Journal of General Virology (1990), 71, 2593-2598. Printed in Great Britain
2593
Monoclonal antibodies to rabbit haemorrhagic disease virus and their use in the diagnosis of infection L. Rodfik,* M. Granhtovfi, L. Vali~ek, B. Smid, T. Vesel~ and Z. Nevorhnkovfi Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czechoslovakia
Hybridomas producing monoclonal antibodies (MAbs)
to rabbit haemorrhagic disease virus (RHDV) were prepared. Using Western blot (WB) analysis, the MAbs obtained were divided into two groups, one reacting with the major structural proteins of Mr 61K and 38K, and the other giving negative reactions. Both groups of MAbs, however, reacted specifically with R H D V in ELISA and by immunoperoxidase (IP) and immunofluorescence (IF) tests with infected ceils. As demonstrated by WB using RHDV-specific MAbs and a MAb
Introduction Rabbit haemorrhagic disease (RHD), which emerged in China in 1984 and in European countries in 1987 to 1988, has a great economic impact, causing 60 to 100~ mortality in rabbit colonies. The causal agent was demonstrated to be a virus (RHDV) with a diameter of 28 to 38 nm (Liu etal., 1984; Deng etal., 1987; Wei etal., 1987; Ohlinger et al., 1989; Smid et al., 1989). Several reports (Liu et al., 1984; Gu et al., 1986; Smid et al., 1990) have described the production of inactivated vaccines with reliable protective effects, and haemagglutination and haemagglutination inhibition tests (HT and HIT respectively) were developed for the demonstration of the virus and specific antibodies (Liu et al., 1984; Pu et al., 1985; D u e t al., 1986; Ohlinger et al., 1989). The earlier, rather controversial reports concerning the classification of RHDV (Liu et al., 1984; Du et al., 1986; Shen et al., 1986; Xu et al., 1988) were followed by several papers suggesting, on the basis of morphology, the size of the major structural proteins and density in CsC1, that RHDV is a calicivirus (Du et al., 1986; Deng et al., 1987 ; Ohlinger et al., 1989; ~mid et al., 1989; Vali~ek et al., 1990). On the other hand, RHDV differs from known caliciviruses in several respects: its haemagglutinating activity and failure in all attempts to propagate it in vitro (Studdert, 1978; Cubitt, 1987; Carter et al., 1989). Although the results obtained hitherto suggest a fair specificity of the HIT and HT, non-specific reactions have been also observed occasionally. HIT is less 0000-9688 © 1990SGM
to feline calicivirus (FCV) strain F9, the major structural (capsid) proteins of R H D V and FCV have very similar sizes (Mr 61K and 38K compared to 62K to 64K and 40K respectively). No cross-reactions of MAbs with proteins of the other virus were observed in WB analysis, ELISA, IP tests or IF. The high specificity and sensitivity of RHDV-specific MAbs make them suitable for the routine IP and IF diagnosis of R H D V in liver cells of rabbits dying after natural or experimental infections.
sensitive and a drawback of both tests is their dependence on a continuous supply of fresh erythrocytes. An ELISA for the determination of antibodies to RHDV in rabbit blood was described recently (Rod~ik et al., 1990), which eliminates the drawbacks of HIT and allows a standard, sensitive and specific assessment of the immune response to infection or vaccination. Hybridomas producing monoclonal antibodies (MAbs) to RHDV proteins were prepared with the aim of replacing H T with a more specific method. Methods for checking the specificity of MAbs as well as their possible use for the diagnosis of RHD are discussed in this paper.
Methods Animals. InbredBALB/cmicewereemployedfor the preparationof hybridomas and ascitic fluids, as well as sourcesof RHDV-negative and -positivesera. Negativerabbit sera were collectedin non-infected colonies, free of naturally acquired antibodies. Organs of normal rabbits and rabbits dyingafter infectionwereused for the preparation of RHDV-negative and -positive tissue sections, impressions and smears for further examination. A reference serum obtained from China, and rabbit hyperimmuneand convalescentsera were used as RHDV-positive controls. Virus purification. RHDV was purified from organ homogenatesof rabbits dyingafterexperimentalinfectionwiththe strain CAPMV-351 [Collection of Animal Pathogenic Microorganisms (CAPM), Brno, Czechoslovakia]. The homogenates were extracted with chloroform andultracentrifugedonto a CsC1cushion, the final step being affinity chromatography(Rod~iket al., 1990).The vaccinestrain F9 (CAPM) of feline calicivirus (FCV) was propagated in the Crandell feline
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L. R o d f k and others
kidney cell line (CRFK) in MEM. After removal of cell debris from the culture medium, the virus was concentrated by ultracentrifugation (Beckman SW28, 90min at 27000r.p.m.) and the pellet was resuspended in 1/100 of the original volume of phosphate-buffered saline (PBS). The suspensions of RHDV or FCV were diluted 1 : 500 or 1:100, respectively, with 0-05 M-carbonate-bicarbonate buffer pH 9.6 lbr ELISA.
Blood sera and conjugates. Specific antibodies were separated from swine hyperimmune sera to rabbit and mouse IgG (SwARIgG, SwAMolgG) by affinity chromatography (CNBr-activated Sepharose 4B, Pharmacia). The IgG fraction was separated from mouse ascitic fluid containing MAbs to RHDV (E7/C3). The antibodies were conjugated with horseradish peroxidase (HRP, RZ = 3-0, Boehringer Mannheim) by the periodate method (Boorsma & Streefkerk, 1979). Stock conjugate solutions (HRP-SwARIgG, HRP-SwAMolgG, H R P - R H D V MAb), containing 2 mg/ml IgG, were diluted 1:1000 to 1:2000 with PBS containing 0.1% Tween 80 and 1X lactalbumin hydrolysate (PBST-LAH) before use in ELISA or Western blot (WB) analysis. The IgG fraction of SwAMolgG separated by ion-exchange chromatography was conjugated with fluorescein isothiocyanate (FITC-SwAMolgG) as described by The & Feltkamp (1970). ELISA. Microtitre plates (Koh-I-Noor) with wells coated with purified RHDV or FCV were incubated with the examined sample (rabbit or mouse serum, ascitic fluids, hybridoma culture medium) at 37 °C for 1 h. A sample of the ascitic fluid of FCV MAb 1G9 was kindly supplied by Dr M. J. Carter of the University of Newcastle-upon-Tyne, U.K. One volume of sample was diluted with one (culture media), or 100 (sera, ascitic fluids) volumes of PBST-LAH and twofold dilution series were prepared for antibody titrations. A second incubation followed with either HRP-SwARIgG (rabbit samples), or H R P SwAMolgG (mouse samples) under the same conditions. The results were read spectrophotometrically at 492 nm (Titertek MCC, Labsystem) 1 h after the addition of substrate solution (5-aminosalicylic acid). Wells containing negative rabbit or mouse