JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1989, p. 1924-19290095-1137/89/091924-06$02.00/0Copyright ©D 1989, American Society for Microbiology

Use of a Group-Reactive and Other Monoclonal Antibodies in an

Enzyme Immunodot Assay for Identification and PresumptiveSerotyping of Aquatic Birnavirusest

PRUDENCE CASWELL-RENO, VIMOLMAS LIPIPUN,t PAUL W. RENO, AND BRUCE L. NICHOLSON*

Department of Microbiology, University of Maine, Orono, Maine 04469

Received 30 January 1989/Accepted 16 May 1989

A panel of monoclonal antibodies (MAbs) was prepared and used to develop an enzyme immunodot assay forthe rapid identification and presumptive serotyping of aquatic birnaviruses. Comparison of the reactionpatterns of these MAbs with representative virus isolates indicated that one MAb recognizes a serogroup-

reactive epitope and can therefore be used for identification of all serogroup A aquatic birnaviruses, thepredominant serotype worldwide. Other MAbs exhibited more restrictive specificities, permitting the presump-tive serotyping of viruses of the three recognized serotypes and the identification of some individual strains.This assay, in which MAbs are used, is more efficient in terms of time, cost, and ease of performance andprovides significant advantages in specificity and standardization compared with currently used tests.

The Birnaviridae are a family of viruses characterized bya bisegmented, double-stranded RNA genome and an unen-

veloped, single-layer, icosahedral capsid approximately 60nm in diameter (5). Members of the family Birnaviridaeinclude the aquatic birnaviruses such as infectious pancre-

atic necrosis virus (IPNV) and other related viruses from fishand shellfish, the avian infectious bursal disease virus, andthe insect Drosophila X virus. Aquatic birnaviruses are theetiological agents of diseases in a variety of economicallyimportant fish species used worldwide in aquaculture andfish farming, such as infectious pancreatic necrosis in salmo-nids and branchionephritis in eels (19, 27). Also, relatedaquatic birnaviruses have been isolated from a variety ofother apparently healthy fish species and marine molluscs (2,7, 8, 18, 20-22, 25, 26).

Previous serological studies of IPNV and other aquaticbirnaviruses have shown that most of these viruses are

antigenically related. On the basis of reciprocal neutraliza-tion titers with rabbit antisera, three major serotypes are

currently recognized (12, 16). However, Hill and Way (B. J.Hill and K. Way, Abstr. First Int. Conf. Eur. Assoc. FishPathol. 1983, p. 10) have presented evidence for six addi-tional serotypes, which, together with the original three,would constitute a major serotype (serogroup A). Four otherisolates have been identified that apparently represent a

minor, unrelated serogroup (serogroup B). The three well-defined serotypes of serogroup A originally were designatedSp, Ab, and VR299, but Hill and Way (Abstr. First Int.Conf. Eur. Assoc. Fish Pathol.) and other investigators (3)have designated this last serotype as West Buxton (WB).The six recently proposed serotypes have been tentativelydesignated Hecht (He), Tellina (Te), Jasper (Ja), Canada 1(Cl), Canada 2 (C2), and Canada 3 (C3). Virtually all aquaticbirnavirus isolates from the United States belong to the WBserotype, whereas four serotypes (Ja, Cl, C2, and C3) have

* Corresponding author.t Publication no. 1378 of the Maine Agriculture Experiment

Station.t Present address: Department of Microbiology, Faculty of Phar-

maceutical Sciences, Chulalongkorn University, Bangkok, Thai-land.

been reported to occur in Canada and four (Sp, Ab, He, andTe) are found in Europe. To date, most isolates from Asiaappear to belong to either the WB or Ab serotype.

Specific and accurate identification of these viruses isrequired for effective control and an understanding of theepidemiology of the diseases that they cause. Various rapidserological tests with polyclonal antisera have been devel-oped for the general identification of one or more aquaticbirnaviruses (4, 9, 13-15, 17, 23). Recently, monoclonalantibodies (MAbs) have been developed and used in theanalysis of birnavirus epitopes and the comparison of dif-ferent isolates and in enzyme-linked immunosorbent assays

(ELISAs) for the identification of some strains of IPNV (3,28). However, most of these rapid assays identify onlycertain strains of virus, and none have been shown to beuniversally applicable in identifying all aquatic birnavirusesfrom a variety of species and geographical areas. Also, theserotype of a birnavirus isolate usually is determined byvirus neutralization tests with polyclonal antisera. In thisreport, we describe the use of a group-reactive MAb in an

enzyme immunodot assay for general virus identification anda panel of MAbs with various specificities for presumptiveserotyping of the aquatic birnaviruses.

