withan input multiplicity of 0.0 [ plaqueforming unit per cell to 2,000 J-tgPAA/ml for cultures with an input mul­tiplicity of 6.0 plaque forming units percell. The toxicity level of PAA in BBcells was evident at 3,000 j.tg PAA/mlof culture medium.

These results indicate that PAA is aneffective and selective agent in the in­hibition of channel catfish virus replica­tion. The fact that 10 times the amountof drug required for other herpesvirussystems is necessary to inhibit CCYshould not be surprising. To date, allherpesvirus-PAA systems reportedhave been of homeothermic mammalsand birds. This report is the first concern­ing a herpesvirus of a poikilothermicspecies. One major implication of thiswork, therefore, is that the action ofPAA may be dependent upon eithertemperature or, relatedly, the physiol­ogy and metabolic rate of the host cell.This is not unusual, as the mode ofaction of this drug is interference withthe enzymes of viral DNA replication.Rate kinetics of the enzyme-substratereaction are directly related to tempera­ture (Lehninger, 1970), and tempera­ture as such a catalytic mechanism hasbeen shown to regulate many life func­tions in poikilothermic species (Swan,1974).

Direct application of such tissue cul­ture data has been investigated for PAAand two human viruses-herpessimplex virus type I (HSY-I) andherpes simplex virus type 2 (HSY-2)

Marine Fisheries Review

(Shipkowitz et aI., 1973). WhenHS Y-2 is inoculated into the denudedskin on the backs of CF strain mice,observable virus lesions are evident in3-5 days. In 11-15 days, virus­inoculated mice develop a flaccid,posterior paralysis resulting in deathwithin 24 hours. Topical application ofPAA in ointment or aqueous fom1 in aminimum concentration of 0.5 percentsignificantly reduced virus-inducedmortality. Equally efficacious, oraladministration of PAA at a minimumdosage of 800 mg/kg/day for 6 daysresulted in 100 percent reduction ofvirus-induced mortality. Using HSY-Iin a rabbit keratitis model system, thesesame authors demonstrated that topicalapplication of 0.5 percent PAA into in­fected eyes reduced virus-inducedcorneal lesions from 5 to 9 days afterinfection (Shipkowitz et aI., 1973).

PAA has been demonstrated to selec­tively inhibit the replication in tissueculture of many different herpesvirusesof homeothermic hosts. In two reportedinstances, this compound has provedeffective against herpesvirus-inducedmorbidity and mortality in animalmodel systems. Results herein re­ported indicate that the replication of apoikilothermic herpesvirus, CCY, canalso be inhibited in tissue culture sys­tems. Logic dictates that oral prepara­tions of PAA in catfish feed next betested to determine if this will sig­nificantly reduce the incidence of mor-

tali ties resulting from experimental in­fection of fry with CCY. If successful,these data would suggest that PAA maybe efficacious as a chemotherapeuticagent in the control of channel catfishvirus disease.

LITERATURE CITED

Barahona, H., M. D. Daniel,J. G. Bekesi, C E.O. Fraser, N. W. King, R. D. Hunt, J. K.Ingalls, and T. C Jones. 1977. In vitro sup­pression of HeqJesvirus sairniri replication byphosphonoacetic acid. Proc. Soc. Exp. BioI.Med. 154:431-434.

Biggs, P. M., G. de-The, and L. N. Payne(editors). 1972. Oncogenesis and herpesvirus­es. IARC Sci. Publ. 2, 515 p.

Duff, R. G., and L. R. Overby. 1975. Specificityof phosphonoacetic acid for inhibition of her­pesviruses. Abstr. Annu. Meet. Am. Soc. Mi­crobiol., p. 240.

Fijan, N. N.. T. L. Wellborn, Jr., and J. P.Naftel. 1970. An acute viral disease of channelcatfish. U.S. Bur. Sport. Fish. Wildl., Tech.Pap. 43, II p.

Huang, E.-S. 1975. Human cytomegalovirus.IV. Specific inhibition of virus-induced DNApolymerase activity and viral ON A replicationby phosphonoacetic acid. J. Virol. 16: 1560­1565.

Lee, L. F., K. Nazerian. S. S. Leinbach, J. M.Reno, and J. A. Boezi. 1976. Effect of phos­phonoacetate on Marek's disease virus replica­tion. J. Natl. Cancer Inst. 56:823-827.

Lehninger, A. L. 1970. Biochemistry. WorthPublishers, Inc .. N.Y., 833 p.

Leinbach, S. S .• J. M. Reno, L. F. Lee. A. F.Isbell, and J. A. Boezi. 1976. Mechanism ofphosphonoacetate inhibition of herpesvirus­induced DNA polymerase. Biochemistry15:426-430.

Mao, J. C.-H., and E. E. Robishaw. 1975. Modeof inhibition of herpes simplex virus DNA

polymerase by phosphonoacetale. Biochemis­try 14:5475-5479

May, D. C, R. L. Miller. and F. Rapp. 1977.The effect of phosphonoacetic acid on the invitro replicalion of varicella-zoster virus. In­tervirology 8:83-91.

Overby, L. R .. E. E. Robishaw,J. B. Schleicher,A. Rueter, N. L. Shipkowitz, and J. C.-H.Mao. 1974. Inhibition of herpes simplex virusreplication by phosphonoacetic acid. Antimic­rob. Agents Chemother. 6:360-365.

Plumb, 1. A. 1972. Channel catfish virus diseasein southern Uniled Slales. Proc. Twenty-fifthAnnu. ConI'. Southeast. Assoc. Game FishComm., p. 489-493.

Rapp, F .. and M. A. Jerkofsky. 1973. Persislenland latenl infections. In A. S. Kaplan (editor),The herpesviruses, p. 271-289. AcademicPress, N. Y.

ShipkowilZ, N. L., R. R. Bo,,\:I' , R. N. Appell.C. W. Nordeen, L. R. Overby, W. R.Roderick, 1. B. Schleicher, and A. M. VonEseh. 1973. Suppression of herpes simplexvirus infection by phosphonoacelic acid. Appl.Microbiol. 26:264-267.

Summers, W. C, and G. Klein. 1976. Inhibitionof Epstein-Barr virus DNA synthesis and lategene expression by phosphonoacetic acid. J.Viral. 18:151-155.

Swan, H. 1974. Thermoregulalion and bioener­getics. Palterns for vertebrate survival. Ameri­can Elsevier Publ. Co., Inc. N.Y., 430 p.

Wolf, K. 1973. Herpesvirus of lower vertebrates.In A. S. Kaplan (editor), The herpesviruses, p.495-520. Academic Press, N.Y.

____, and R. W. Darlington. 1971. Chan­nel catfish virus: a new herpesvirus of ictaluridfish. 1. Virol. 8:525-533.

____, , T. Nagabayashi, andM. C. Quimby. 1976. Herpesvirus salrnonis:characterization of a new pathogen from rain­bow troul (Sa/rno gairdneri). Abstr. Annu.Meet. Am. Soc. Microbiol., p. 235.

Yajima, Y., A. Tanaka, and M. Nonoyama.1976. Inhibition of productive replication ofEpstein-Barr virus DNA by phosphonoaceticacid. Virology 71 :352-354.

October /978

MFR Paper 1339. From Marine Fisheries Review, Vol. 40, No. 10, October1978. Copies of this paper, in limited numbers, are available from 0822, UserServices Branch, Environmental Information Genter, NOAA, Rockville, MD20852. Copies of Marine Fisheries Review are available from the Superinten­dent of Documents, U.S. Government Printing Office, Washington, DC 20402for $1. 10 each.

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