sera served as controls and rows of wells containing PBST LAH alone as blanks (RodAk et al., 1990). The highest dilution giving an absorbance higher than 0.1 was considered as the final antibody titre. Hybridomas and tests on MAbs. Inbred BALB/c mice were repeatedly inoculated subcutaneously with an emulsion of purified RHDV in AISpan-Oil adjuvant (Sevac). Three to 4 days prior to fusion, the mice were inoculated intraperitoneally with a suspension of RHDV in PBS. Spleen cells were fused with myeloma cells (SP 2/0; Institute of Molecular Genetics, Prague, Czechoslovakia) at a ratio of 5 : 1 in 50% polyethylene glycol 1500 in serum-free Dulbecco's MEM (DMEM, Serva) (Galfr6 & Milstein, 1981). Culture media from the hybridomas grown in D M E M - H A T were examined for the presence of antibodies by ELISA 10 to 12 days later. Antibody-producing hybridomas were cloned and transferred into the complete medium RPMI 1640 and antibody production was tested again by ELISA and Western blotting. Selected hybridomas were injected intraperitoneally into Pristane (Sigma)-primed BALB/c mice (1 x 106 cells per animal). Ascitic fluids were collected 10 days later and stored at 4 °C with 0-1% sodium azide as preservative. Specific immune rabbit sera to individual types of heavy and light chains of mouse immunoglobulins (ICN Immunobiochemicals) were used for the exact classification of the MAbs. The concentration of immunoglobulins in ascitic fluids was determined by radial immunodiffusion in 1.5 % agarose gels. Immunoglobulins, which bad been separated from the same ascitic fluids and whose IgG concentration had been determined spectrophotometricaUy (E~8~ = 14), were used as standards. PAGE and WB analysis. The major structural proteins of RHDV and FCV were determined by discontinuous SDS-PAGE under reducing
conditions in a 10~ gel as described by Laemmli (1970). Low M r standards (Pharmacia) were used for the determination of Mr. After electrophoresis, the gel was divided into two parts. One was stained with silver (Heukeshoven & Dernick, 1985) and proteins from the other one were transferred onto a nitrocellulose membrane (NC) (Sartorius SM 113) and used for WB analysis. Strips of NC were incubated with rabbit or mouse sera or ascitic fluids, diluted 1:1000 with PBST-LAH, at 20 °C for 1 h and subsequently with HRP-SwARIgG or H R P SwAMolgG under the same conditions. After a short period of staining in substrate solution [3,Y-diaminobenzidine (DAB), Merck], the reaction was stopped with 0.5 % sodium azide and the strips were dried and photographed.
Demonstration of virus by immunoperoxidase (IP) and immunofluorescence (IF) tests. Cryostat sections, impressions and smears of liver, kidney, spleen and lung of several tens of rabbits dying after natural or experimental infections were examined. The dried samples were fixed with acetone at 20 °C for 15 min. For the IP test, the samples were preincubated for 15 to 30 min in PBS containing 0.1% sodium azide and 0-3~ hydrogen peroxide to inhibit the activity of endogenous peroxidase (Li et al., 1987). This step was followed by incubation in a wet chamber with MAbs (ascitic fluids) in a series of 10-fold dilutions (102 to 105) with PBST LAH at 20 °C for 30 min. Parallel samples incubated with serial dilutions of negative mouse serum served as controls. After rinsing with PBS (three times for 5 min), the samples were incubated under the same conditions with HRP-SwAMolgG or FITC-SwAMolgG diluted l : 10 to 1:200. The samples for the indirect IP test were examined under a conventional microscope after a short incubation with the substrate solution (DAB) and a fluorescence microscope (Fluoval) was used for the indirect IF test. Other samples were incubated (30 min at 20 °C) with H R P - R H D V MAb diluted 1:20, 1:50 and 1:100 with PBST-LAH for direct IP demonstration of RHDV. Samples of organs of a healthy, non-infected rabbit were used as controls. The same procedure was used for the indirect IP demonstration of FCV in monolayers of the cell line CRFK 6 h after infection with the strain F9. Parallel samples incubated with normal mouse serum and samples of non-infected cells served as controls.
Results A n t i b o d y titration by E L I S A The reference serum to RHDV obtained from China, and convalescent and hyperimmune rabbit sera with E L I S A a n t i b o d y t i t r e s o f 2 5 6 0 0 t o 2 0 4 8 0 0 ( T a b l e 1) served as standards in ELISA and WB analysis. The highest dilutions giving positive absorbance values (>0.1) were 12800 and 102400 to 819200 for the mouse s e r u m a n d a s c i t i c fluid ( R H D V M A b s ) s a m p l e s , r e s p e c t ively. N o a n t i b o d i e s t o R H D V w e r e d e m o n s t r a b l e i n t h e normal mouse serum and the ascitic fluid of FCV MAb 1 G 9 b y E L I S A i n a n y d i l u t i o n ( T a b l e 1). High titres of antibodies to FCV were found in the ascitic fluid containing FCV MAb 1G9 (1638400), w h e r e a s n e g a t i v e r e s u l t s w e r e o b t a i n e d w i t h all R H D V M A b - c o n t a i n i n g s a m p l e s ( T a b l e 1). O f t h e r a b b i t s e r a examined, only those collected from vaccinated animals showed positive titres of antibodies to FCV. However,
Monoclonal antibodies to R H D V
n o n e of the E L I S A - p o s i t i v e r a b b i t samples reacted in W B or IP with F C V (Table 1, Fig. 3).
1 R
F
F
2 R
F
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3 R
F
R
(b)
P A G E and Western blotting Proteins of R H D V a n d F C V were analysed after f r a c t i o n a t i o n by P A G E . O n e part of the gel was stained with silver a n d the other was used for W B analysis. Strips of N C m e m b r a n e s c o n t a i n i n g both R H D V a n d F C V proteins were i n c u b a t e d with R H D V M A b F 2 a n d / o r F C V M A b 1G9. T h e results of c o m p a r a t i v e e x a m i n a t i o n s both confirmed the specificity of M A b s a n d d e m o n s t r a t e d the similarity in size of the R H D V a n d F C V major proteins ( R H D V , Mr values of 61K a n d 3 8 K ; F C V , Mr values of 62K to 64K a n d 40K) (Fig. 1). Only three of the five R H D V M A b s reacted with the m a j o r R H D V proteins of Mr 61K a n d 38K. T h e specificity of the reactions was confirmed by parallel W B analysis of the r a b b i t sera. N e i t h e r F C V M A b 1G9 nor a n y of the negative control sera reacted with R H D V (Table 1, Fig. 1 a n d 2) a n d n o n e of the R H D V - p o s i t i v e r a b b i t sera or R H D V M A b s reacted with F C V proteins. F C V M A b 1G9 reacted strongly with the F C V p r o t e i n of Mr 62K to 64K, whereas the r e a c t i o n with the 40K p r o t e i n was m a r k e d l y weaker (Table 1, Fig. 1 a n d 3).