MATERIALS AND METHODS

Viruses. The viruses used are listed in Table 1. The typestrains of the WB, Ab, and Sp birnavirus serotypes, variousWB and Sp representative strains, and AS virus were fromstock preparations maintained in our laboratory for manyyears. The Cl, C2, C3, Ja, He, and Te type strains were

provided by B. Hill, MAFF, Fish Diseases Laboratory,Weymouth, England. Representative isolates of the Abserotype were obtained from J. L. Wu and Y. L. Hsu,Academia Sinica, and C. F. Lo, Fu-Jen University, Taipei,Republic of China.Each virus was cloned by terminal dilution or plaque

purification and subsequently propagated in CHSE-214 cells,an established cell line originated from Chinook salmonembryos (10). All viruses were purified as previously de-scribed (3). Briefly, virus-infected cells were removed fromcell culture supernatants by low-speed centrifugation, sus-

pended in TNE buffer (0.01 M Tris hydrochloride, 0.1 M

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MAbs FOR IDENTIFYING AQUATIC BIRNAVIRUSES 1925

TABLE 1. Aquatic birnaviruses

Virus

West Buxton (WB)(type strain)

Dry MillsGilbertVR299BerlinBuhlRenoTaiwanMenhadenStriped bass

Sp (type strain)Bonnamyd'HonninchtonOV-2OV-7

Ab (type strain)EEVElSPVCV-1CV-HB-1CV-TS-1

Hecht (type strain)

Tellina (type strain)

Canada 1 (type strain)AS

Canada 2 (type strain)

Canada 3 (type strain)

Jasper (type strain)

Countryof origina

USA

USAUSAUSAUSAUSAUSATaiwan, ROCUSAUSA

DenmarkFranceFranceEnglandEngland

DenmarkJapanTaiwan, ROCTaiwan, ROCTaiwan, ROCTaiwan, ROCTaiwan, ROC

FRG

England

CanadaCanada

Canada

Canada

Canada

Animalof origin

Trout

TroutTroutTroutTroutTroutTroutTroutMenhadenStriped bass

TroutTroutTroutOysterOyster

TroutEelEelPerchClamClamClam

Tellina

TroutAtlantic salmon

Trout

Arctic char

Trout

a Abbreviations: USA, United States of America; ROC, Republic of China;FRG, Federal Republic of Germany.

NaCI, 1 mM EDTA [pH 7.5]), and extracted with an equalvolume of Freon 113 (Du Pont Co., Wilmington, Del.) torecover cell-associated virus. This was combined with re-leased virus, which had been precipitated from the cell-freesupernatants with polyethylene glycol (molecular weight,20,000). Virus was then layered over a step gradient consist-ing of 20% (wt/wt) sucrose, 30% (wt/vol) CsCl, and 40%(wt/vol) CsCI and centrifuged for 2 h at 143,000 x g.Fractions containing virus then were banded a second timeby isopycnic centrifugation on a self-forming CsCl (initialdensity, 1.33 g/cm3) gradient for 16 h at 113,000 x g.Fractions containing virus were dialyzed in buffer, filteredthrough a filter membrane (pore size, 0.22 ,um), and storedfrozen in liquid nitrogen.

Production of MAbs. The production and characterizationof MAbs Wl to W5 against WB IPNV have been describedpreviously (3). MAbs El, E2, E3, E5, and E6 were preparedagainst European eel virus, a representative of the Abserotype, and MAb AS-1 was prepared against AS IPNV, arepresentative of the Cl serotype, by the procedures ofCaswell-Reno et al. (3). Briefly, 8-week-old BALB/c micewere injected intraperitoneally with purified virus (5 x 10850% tissue culture infective doses [TCID50]; approximately0.8 ,ug of protein) in complete Freund adjuvant and then 5

weeks later intraperitoneally and intravenously with boost-ing doses of purified virus (1 x 109 TCID50; approximately1.6 ,ug of protein each) without adjuvant. Spleen cells wereharvested 4 days later from mice with high antibody titersand fused with Sp2/0-Ag-14 mouse myeloma cells at a ratioof 10:1 by using polyethylene glycol. Each well of 96-wellplates was seeded with approximately 2.7 x 105 total cells in0.1 ml of HAT growth medium (GM-HAT) consisting ofDulbecco modified Eagle medium with D-glucose (4.5 mg/ml), NCTC 109 (10%, vol/vol), bovine pancreatic insulin (0.2,ugIml), oxalacetic acid (1 mM), pyruvic acid (0.45 mM), fetalbovine serum (15%, vol/vol), hypoxanthine (0.1 mM), ami-nopterin (4 x 10-4 mM), and thymidine (1.6 x 10-2 mM) andincubated at 37°C in a humidified 10% C02 atmosphere.Hybridomas producing virus-specific antibody were adaptedto HAT-free GM, expanded, cloned at least twice, andstored frozen in liquid nitrogen.Immunodot assay. The dot immunobinding assay was

adapted from the method of Hawkes et al. (6). All viruspreparations consisted of infected-cell culture supernatantsthat had been clarified by centrifugation at 1,000 rpm (100 xg) for 10 min. Aliquots of virus (2 ,ul) at a minimumconcentration of 107 TCID5d/ml were dotted onto nitrocellu-lose strips (Bio-Rad Laboratories, Richmond, Calif.) and airdried at room temperature. In some cases, the use of virusconcentrations up to 1010 TCID5dml was tested, with nodifference in reaction patterns observed in comparison withthose with the minimum concentration. After the strips hadbeen washed four times with Tris-buffered saline (TBS) (pH7.4), the remaining nonspecific protein-binding sites wereblocked with 3% (wt/vol) bovine serum albumin and 10%(vol/vol) calf serum. Individual strips were then incubatedfor 3 h at room temperature in each MAb (hybridoma cellculture supernatants) with titers two to four times theminimum previously determined to give positive results withhomologous virus. After being washed and subjected to theblocking procedure as above, the strips were incubated for 2h with goat anti-mouse immunoglobulin conjugated withhorseradish peroxidase, washed, and incubated for 15 min insubstrate (0.5 mg of 4-chloronaphthol and 10 pil of 3% H202in 1 ml of TBS). A positive reaction was indicated by avisible purple developing in 2 to 15 min. Occasionally, withsome isolates exhibiting weakly positive reactions with someMAbs, the contrast of the colored dot with the backgroundappeared less distinct after drying. Therefore, for maximumsensitivity in such cases, results were read immediately.To confirm the specificity of positive reactions, we used

two types of negative controls (data not shown). The antigencontrols were negative reactions with unrelated antigensapplied as dots to the strips. These included (i) uninfectedCHSE-214 cell culture supernatant; (ii) equivalent concen-trations of infectious hematopoietic necrosis virus, a fishrhabdovirus; and (iii) equivalent concentrations of infectiousbursal disease virus, an unrelated birnavirus. In addition, thespecificity of these MAbs for aquatic birnaviruses previouslyhad been demonstrated by the lack of reaction in an ELISAwith equivalent amounts of a purified double-stranded RNAmycovirus (Rhs 717) from Rhizoctonia solani (24) and puri-fied Rhs 717 mycovirus double-stranded RNA. The antibodycontrol was the lack of reaction with virus dots of a knownbirnavirus-negative MAb (EO), used at dilutions comparableto those of birnavirus-specific MAbs.

RESULTSA set of five aquatic birnavirus MAbs (Wl to W5) with

different epitope specificities have been developed and char-

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1926 CASWELL-RENO ET AL.

.MAbE5 E2 E3 EO

m-- m-

FIG. 1. Typical reactions of representative aquatic birnaviruseswith several MAbs in the immunodot assay. Abbreviations: EEV,European eel virus; Ab, Ab IPNV; Sp, Sp-IPNV; UM, culturesupernatants from uninfected cell monolayer; E5, E2, and E3,birnavirus-specific MAbs; EO, birnavirus-negative hybridoma cellculture supernatant.