Demonstration of R H D V and F C V by IF and IP tests E x a m i n a t i o n s of organs of r a b b i t s d y i n g after n a t u r a l or e x p e r i m e n t a l R H D infections have s h o w n repeatedly that liver c o n t a i n e d more virus t h a n other organs a n d
Fig. 1. SDS-PAGE of samples fractionated under reducing conditions in a 10% gel. One part of the gel (a), containing the Mf standards (lane S), RHDV (lane R) and FCV (lane F), was stained with silver. The other part of the gel (b), containing RHDV (R) and FCV (F), was examined by WB. Strips of NC membrane were incubated with RHDV MAb F2 (lane 1), FCV MAb 1G9 (lane 2), and with a mixture of both MAbs (lane 3). Arrowheads show the major viral antigens on the silverstained gel.
that smears gave more reliable results t h a n impressions or sections. Therefore liver smears were used preferentially for routine e x a m i n a t i o n s . R H D V could be observed by indirect I P a n d I F up to the highest d i l u t i o n of M A b s used (10-5), b u t the n u m b e r of infected cells a n d the intensity of s t a i n i n g were indirectly p r o p o r t i o n a l to the dilution. Ascitic fluids a n d
Table 1. Specificity of rabbit sera, mouse sera and MAbs in reactions with R H D V and FCV, tested by ELISA, WB and IP
demonstration of viruses in infected cells Reaction with RHDV Sample Rabbit blood sera
Mouse sera MAbs (ascitic fluid)
Immunization (IgG concentration)*
Sample no.
Directed to
ELISA titre
3 x v. Unknown i. + i. v. + chall. Neg. control 3 x v. Neg. control 7.6 mg/ml 2.65 mg/ml 3.77 mg/ml 1.86 mg/ml 5.4 mg/ml
7/88-4 China 2786 2823 Neg. Pos. Neg. E9 C3 F2 E7/D10 E7/C3
RHDV RHDV RHDV RHDV Neg. RHDV Neg. RHDV RHDV RHDV RHDV RHDV
204 800 51200 25 600 51200 Neg. 12800 Neg. 819200 102400 204800 102400 204800
ND
1G9
FCV
Neg.
WB + + + +
+ +~ q- q++ ++ q- + --}+++ +++ ++ -
Reaction with FCV IP?
+++
ND§ ND ND ND +++ +++ +++ +++ +++ +++ -
* Infection (i.), challenge (chall.), vaccination (v.). t IP, Direct and indirect IP demonstration of the virus in infected cells. :~The symbols ' - ' and ' + + + ' represent negative and strongly positive reactions, respectively. § ND, Not determined.
ELISA titre
WB
3200 Neg. Neg. 400 Neg. ND Neg. Neg. Neg. Neg. Neg. Neg.
---ND -
1638400
+++
IP
ND ND
ND
+++
2596
S
L. Rod~tk and others
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1
2
3
4
5
6
8
7
9
10
11 12
(b)
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.............
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Fig. 2 S
F
1
2
3
4
5
7
6
8
9
10
11 12
(b)
!I!i"i
Fig. 4. Indirect IP demonstration of RHDV in a liver tissue smear of an infected rabbit, using MAb F2 to RHDV. R H D V is visible in the cytoplasm of freshly infected cells as a ring of dense granules (full arrowheads); non-infected cells are pale (open arrowhead). Bar marker represents 25 ~tm.
,
iiIiii
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i~'iiii :;=,;::iiiI~;ii,Iiiii!i' ! ! )?'i!i!i'iI;=== Q~=I I 1,,11!,,',ii=,1';!iii II:l£Iim =' i i I " ~,i i ==!i!:'"i1:: i: i!ii= iii:,i! i=:ii i ~ = ~ ~ IIII
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Fig. 3 Fig. 2 and Fig. 3. S D S - P A G E of R H D V (R, Fig. 2) and FCV (F, Fig. 3) fractionated under reducing conditions in a 10~ gel. One part of each gel (a), containing the Mr standards (lane S) or the virus (lanes R and F), was stained with silver. Other parts of the gels (b), containing R H D V (Fig. 2) and FCV (Fig. 3) only, were examined by WB. Strips of NC membrane in Fig. 2 and 3 were incubated with the same rabbit (lanes 1 to 4), or mouse (lanes 5 to 12) samples. Rabbit hyperimmune anti-RHDV serum 7/88/4 (lanes 1), Chinese RHDV-positive rabbit serum (lanes 2), negative rabbit serum (lanes 3), control without rabbit serum (lanes 4), RHDV MAb E9 (lanes 5), R H D V MAb C3 (lanes 6), R H D V MAb F2 (lanes 7), MAb R H D V E7/D10 (lanes 8), MAb R H D V E7/C3 (lanes 9), FCV MAb 1G9 (lanes 10), negative mouse serum (lanes 11), control without mouse serum or ascitic fluid (lanes 12). Arrowheads show the major viral antigens on the silver-stained gels. Further details are given in Table 1.
conjugated SwAMolgG diluted 1:1000 and 1:100 to 1:200, respectively, were used in routine examinations. The results of incubation of positive control smears (impressions, sections) with the negative mouse serum, or FCV MAb 1G9 were uniformly negative (Table 1). Identical results were obtained by the direct IP test. FCV was demonstrable in the C R F K cell line 6 h after infection with FCV 9 by the indirect IP and IF tests only in cultures incubated with FCV MAb 1G9 (Fig. 5). All
Fig. 5. Indirect IP demonstration of FCV in the C R F K cell line 6 h after infection using MAb 1G9 to FCV. FCV is visible as dense granules in the cytoplasm of infected cells (arrowheads). Bar marker represents 25 ~tm.
cultures incubated with RHDV-positive rabbit sera or RHDV MAbs were negative (Table 1). Diffusely stained cells in various stages of degeneration, as well as freshly infected cells with pronounced positive reactions in the form of granules or a ring in the cytoplasm only (Fig. 4 and 5), were seen in the IP test, which was more suitable for visual evaluation than IF.