acterized previously (3). To enlarge the available library ofsuch antibodies, we developed additional MAbs in thisstudy. Six of these new MAbs were selected for use indeveloping enzyme immunoassays. All six new MAbs wereimmunoglobulin G (IgG) with K light chains and representedthe following isotypes: El, IgG2a; E2, IgG2a; E3, IgG2b;E5, IgG1; E6, IgGl; and AS-1, IgGl. Also, all the new MAbswere shown to recognize different epitopes by differences inreaction patterns with various isolates and/or radioimmuneprecipitation of individual viral proteins (V. Lipipun, Ph.D.thesis, University of Maine, Orono, 1988). A simple immu-nodot test involving the use of the five previously availableand six new MAbs was developed for the rapid identificationof aquatic birnaviruses from infected-cell culture superna-tants. The test protocol was designed by determining theoptimal concentrations of reagents and reaction times foreach homologous virus-MAb pair. Preliminary studies of thesensitivity of this assay with a more limited selection ofMAbs and viruses demonstrated that supernatants frominfected-cell cultures exhibiting extensive cytopathic effectand a virus titer of 2104 TCID5dptl gave clearly visiblepositive results (11). The appearance of a typical assay isshown in Fig. 1. In this case three related birnaviruses were

distinguished by different reaction patterns with threeMAbs.

This immunodot assay was used to compare the reactivityof the panel of 11 MAbs with the type strains and a varietyof other representative isolates of the three widely recog-nized aquatic birnavirus serotypes (WB, Sp, and Ab). Theresults are shown in Table 2. Several MAbs had a wide rangeof cross-reactivity, whereas others reacted only with certainserotypes or isolates. Epitopes recognized by MAbs AS-1,El, and E5 appear to be highly conserved among these

viruses; these epitopes were present on all birnavirusestested from the three established serotypes, including iso-lates from North America, Europe, and Asia. Other MAbsexhibited more specific patterns of cross-reactions. Severalreacted only with representatives of a single serotype; MAbsW4 and W5 reacted with all serotype WB viruses, and MAbE2 reacted with all serotype Ab viruses. Several MAbsreacted with some or all isolates from two of the threeserotypes. MAb E3 reacted with all isolates of serotype Abexcept Ab virus itself, the type strain for this serotype. All ofthe Sp serotype viruses exhibited identical reaction patterns,whereas at least two distinct strains were apparently repre-sented within each of the WB and Ab serotypes. Overall, theserotype of the viruses tested belonging to the three estab-lished serotypes (WB, Sp, and Ab) of serogroup A could bedetermined presumptively by the distinctive reaction pat-terns of these viruses with the entire panel of MAbs. Asummary scheme for presumptive serotyping of these vi-ruses is illustrated in Fig. 2A.A variety of isolates representing the six newly proposed

aquatic birnavirus serotypes (Hill and Way, Abstr. First Int.Conf. Eur. Assoc. Fish Pathol.) were not available; how-ever, the type strains of each of these serotypes as well as

one other Cl serotype virus (AS virus) were tested forreactivity with the panel of MAbs (Table 2). As with thethree well-established serotypes, MAbs AS-1 and E5 reactedwith all six serotypes, whereas other MAbs discriminatedamong viruses. Interestingly, MAb El, which reacted withall representatives ofthe three recognized serotypes, reactedwith only three of the newly proposed serotypes. The typestrains of four (Ja, Te, He, and Cl) of these proposedserotypes not only could be distinguished from each otherand from the other two by their unique patterns of MAbcross-reactivity but also were different from the three origi-nal serotypes. Of these, only the serotype pair of C2 and C3could not be distinguished from each other in this assay. Thereaction patterns of these six serotypes are summarized inFig. 2B as a presumptive serotyping scheme.

DISCUSSION

The results presented in this report describe a simpleimmunodot assay in which a panel of MAbs is used for therapid identification of aquatic birnaviruses from infected-cellculture supernatants. MAb AS-1 reacted with all isolatesfrom serogroup A tested in this study, indicating that thisMAb recognizes a group-reactive epitope. This is supportedby results of preliminary investigations showing that thisMAb also reacts with all viruses from a diverse group ofuntyped birnaviruses recently isolated from a variety of fishand mollusc species in Asia (Lipipun, Ph.D. thesis). There-fore, general identification of serogroup A aquatic birnavi-ruses can be made in a few hours by using this assay withMAb AS-1. This is particularly significant, since all but 4 ofalmost 200 aquatic birnavirus isolates worldwide belong toserogroup A (Hill and Way, Abstr. First Int. Conf. Eur.Assoc. Fish Pathol.). Previously developed rapid identifica-tion tests used antibodies that were applicable only tospecific serotypes or even individual strains (3, 9, 13, 14, 17,23, 28). Use of MAb AS-1 would probably make these otherassays more universally applicable. MAb E5 also reactedwith all virus isolates tested in this study; however, it doesnot react with two recently isolated, uncharacterized aquaticbirnaviruses from Asia (Lipipun, Ph.D. thesis). That thesetwo new isolates are serogroup A aquatic birnaviruses isindicated by their reaction with MAb AS-1. Therefore, MAb