Discussion Five clones of hybridomas, producing MAbs to RHDV, were prepared and tested. Some of the MAbs reacted with the dominant RHDV proteins, Mr 61K and 38K, in WB analysis, but others did not. However, all MAbs reacted intensely and highly specifically with native RHDV proteins, as demonstrated by high ELISA antibody titres in all samples of ascitic fluid, as well as by
Monoclonal antibodies to R H D V
their suitability for IP and IF demonstration of the virus in tissues of rabbits dying after natural or experimental infections. Therefore, we suppose the MAbs that were non-reactive with the dominant proteins in WB react with antigenic structures which are damaged during the treatment of the virus for PAGE under reducing conditions. The ELISA technique described earlier (Rodfik et al., 1990) proved fully suitable for the examination of hybridoma culture media. The possibility of using HIT for this purpose is rather unlikely. ELISA detected very low concentrations of antibodies, released into culture media by several tens of hybridoma cells only, even during first screening tests. Cross reactions of RHDV MAbs and FCV MAbs with the dominant proteins of the other virus were observed neither in WB, nor in IP or IF tests. Antigenic differences between the two viruses were also confirmed by the non-reactivity of convalescent rabbit sera with the FCV antigen. Cross-reactions were observed only when sera of vaccinated rabbits and the FCV antigen were used as components in the ELISA (Table 1). However these sera did not react with the FCV antigen in WB, IP or IF tests. Positive reactions of the sera are therefore interpreted as the result of a reaction of non-specific antibodies with traces of cell and protein antigens present in the incompletely purified FCV antigen. The correct classification of RHDV has been complicated by its haemagglutinating activity, failure to propagate in vitro and by a number of conflicting results obtained by different authors (Liu et al., 1984; Cao et al., 1986; Shen et al., 1986; Wei et al., 1987; Xu et al., 1988; Ohlinger et al., 1989; ~mid et al., 1989). The causal agent of RHD has most often been classified as a parvo-, picorna-, or calicivirus. Its ultrastructure (~mid et al., 1989; Ohlinger et al., 1989; Vali~ek et al., 1990) and the presence and size of the dominant protein (61K) classes RHDV with caliciviruses. Such classification is supported by the finding that both RHDV and FCV contain dominant (capsid) proteins with very similar Mr values (60K to 61K and 62K to 64K), typical for caliciviruses (Carter et al., 1989; Capucci et al., 1990; Rodfik et al., 1990). Moreover, using IP and IF tests both RHDV and FCV were detectable in only the cytoplasm of freshly infected cells, whereas parvovirus is located in the nucleus. The suitability of MAbs for the IP and IF diagnosis of RHDV was confirmed by the results from examinations of tissue samples collected from rabbits dying after natural or experimental infections. The specificity of both methods was fully confirmed by electron microscopic findings. Routine use of RHDV MAbs for IP and IF diagnosis of this dangerous rabbit disease can be highly recommended. Both techniques replace HT as the
2597
only method commonly used for virus demonstration, being simpler and possessing a much higher sensitivity and specificity. The use of electron microscopy will remain limited to laboratories possessing the necessary equipment and qualified staff. We would like to thank Dr M. J. Carter, University of Newcastle upon Tyne, U.K., for the kind supply of monoclonal antibodies to feline calicivirus and to Dr X. X. Wang, National Control Institute of Veterinary Bioproducts and Pharmaceuticals, Beijing, China, for the RHDV-positive rabbit serum. We are indebted to Mrs A. Farnikovfi, Mrs H. Kfipa~ovfi, Mrs J. Mrvovfi and Mrs Z. Mikulfigkov~. for their excellent technical assistance.
References BOORSMA, D. M. & STREEFKERK, J. G. (1979). Periodate or glutaraldehyde for preparing peroxidase conjugates? Journal of Immunological Methods 30, 245-255. CAO, S. Z., LIU, S. G., GAN, M. H., LIU, R. P., CAI, S. W. t~. LIU, S. F. (1986). A preliminary report on viral haemorrhagic pneumonia (tentative name) in rabbits. Chinese Journal of Veterinary Medicine 12, 9-11.
CAPUCCI, L., SCICLUNA,M. T., LAVAZZA,A. & BROCCHI, E. (1990). Purificazione e caratterizzazione dell'agente eziologico della malattia emorragica virale del coniglio. Estratto da Selezione Veterinaria 31, 301-312.
CARTER,i . J., ROUTLEDGE,E. G. & TOMS,G. L. (1989). Monoclonal antibodies to feline calicivirus. Journal of General Virology 70, 2197-2200. CUBITT, W. D. (1987). The candidate caliciviruses. In Novel Diarrhoea Viruses, CIBA Foundation Symposium 128, pp. 128-143. Edited by G. Bock & J. Whelan. Chichester: John Wiley & Sons.
DENG, R., Xu, W., DU, N., YANG,X. & Wu, S. (1987). Characteristics of rabbit haemorrhagic disease virus. Journal of Nanjing Agricultural University 6, ll0-114. Du, N., Xu, W. & LIU, S. (1986). A preliminary report on the identification of a new virus - rabbit haemorrhagic disease virus.
Chinese Journal of Virology 2, 146-151. GALFRI~, G. & MILSTEIN, C. (1981). Preparation of monoclonal antibodies. Strategies and procedures. Methods in Enzymology 73, 3-46. Go, Z. D., WANG, X. X., LI, Q. z. & SUN, F. F. (1986). An inactivated vaccine against haemorrhagic pneumonia in rabbits. Chinese Journal of Veterinary Medicine 12, 50-51. HEUKESHOVEN, J. & DERNICK, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685.
L!, C. Y., ZIESMER,S. C. d~;LAZCANO-VILLAREAL,O. (1987). Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method. Journal of Histochemistry and Cytochemistry 35, 1457-1460. LIU, S. J., XUE, H. P., PU, B. Q. & QIAN, N. H. (1984). A new viral
disease in rabbits. Animal Husbandry and Veterinary Medicine 16, 253-255. PO, B. Q., QIAN, H. H. & CuI, S. J. (1985). Micro HA test for the detection of antibody titres to so-called "haemorrhagic pneumonia" in rabbits. Chinese Journal of Veterinary Medicine 11, 16-17. OHLINGER, V. F., HAAS, B., AHL, R. & WEILAND, F. (1989). Die infekti6se h/imorrhagische Krankheit der Kaninchen - eine durch ein Calicivirus verursachte Tierseuche. Tieriirztliche Umschau 44, 284-294.
ROD.~K,L., SMiD,B., VALi~EK,L., VESEL~,T., gT~P~NEK,J., HAMPL,J. JUR.~K, E. (1990). Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic disease virus and determination
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of its major structural proteins. Journal of General Virology 71, 1075-1080. SHEN, X., PENG, H., SHEN,J., PENG, B., KUNG, T., CHEN, Y., FAN, Y., Yu, M., ZHAO, H., HOU, Z., XO, Q. & CHEN,J. (1986). Studies on viral haemorrhagic disease of rabbit. II. Purification, electron microscopy and nucleic acid of rabbit haemorrhagic virus. Chinese Journal of Virology 2, 323-328. ~MiD, B., VALi(~EK,L., ~TEP/kNEK,J., JUR~.K, E. tg. ROD:d~, L. (1989). Experimental transmission and electron microscopic demonstration of the virus of haemorrhagic disease of rabbits in Czechoslovakia. Journal of Veterinary Medicine B36, 237-240. gMiD, B., VALi(~EK,L., ROD.~K, L., ~TF.PANEK,J. & JURKK,E. (1990). Rabbit haemorrhagic disease :.an investigation of some properties of the virus and evaluation of an inactivated vaccine. Veterinary Microbiology (in press). STUDDERT, M. J. (1978). Caliciviruses. Brief Review. Archives of Virology 58, 157-191.
THE, T. M. & FELTKAMP,T. E. W. (1970). Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology 17, 865-872. VALi~EK, L., SMiD, B., ROD.~.K,L. & KUDRNA,J. (1990). Electron and immunoelectron microscopy of rabbit haemorrhagic disease virus (RHDV). Archives of Virology (in press)~ WEI, J. S., Yu, N. S., YANG,Y. F., ZHANG,X. S., LONG, P. R. & SHEN, J. R. (1987). Investigation on a viral haemorrhagic disease in rabbits in Juunan Province. Chinese Journal of Veterinary Science and Technology 8, 20-24. Xu, W., DE, N. & LIU, S. (1988). A new virus isolated from haemorrhagic disease in rabbits. In Proceedings of the 4th Worm Rabbit Congress, pp. 456461. Budapest.
(Received 21 May 1990; Accepted 20 July 1990)
2593
Monoclonal antibodies to rabbit haemorrhagic disease virus and their use in the diagnosis of infection L. Rodfik,* M. Granhtovfi, L. Vali~ek, B. Smid, T. Vesel~ and Z. Nevorhnkovfi Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czechoslovakia
Hybridomas producing monoclonal antibodies (MAbs)
to rabbit haemorrhagic disease virus (RHDV) were prepared. Using Western blot (WB) analysis, the MAbs obtained were divided into two groups, one reacting with the major structural proteins of Mr 61K and 38K, and the other giving negative reactions. Both groups of MAbs, however, reacted specifically with R H D V in ELISA and by immunoperoxidase (IP) and immunofluorescence (IF) tests with infected ceils. As demonstrated by WB using RHDV-specific MAbs and a MAb
Introduction Rabbit haemorrhagic disease (RHD), which emerged in China in 1984 and in European countries in 1987 to 1988, has a great economic impact, causing 60 to 100~ mortality in rabbit colonies. The causal agent was demonstrated to be a virus (RHDV) with a diameter of 28 to 38 nm (Liu etal., 1984; Deng etal., 1987; Wei etal., 1987; Ohlinger et al., 1989; Smid et al., 1989). Several reports (Liu et al., 1984; Gu et al., 1986; Smid et al., 1990) have described the production of inactivated vaccines with reliable protective effects, and haemagglutination and haemagglutination inhibition tests (HT and HIT respectively) were developed for the demonstration of the virus and specific antibodies (Liu et al., 1984; Pu et al., 1985; D u e t al., 1986; Ohlinger et al., 1989). The earlier, rather controversial reports concerning the classification of RHDV (Liu et al., 1984; Du et al., 1986; Shen et al., 1986; Xu et al., 1988) were followed by several papers suggesting, on the basis of morphology, the size of the major structural proteins and density in CsC1, that RHDV is a calicivirus (Du et al., 1986; Deng et al., 1987 ; Ohlinger et al., 1989; ~mid et al., 1989; Vali~ek et al., 1990). On the other hand, RHDV differs from known caliciviruses in several respects: its haemagglutinating activity and failure in all attempts to propagate it in vitro (Studdert, 1978; Cubitt, 1987; Carter et al., 1989). Although the results obtained hitherto suggest a fair specificity of the HIT and HT, non-specific reactions have been also observed occasionally. HIT is less 0000-9688 © 1990SGM
to feline calicivirus (FCV) strain F9, the major structural (capsid) proteins of R H D V and FCV have very similar sizes (Mr 61K and 38K compared to 62K to 64K and 40K respectively). No cross-reactions of MAbs with proteins of the other virus were observed in WB analysis, ELISA, IP tests or IF. The high specificity and sensitivity of RHDV-specific MAbs make them suitable for the routine IP and IF diagnosis of R H D V in liver cells of rabbits dying after natural or experimental infections.
sensitive and a drawback of both tests is their dependence on a continuous supply of fresh erythrocytes. An ELISA for the determination of antibodies to RHDV in rabbit blood was described recently (Rod~ik et al., 1990), which eliminates the drawbacks of HIT and allows a standard, sensitive and specific assessment of the immune response to infection or vaccination. Hybridomas producing monoclonal antibodies (MAbs) to RHDV proteins were prepared with the aim of replacing H T with a more specific method. Methods for checking the specificity of MAbs as well as their possible use for the diagnosis of RHD are discussed in this paper.
Methods Animals. InbredBALB/cmicewereemployedfor the preparationof hybridomas and ascitic fluids, as well as sourcesof RHDV-negative and -positivesera. Negativerabbit sera were collectedin non-infected colonies, free of naturally acquired antibodies. Organs of normal rabbits and rabbits dyingafter infectionwereused for the preparation of RHDV-negative and -positive tissue sections, impressions and smears for further examination. A reference serum obtained from China, and rabbit hyperimmuneand convalescentsera were used as RHDV-positive controls. Virus purification. RHDV was purified from organ homogenatesof rabbits dyingafterexperimentalinfectionwiththe strain CAPMV-351 [Collection of Animal Pathogenic Microorganisms (CAPM), Brno, Czechoslovakia]. The homogenates were extracted with chloroform andultracentrifugedonto a CsC1cushion, the final step being affinity chromatography(Rod~iket al., 1990).The vaccinestrain F9 (CAPM) of feline calicivirus (FCV) was propagated in the Crandell feline
2594
L. R o d f k and others
kidney cell line (CRFK) in MEM. After removal of cell debris from the culture medium, the virus was concentrated by ultracentrifugation (Beckman SW28, 90min at 27000r.p.m.) and the pellet was resuspended in 1/100 of the original volume of phosphate-buffered saline (PBS). The suspensions of RHDV or FCV were diluted 1 : 500 or 1:100, respectively, with 0-05 M-carbonate-bicarbonate buffer pH 9.6 lbr ELISA.
Blood sera and conjugates. Specific antibodies were separated from swine hyperimmune sera to rabbit and mouse IgG (SwARIgG, SwAMolgG) by affinity chromatography (CNBr-activated Sepharose 4B, Pharmacia). The IgG fraction was separated from mouse ascitic fluid containing MAbs to RHDV (E7/C3). The antibodies were conjugated with horseradish peroxidase (HRP, RZ = 3-0, Boehringer Mannheim) by the periodate method (Boorsma & Streefkerk, 1979). Stock conjugate solutions (HRP-SwARIgG, HRP-SwAMolgG, H R P - R H D V MAb), containing 2 mg/ml IgG, were diluted 1:1000 to 1:2000 with PBS containing 0.1% Tween 80 and 1X lactalbumin hydrolysate (PBST-LAH) before use in ELISA or Western blot (WB) analysis. The IgG fraction of SwAMolgG separated by ion-exchange chromatography was conjugated with fluorescein isothiocyanate (FITC-SwAMolgG) as described by The & Feltkamp (1970). ELISA. Microtitre plates (Koh-I-Noor) with wells coated with purified RHDV or FCV were incubated with the examined sample (rabbit or mouse serum, ascitic fluids, hybridoma culture medium) at 37 °C for 1 h. A sample of the ascitic fluid of FCV MAb 1G9 was kindly supplied by Dr M. J. Carter of the University of Newcastle-upon-Tyne, U.K. One volume of sample was diluted with one (culture media), or 100 (sera, ascitic fluids) volumes of PBST-LAH and twofold dilution series were prepared for antibody titrations. A second incubation followed with either HRP-SwARIgG (rabbit samples), or H R P SwAMolgG (mouse samples) under the same conditions. The results were read spectrophotometrically at 492 nm (Titertek MCC, Labsystem) 1 h after the addition of substrate solution (5-aminosalicylic acid). Wells containing negative rabbit or mouse sera served as controls and rows of wells containing PBST LAH alone as blanks (RodAk et al., 1990). The highest dilution giving an absorbance higher than 0.1 was considered as the final antibody titre. Hybridomas and tests on MAbs. Inbred BALB/c mice were repeatedly inoculated subcutaneously with an emulsion of purified RHDV in AISpan-Oil adjuvant (Sevac). Three to 4 days prior to fusion, the mice were inoculated intraperitoneally with a suspension of RHDV in PBS. Spleen cells were fused with myeloma cells (SP 2/0; Institute of Molecular Genetics, Prague, Czechoslovakia) at a ratio of 5 : 1 in 50% polyethylene glycol 1500 in serum-free Dulbecco's MEM (DMEM, Serva) (Galfr6 & Milstein, 1981). Culture media from the hybridomas grown in D M E M - H A T were examined for the presence of antibodies by ELISA 10 to 12 days later. Antibody-producing hybridomas were cloned and transferred into the complete medium RPMI 1640 and antibody production was tested again by ELISA and Western blotting. Selected hybridomas were injected intraperitoneally into Pristane (Sigma)-primed BALB/c mice (1 x 106 cells per animal). Ascitic fluids were collected 10 days later and stored at 4 °C with 0-1% sodium azide as preservative. Specific immune rabbit sera to individual types of heavy and light chains of mouse immunoglobulins (ICN Immunobiochemicals) were used for the exact classification of the MAbs. The concentration of immunoglobulins in ascitic fluids was determined by radial immunodiffusion in 1.5 % agarose gels. Immunoglobulins, which bad been separated from the same ascitic fluids and whose IgG concentration had been determined spectrophotometricaUy (E~8~ = 14), were used as standards. PAGE and WB analysis. The major structural proteins of RHDV and FCV were determined by discontinuous SDS-PAGE under reducing
conditions in a 10~ gel as described by Laemmli (1970). Low M r standards (Pharmacia) were used for the determination of Mr. After electrophoresis, the gel was divided into two parts. One was stained with silver (Heukeshoven & Dernick, 1985) and proteins from the other one were transferred onto a nitrocellulose membrane (NC) (Sartorius SM 113) and used for WB analysis. Strips of NC were incubated with rabbit or mouse sera or ascitic fluids, diluted 1:1000 with PBST-LAH, at 20 °C for 1 h and subsequently with HRP-SwARIgG or H R P SwAMolgG under the same conditions. After a short period of staining in substrate solution [3,Y-diaminobenzidine (DAB), Merck], the reaction was stopped with 0.5 % sodium azide and the strips were dried and photographed.