EEV

EEV

Ab

AbVIRUS

Sp

Sp

UM

UM

e,-

-M. M- M a-

..j_

m.m

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TABLE 2. Reaction of MAbs with representative aquatic birnaviruses

Reaction of following MAba:Virus

AS-1 Wl W2 W3 W4 W5 El E2 E3 E5 E6

Established serotypesWest Buxton (WB)WB (type strain)Dry MillsTaiwanGilbertVR 299BerlinMenhadenStriped bassBuhlReno

SpSp (type strain)OV-2OV-7Bonnamyd'Honninchton

+ + + + + + + - - + ++ + + + + + + - - + ++ + + + + + + - - + ++ + + + + + + - _ + ++ - - + + + + - - + +

+ - - + + + + - - + +

+ - - + + + + - - + +

+ - - + + + + - - + +

+ - - + + + + - - + +

+ - -+ + + + - - + +

+ _ _ + _ _ + __+_

+ - -+ - _+ __+_

+ - - +

+ ++ - - +

_- + _ _ +_

_- + _ +_

_- + _ _ +_

AbAb (type strain)EEVElSCV-1CV-HB-1CV-TS-1

Proposed serotypesHecht (He)He (type strain)

Tellina (Te)Te (type strain)

Canada 1 (Cl)Cl (type strain)AS

Canada 2 (C2)C2 (type strain)

Canada 3 (C3)C3 (type strain)

Jasper (Ja)Ja (type strain)

+ + + - - - + + - + +

+ + + _ _ _ ~~~++ + + +

+ + + - - - + + + + ++ + + - - - + + + + +

+ + + - - - + + + + ++ + + - - - + + + + +

+_ ____ +_

+ + -- - - + + - + +

+:

+

_ _ + + - + +

_- + + - + +

-- + +

_- + +

+ + + + + + _ _ + _

a Results from at least three experiments. Symbols: +, positive reaction; ±, weakly positive reaction; -, no reaction.

E5, although not universally applicable, may also be usefulin general screening assays, particularly for virus isolatesfrom North America and Europe.

Furthermore, this assay can be used for presumptiveserotyping of members of the three established serotypes(WB, Sp, and Ab) of birnavirus serogroup A by their uniquepatterns of reaction with the entire MAb panel (Fig. 2A).Currently, these viruses are usually serotyped by performingneutralization tests of each isolate with polyclonal antiseraand reference viruses for each serotype. The results alsoindicate that this panel of MAbs may be useful in thepresumptive serotyping of isolates form the six newly pro-

posed serotypes. The type strains of four (JA, Te, He, andCl) of these new serotypes could be distinguished from eachother as well as from the other two (C2 and C3) and the threeoriginal, well-defined serotypes. The type strains of sero-types C2 and C3 reacted with only three MAbs and could notbe distinguished from each other with these MAbs; however,the reaction patterns of these two viruses were distinct fromthose of each of the other seven serotypes. The development

of additional MAbs specific for one or both of these virusesshould enable the specific identification of these two sero-types. Whether the patterns of cross-reactions of these typestrains are representative of the serotype must be deter-mined by comparison with other representative isolates, aswas done with WB, Sp, and Ab.

Current general screening assays for identifying fish virusisolates most often use virus neutralization tests with un-standardized polyclonal antisera, although the use of ELISAand other rapid assays is becoming more widespread. Thisimmunodot assay with MAbs provides distinct advantagesover these other assays in ease of performance, cost, and/orstandardization. Both the assay system itself and the use ofMAbs in place of polyclonal antisera contribute to theseadvantages. For instance, the use of a standardized amountof a broadly reacting MAb such as AS-1 will identify allknown serotypes of the major aquatic birnavirus serogroupmore effectively than will the use of unstandardized poly-clonal antibody mixtures which exhibit different reactivitiesfor viruses of different serotypes. In serotyping viruses, the

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1928 CASWELL-RENO ET AL.