Demonstration of virus by immunoperoxidase (IP) and immunofluorescence (IF) tests. Cryostat sections, impressions and smears of liver, kidney, spleen and lung of several tens of rabbits dying after natural or experimental infections were examined. The dried samples were fixed with acetone at 20 °C for 15 min. For the IP test, the samples were preincubated for 15 to 30 min in PBS containing 0.1% sodium azide and 0-3~ hydrogen peroxide to inhibit the activity of endogenous peroxidase (Li et al., 1987). This step was followed by incubation in a wet chamber with MAbs (ascitic fluids) in a series of 10-fold dilutions (102 to 105) with PBST LAH at 20 °C for 30 min. Parallel samples incubated with serial dilutions of negative mouse serum served as controls. After rinsing with PBS (three times for 5 min), the samples were incubated under the same conditions with HRP-SwAMolgG or FITC-SwAMolgG diluted l : 10 to 1:200. The samples for the indirect IP test were examined under a conventional microscope after a short incubation with the substrate solution (DAB) and a fluorescence microscope (Fluoval) was used for the indirect IF test. Other samples were incubated (30 min at 20 °C) with H R P - R H D V MAb diluted 1:20, 1:50 and 1:100 with PBST-LAH for direct IP demonstration of RHDV. Samples of organs of a healthy, non-infected rabbit were used as controls. The same procedure was used for the indirect IP demonstration of FCV in monolayers of the cell line CRFK 6 h after infection with the strain F9. Parallel samples incubated with normal mouse serum and samples of non-infected cells served as controls.
Results A n t i b o d y titration by E L I S A The reference serum to RHDV obtained from China, and convalescent and hyperimmune rabbit sera with E L I S A a n t i b o d y t i t r e s o f 2 5 6 0 0 t o 2 0 4 8 0 0 ( T a b l e 1) served as standards in ELISA and WB analysis. The highest dilutions giving positive absorbance values (>0.1) were 12800 and 102400 to 819200 for the mouse s e r u m a n d a s c i t i c fluid ( R H D V M A b s ) s a m p l e s , r e s p e c t ively. N o a n t i b o d i e s t o R H D V w e r e d e m o n s t r a b l e i n t h e normal mouse serum and the ascitic fluid of FCV MAb 1 G 9 b y E L I S A i n a n y d i l u t i o n ( T a b l e 1). High titres of antibodies to FCV were found in the ascitic fluid containing FCV MAb 1G9 (1638400), w h e r e a s n e g a t i v e r e s u l t s w e r e o b t a i n e d w i t h all R H D V M A b - c o n t a i n i n g s a m p l e s ( T a b l e 1). O f t h e r a b b i t s e r a examined, only those collected from vaccinated animals showed positive titres of antibodies to FCV. However,
Monoclonal antibodies to R H D V
n o n e of the E L I S A - p o s i t i v e r a b b i t samples reacted in W B or IP with F C V (Table 1, Fig. 3).
1 R
F
F
2 R
F
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3 R
F
R
(b)
P A G E and Western blotting Proteins of R H D V a n d F C V were analysed after f r a c t i o n a t i o n by P A G E . O n e part of the gel was stained with silver a n d the other was used for W B analysis. Strips of N C m e m b r a n e s c o n t a i n i n g both R H D V a n d F C V proteins were i n c u b a t e d with R H D V M A b F 2 a n d / o r F C V M A b 1G9. T h e results of c o m p a r a t i v e e x a m i n a t i o n s both confirmed the specificity of M A b s a n d d e m o n s t r a t e d the similarity in size of the R H D V a n d F C V major proteins ( R H D V , Mr values of 61K a n d 3 8 K ; F C V , Mr values of 62K to 64K a n d 40K) (Fig. 1). Only three of the five R H D V M A b s reacted with the m a j o r R H D V proteins of Mr 61K a n d 38K. T h e specificity of the reactions was confirmed by parallel W B analysis of the r a b b i t sera. N e i t h e r F C V M A b 1G9 nor a n y of the negative control sera reacted with R H D V (Table 1, Fig. 1 a n d 2) a n d n o n e of the R H D V - p o s i t i v e r a b b i t sera or R H D V M A b s reacted with F C V proteins. F C V M A b 1G9 reacted strongly with the F C V p r o t e i n of Mr 62K to 64K, whereas the r e a c t i o n with the 40K p r o t e i n was m a r k e d l y weaker (Table 1, Fig. 1 a n d 3).
Demonstration of R H D V and F C V by IF and IP tests E x a m i n a t i o n s of organs of r a b b i t s d y i n g after n a t u r a l or e x p e r i m e n t a l R H D infections have s h o w n repeatedly that liver c o n t a i n e d more virus t h a n other organs a n d
Fig. 1. SDS-PAGE of samples fractionated under reducing conditions in a 10% gel. One part of the gel (a), containing the Mf standards (lane S), RHDV (lane R) and FCV (lane F), was stained with silver. The other part of the gel (b), containing RHDV (R) and FCV (F), was examined by WB. Strips of NC membrane were incubated with RHDV MAb F2 (lane 1), FCV MAb 1G9 (lane 2), and with a mixture of both MAbs (lane 3). Arrowheads show the major viral antigens on the silverstained gel.
that smears gave more reliable results t h a n impressions or sections. Therefore liver smears were used preferentially for routine e x a m i n a t i o n s . R H D V could be observed by indirect I P a n d I F up to the highest d i l u t i o n of M A b s used (10-5), b u t the n u m b e r of infected cells a n d the intensity of s t a i n i n g were indirectly p r o p o r t i o n a l to the dilution. Ascitic fluids a n d
Table 1. Specificity of rabbit sera, mouse sera and MAbs in reactions with R H D V and FCV, tested by ELISA, WB and IP
demonstration of viruses in infected cells Reaction with RHDV Sample Rabbit blood sera
Mouse sera MAbs (ascitic fluid)
Immunization (IgG concentration)*
Sample no.
Directed to
ELISA titre
3 x v. Unknown i. + i. v. + chall. Neg. control 3 x v. Neg. control 7.6 mg/ml 2.65 mg/ml 3.77 mg/ml 1.86 mg/ml 5.4 mg/ml
7/88-4 China 2786 2823 Neg. Pos. Neg. E9 C3 F2 E7/D10 E7/C3
RHDV RHDV RHDV RHDV Neg. RHDV Neg. RHDV RHDV RHDV RHDV RHDV
204 800 51200 25 600 51200 Neg. 12800 Neg. 819200 102400 204800 102400 204800
ND
1G9
FCV
Neg.
WB + + + +
+ +~ q- q++ ++ q- + --}+++ +++ ++ -
Reaction with FCV IP?
+++
ND§ ND ND ND +++ +++ +++ +++ +++ +++ -
* Infection (i.), challenge (chall.), vaccination (v.). t IP, Direct and indirect IP demonstration of the virus in infected cells. :~The symbols ' - ' and ' + + + ' represent negative and strongly positive reactions, respectively. § ND, Not determined.
ELISA titre
WB
3200 Neg. Neg. 400 Neg. ND Neg. Neg. Neg. Neg. Neg. Neg.
---ND -
1638400
+++
IP
ND ND
ND
+++
2596
S
L. Rod~tk and others
R
1
2
3
4
5
6
8
7
9
10
11 12
(b)
(a)
.....
N N ~, ~!iii
liifl
...........
N .............. ~iii ~';: ==~== !!!!ii I,,=~=I.......... I==#i# ~::~ %'~ ...... i i! iiii II!iiii ............ i::!! i~: ......... I I~II i q=iiiiih
.............
i!iilii
!!11!=!!,iili;iii ~== IIIUi Ii 111i I
iiii ~i iiiili!I . i ! ! % i!Iii ii, i ii=i ii!iliiiiii Ii : ~i~', ==~.,.,,,,
Fig. 2 S
F
1
2
3
4
5
7
6
8
9
10
11 12
(b)
!I!i"i
Fig. 4. Indirect IP demonstration of RHDV in a liver tissue smear of an infected rabbit, using MAb F2 to RHDV. R H D V is visible in the cytoplasm of freshly infected cells as a ring of dense granules (full arrowheads); non-infected cells are pale (open arrowhead). Bar marker represents 25 ~tm.
,
iiIiii
===~iiI
®I
5"N
N
i!!','1i~:
i~'iiii :;=,;::iiiI~;ii,Iiiii!i' ! ! )?'i!i!i'iI;=== Q~=I I 1,,11!,,',ii=,1';!iii II:l£Iim =' i i I " ~,i i ==!i!:'"i1:: i: i!ii= iii:,i! i=:ii i ~ = ~ ~ IIII
: ',
: ii :iii[ : -
!
]ii::]ilii = I.'ii= I i~ i=ii:i~Ii i:~! i!iii#"ii I~ii=ii, iili!IIii1]iliiilii ............ ]!
Fig. 3 Fig. 2 and Fig. 3. S D S - P A G E of R H D V (R, Fig. 2) and FCV (F, Fig. 3) fractionated under reducing conditions in a 10~ gel. One part of each gel (a), containing the Mr standards (lane S) or the virus (lanes R and F), was stained with silver. Other parts of the gels (b), containing R H D V (Fig. 2) and FCV (Fig. 3) only, were examined by WB. Strips of NC membrane in Fig. 2 and 3 were incubated with the same rabbit (lanes 1 to 4), or mouse (lanes 5 to 12) samples. Rabbit hyperimmune anti-RHDV serum 7/88/4 (lanes 1), Chinese RHDV-positive rabbit serum (lanes 2), negative rabbit serum (lanes 3), control without rabbit serum (lanes 4), RHDV MAb E9 (lanes 5), R H D V MAb C3 (lanes 6), R H D V MAb F2 (lanes 7), MAb R H D V E7/D10 (lanes 8), MAb R H D V E7/C3 (lanes 9), FCV MAb 1G9 (lanes 10), negative mouse serum (lanes 11), control without mouse serum or ascitic fluid (lanes 12). Arrowheads show the major viral antigens on the silver-stained gels. Further details are given in Table 1.
conjugated SwAMolgG diluted 1:1000 and 1:100 to 1:200, respectively, were used in routine examinations. The results of incubation of positive control smears (impressions, sections) with the negative mouse serum, or FCV MAb 1G9 were uniformly negative (Table 1). Identical results were obtained by the direct IP test. FCV was demonstrable in the C R F K cell line 6 h after infection with FCV 9 by the indirect IP and IF tests only in cultures incubated with FCV MAb 1G9 (Fig. 5). All
Fig. 5. Indirect IP demonstration of FCV in the C R F K cell line 6 h after infection using MAb 1G9 to FCV. FCV is visible as dense granules in the cytoplasm of infected cells (arrowheads). Bar marker represents 25 ~tm.
cultures incubated with RHDV-positive rabbit sera or RHDV MAbs were negative (Table 1). Diffusely stained cells in various stages of degeneration, as well as freshly infected cells with pronounced positive reactions in the form of granules or a ring in the cytoplasm only (Fig. 4 and 5), were seen in the IP test, which was more suitable for visual evaluation than IF.