A

VirusSerotype

WB

SP

AB

Monoclonal Antlibody (MAb)

I W2l E E E E

B

HE

C2 * _

FIG. 2. Scheme for presumptive serotype identification ofaquatic birnaviruses in the immunodot assay with MAbs. (A)Recognized serotypes. Reaction patterns based on tests of repre-sentatives of each serotype previously serotyped by reciprocal virusneutralization tests with polyclonal antisera. Symbols: *, alwayspositive; O, always negative; ÉD, variable reaction with differentviruses. (B) Proposed new serotypes. The reaction pattern is basedon a test with type strains only.

use of a panel of MAbs offers obvious advantages in speci-ficity over the use of polyclonal antisera. Also, the immun-odot assay described in this report has distinct advantagesover the available MAb-based ELISA (3) for identifyingaquatic birnaviruses. For maximum sensitivity and speci-ficity, the ELISA uses either polyclonal antisera as an initialcapture antibody system to remove virus from infected-cellculture supernatants or purified virus bound directly to thesolid phase. This immunodot assay, however, is performedby using clarified supernatants taken directly from infected-cell cultures. The two assays have comparable levels ofsensitivity.

In addition to identification and presumptive serotyping,these MAbs provide some interesting information about theantigenic characteristics of these aquatic birnaviruses. Theviruses tested within each of the three recognized serotypes(WB, Sp, and Ab) were relatively homogeneous in antigeniccharacteristics. All representatives of the Sp serotype exhib-ited identical patterns of reaction with these MAbs. Twodistinct sets of WB serotype isolates were apparent fromtheir reaction or lack of reaction with two MAbs (Wl andW2). The Ab serotype viruses also showed two reactionpatterns. A previous report (11) demonstrated that the Abtype strain differed from at least one other Ab serotype virus(European eel virus) by its failure to bind MAb E3, as is alsoshown here. In the present study, failure to react with thissingle MAb was the only feature distinguishing the Ab typestrain virus from European eel virus, even with this largerpanel of MAbs. Interestingly, all of the other Ab serotypeviruses tested were identical to European eel virus. It shouldbe noted that MAb W2 exhibited a positive reaction with Abvirus in this immunodot assay, although in a previous studyit failed to bind to this virus in an ELISA (3). The exactreason for this apparent anomaly is not clear; however, it iswell known that the manner in which epitopes are presented

(e.g., attachment of antigen to polystyrene versus nitrocel-lulose substrates) can markedly affect the binding of certainMAbs. Such conformational changes in a virus when boundto plastic have been demonstrated for tobacco mosaic virus(1). Moreover, it has been shown that certain aquatic birna-virus MAbs which gave marginal or equivocal results in anELISA when the virus was adsorbed directly to plasticreacted much more strongly when the virus was attached byan intermediary layer of capture antibody (3).The MAb reaction pattern of Jasper virus, the type strain

for the proposed Ja serotype, was particularly interesting.This virus appeared to be closely related antigenically toviruses of the WB serotype with this panel of MAbs. Indeed,Jasper virus could be distinguished from four of the WBserotype viruses only by its failure to react with MAb E6. Itshould be noted that although most of the other recognizedand proposed serotypes are represented by numerous iso-lates and/or strains from diverse host species and geograph-ical areas, the proposed Ja serotype is represented only bythis single isolate. Perhaps, therefore, the antigenic charac-teristics of this virus and the establishment of a separate Jaserotype also should be investigated in more detail.Also interesting was the fact that several pairs of viruses

isolated from different species and widely separated geo-graphical areas were distinguished only by the presence orabsence of one or two epitopes defined by these MAbs. Forexample, Canada 1 virus from salmonid fish in Canada wasdistinguished from Tellina virus from a bivalve mollusc inEurope only by the failure of the former to bind MAb Wl.Therefore, these MAbs also may be useful probes forinvestigating the function(s) of individual epitopes in virusinfectivity, host specificity, and epidemiology.

In summary, we report the development of a panel ofMAbs, including one recognizing a group-reactive epitope,and the use of these MAbs in a simple and rapid immunodotassay for the general identification and presumptive serotyp-ing of aquatic birnaviruses. The assay requires about 2 h ofhands-on time, and results can be obtained without special-ized equipment in approximately 6 h. The specificity andavailability of these MAbs, combined with the simplicity ofthe immunodot test, should make this an important assaysystem in aquatic animal disease diagnosis and research.

ACKNOWLEDGMENTS

This research was supported by grants from the National Oceanicand Atmospheric Administration University of Maine Sea GrantCollege Program (no. R/FMD-177), the National Science Founda-tion (no. INT-810447), the Maine Agriculture Experiment Station,and the University of Maine Center for Marine Studies.The technical assistance of C. Turner in developing MAb AS-1 is

greatly appreciated.

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