Discussion Five clones of hybridomas, producing MAbs to RHDV, were prepared and tested. Some of the MAbs reacted with the dominant RHDV proteins, Mr 61K and 38K, in WB analysis, but others did not. However, all MAbs reacted intensely and highly specifically with native RHDV proteins, as demonstrated by high ELISA antibody titres in all samples of ascitic fluid, as well as by
Monoclonal antibodies to R H D V
their suitability for IP and IF demonstration of the virus in tissues of rabbits dying after natural or experimental infections. Therefore, we suppose the MAbs that were non-reactive with the dominant proteins in WB react with antigenic structures which are damaged during the treatment of the virus for PAGE under reducing conditions. The ELISA technique described earlier (Rodfik et al., 1990) proved fully suitable for the examination of hybridoma culture media. The possibility of using HIT for this purpose is rather unlikely. ELISA detected very low concentrations of antibodies, released into culture media by several tens of hybridoma cells only, even during first screening tests. Cross reactions of RHDV MAbs and FCV MAbs with the dominant proteins of the other virus were observed neither in WB, nor in IP or IF tests. Antigenic differences between the two viruses were also confirmed by the non-reactivity of convalescent rabbit sera with the FCV antigen. Cross-reactions were observed only when sera of vaccinated rabbits and the FCV antigen were used as components in the ELISA (Table 1). However these sera did not react with the FCV antigen in WB, IP or IF tests. Positive reactions of the sera are therefore interpreted as the result of a reaction of non-specific antibodies with traces of cell and protein antigens present in the incompletely purified FCV antigen. The correct classification of RHDV has been complicated by its haemagglutinating activity, failure to propagate in vitro and by a number of conflicting results obtained by different authors (Liu et al., 1984; Cao et al., 1986; Shen et al., 1986; Wei et al., 1987; Xu et al., 1988; Ohlinger et al., 1989; ~mid et al., 1989). The causal agent of RHD has most often been classified as a parvo-, picorna-, or calicivirus. Its ultrastructure (~mid et al., 1989; Ohlinger et al., 1989; Vali~ek et al., 1990) and the presence and size of the dominant protein (61K) classes RHDV with caliciviruses. Such classification is supported by the finding that both RHDV and FCV contain dominant (capsid) proteins with very similar Mr values (60K to 61K and 62K to 64K), typical for caliciviruses (Carter et al., 1989; Capucci et al., 1990; Rodfik et al., 1990). Moreover, using IP and IF tests both RHDV and FCV were detectable in only the cytoplasm of freshly infected cells, whereas parvovirus is located in the nucleus. The suitability of MAbs for the IP and IF diagnosis of RHDV was confirmed by the results from examinations of tissue samples collected from rabbits dying after natural or experimental infections. The specificity of both methods was fully confirmed by electron microscopic findings. Routine use of RHDV MAbs for IP and IF diagnosis of this dangerous rabbit disease can be highly recommended. Both techniques replace HT as the
2597
only method commonly used for virus demonstration, being simpler and possessing a much higher sensitivity and specificity. The use of electron microscopy will remain limited to laboratories possessing the necessary equipment and qualified staff. We would like to thank Dr M. J. Carter, University of Newcastle upon Tyne, U.K., for the kind supply of monoclonal antibodies to feline calicivirus and to Dr X. X. Wang, National Control Institute of Veterinary Bioproducts and Pharmaceuticals, Beijing, China, for the RHDV-positive rabbit serum. We are indebted to Mrs A. Farnikovfi, Mrs H. Kfipa~ovfi, Mrs J. Mrvovfi and Mrs Z. Mikulfigkov~. for their excellent technical assistance.
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CAPUCCI, L., SCICLUNA,M. T., LAVAZZA,A. & BROCCHI, E. (1990). Purificazione e caratterizzazione dell'agente eziologico della malattia emorragica virale del coniglio. Estratto da Selezione Veterinaria 31, 301-312.
CARTER,i . J., ROUTLEDGE,E. G. & TOMS,G. L. (1989). Monoclonal antibodies to feline calicivirus. Journal of General Virology 70, 2197-2200. CUBITT, W. D. (1987). The candidate caliciviruses. In Novel Diarrhoea Viruses, CIBA Foundation Symposium 128, pp. 128-143. Edited by G. Bock & J. Whelan. Chichester: John Wiley & Sons.
DENG, R., Xu, W., DU, N., YANG,X. & Wu, S. (1987). Characteristics of rabbit haemorrhagic disease virus. Journal of Nanjing Agricultural University 6, ll0-114. Du, N., Xu, W. & LIU, S. (1986). A preliminary report on the identification of a new virus - rabbit haemorrhagic disease virus.
Chinese Journal of Virology 2, 146-151. GALFRI~, G. & MILSTEIN, C. (1981). Preparation of monoclonal antibodies. Strategies and procedures. Methods in Enzymology 73, 3-46. Go, Z. D., WANG, X. X., LI, Q. z. & SUN, F. F. (1986). An inactivated vaccine against haemorrhagic pneumonia in rabbits. Chinese Journal of Veterinary Medicine 12, 50-51. HEUKESHOVEN, J. & DERNICK, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685.
L!, C. Y., ZIESMER,S. C. d~;LAZCANO-VILLAREAL,O. (1987). Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method. Journal of Histochemistry and Cytochemistry 35, 1457-1460. LIU, S. J., XUE, H. P., PU, B. Q. & QIAN, N. H. (1984). A new viral
disease in rabbits. Animal Husbandry and Veterinary Medicine 16, 253-255. PO, B. Q., QIAN, H. H. & CuI, S. J. (1985). Micro HA test for the detection of antibody titres to so-called "haemorrhagic pneumonia" in rabbits. Chinese Journal of Veterinary Medicine 11, 16-17. OHLINGER, V. F., HAAS, B., AHL, R. & WEILAND, F. (1989). Die infekti6se h/imorrhagische Krankheit der Kaninchen - eine durch ein Calicivirus verursachte Tierseuche. Tieriirztliche Umschau 44, 284-294.
ROD.~K,L., SMiD,B., VALi~EK,L., VESEL~,T., gT~P~NEK,J., HAMPL,J. JUR.~K, E. (1990). Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic disease virus and determination
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of its major structural proteins. Journal of General Virology 71, 1075-1080. SHEN, X., PENG, H., SHEN,J., PENG, B., KUNG, T., CHEN, Y., FAN, Y., Yu, M., ZHAO, H., HOU, Z., XO, Q. & CHEN,J. (1986). Studies on viral haemorrhagic disease of rabbit. II. Purification, electron microscopy and nucleic acid of rabbit haemorrhagic virus. Chinese Journal of Virology 2, 323-328. ~MiD, B., VALi(~EK,L., ~TEP/kNEK,J., JUR~.K, E. tg. ROD:d~, L. (1989). Experimental transmission and electron microscopic demonstration of the virus of haemorrhagic disease of rabbits in Czechoslovakia. Journal of Veterinary Medicine B36, 237-240. gMiD, B., VALi(~EK,L., ROD.~K, L., ~TF.PANEK,J. & JURKK,E. (1990). Rabbit haemorrhagic disease :.an investigation of some properties of the virus and evaluation of an inactivated vaccine. Veterinary Microbiology (in press). STUDDERT, M. J. (1978). Caliciviruses. Brief Review. Archives of Virology 58, 157-191.
THE, T. M. & FELTKAMP,T. E. W. (1970). Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology 17, 865-872. VALi~EK, L., SMiD, B., ROD.~.K,L. & KUDRNA,J. (1990). Electron and immunoelectron microscopy of rabbit haemorrhagic disease virus (RHDV). Archives of Virology (in press)~ WEI, J. S., Yu, N. S., YANG,Y. F., ZHANG,X. S., LONG, P. R. & SHEN, J. R. (1987). Investigation on a viral haemorrhagic disease in rabbits in Juunan Province. Chinese Journal of Veterinary Science and Technology 8, 20-24. Xu, W., DE, N. & LIU, S. (1988). A new virus isolated from haemorrhagic disease in rabbits. In Proceedings of the 4th Worm Rabbit Congress, pp. 456461. Budapest.
(Received 21 May 1990; Accepted 20 July 1990)
