VIROLOGY
191, 272-281
(1992)
Production and Characterization of Monoclonal Antibodies to Porcine Group C Rotaviruses Cross-Reactive with Group A Rotaviruses HIROSHI TSUNEMITSU,**t CLEM K. OJEH,t+ BAOMING JIANG,?’ RON A. SIMKINS,t PEGGY A. WEILNAU,t AND LINDA J. SAIFt*’ ‘Hokkaido
Prefectural Shintoku Animal Husbandry Experiment Station, Shintoku, Hokkaido 08 1, Japan; tFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 4469 1; and *Department of Veterinary Microbiology and Parasitology, University of Ibadan, lbadan, Nigeria Received May 26, 1992; accepted July 21, 1992
Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41 -kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is 0 1992 Academic Press, Inc. recognized by certain MAbs in various serologic assays.
INTRODUCTION
sumoto et a/., 1989; Penaranda et al., 1989; Ushijima et al., 1989; Caul et a/., 1990), and more recently in cattle (Tsunemitsu et al., 1991) with diarrhea. Preliminary serological surveys indicated that gp C rotaviruses were prevalent in swine in North America, Australia, and Europe (Bridger et al., 1985; Nagesha et a/., 1988; Saif, 1990). Recently, a large-scale outbreak of diarrhea caused by gp C rotavirus in children in Japan has been reported (Matsumoto eta/., 1989). Studies of the viral structural proteins of porcine gp C rotavirus have revealed that the 41 -kDa inner shell protein is the counterpart to VP6 of gp A rotavirus and contains common group antigens (Bremont et al., 1988; Jiang et a/., 1990; Ojeh et al., 1991, 1992). At present only porcine and bovine gp C rotavirus have been successfully propagated in a monkey kidney cell line (MA104) (Terrett and Saif, 1987; Saif et a/., 1988; Tsunemitsu et a/., 1991). Although the failure of in vitro propagation of other non-gp A rotaviruses and limited quantities of samples containing non-gp A rotaviruses have hampered antigenic comparisons, most reports suggest that no common antigens exist between rotaviruses from different serogroups (Pedley et al., 1983, 1986; Snodgrass et al., 1984; Saif and Theil, 1985). A prior report from our laboratory confirmed that three monoclonal antibodies (MAbs) produced to the 41 -kDa protein of porcine gp C rotavirus failed to react with several gp A and B rotaviruses in CCIF and ELISA tests (Ojeh et al., 199 1, 1992). However, we previously
Rotaviruses are classified as a genus within the family Reoviridae, based on a characteristic morphology and a genome containing 11 segments of doublestranded (ds) RNA within a double-shelled capsid. At present, rotaviruses are assigned to seven groups (A to G), on the basis of serologic and genome analysis (Pedley et al., 1983, 1986; McNulty et a/., 1984; Snodgrass et al., 1984; Saif and Theil, 1985; Saif, 1990). Each group (gp) of rotaviruses shares its own common group antigen and showssimilarelectrophoretic mobilities of the ds RNA segments. The gp A rotaviruses are widespread, are a major cause of diarrhea in infants and neonatal animals, and compared with non-gp A rotaviruses, are well studied and characterized (Flewett and Woode, 1978; McNulty, 1978). A common group antigen located on the VP6 protein of the inner capsid provides the basis for immunoassays used to detect gp A rotaviruses from different species (Estes and Cohen, 1989; Kapikian and Chanock, 1990). The gp C rotaviruses have been found in swine (Saif et al., 1980; Bohl et a/., 1982; Janke et al., 1990), humans (Bridger et a/., 1986; Mat-
’ Present address: Viral Gastroenteritis Unit, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, GA 30333. ’ To whom reprint requests should be addressed. 0042-6822/92
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction I” any form reserved.
272
ISOLATION
OF GROUP A AND C ROTAVIRUS
reported minor cross-reactivity between gp A and C rotaviruses in indirect immunofluorescent (IF) tests by using hyperimmune polyclonal antisera and gp C rotaviruses propagated in MA1 04 cells (Tsunemitsu e[ al., 1991). In the present study, we produced and characterized MAbs reactive to both gp A and C rotaviruses, confirming the presence of a common antigen shared by both rotavirus serogroups. MATERIALS Rotaviruses
AND METHODS
and hyperimmune
antisera
The Cowden strain of porcine gp C rotavirus was propagated in MA1 04 cells or gnotobiotic pigs (Bohl et a/., 1982; Terrett and Saif, 1987; Saif et al., 1988). Six non-cell-culture-adapted field strains of porcine gp C rotaviruses, each having a distinct ds RNA electropherotype and designated NB, WH, Ah, HF, KH, and Wi, were propagated in gnotobiotic pigs (Jiang et al., 199 1). The Shintoku strain of bovine gp C rotavirus was propagated in MA1 04 cells or colostrum-deprived calves (Tsunemitsu eta/., 1991). Human gp C rotavirus (88-261 strain) was provided as a stool specimen collected from a child with diarrhea by M. Oseto and Y. Yamashita, Ehime Prefectural Institute of Public Health, Ehime, Japan. The porcine (OSU and Gottfried strains), bovine (NCDV strain), and human (Wa strain) gp A rotaviruses were propagated in MA1 04 cells and gnotobiotic pigs (Kang et al., 1989). Hyperimmune antisera to bovine gp A rotavirus (NCDV) and porcine gp C rotavirus (Cowden) were prepared in gnotobiotic pigs (Bohl eT al., 1982; Saif et a/., 1988). lmmunoglobulin (lg) G was purified from these antisera on protein A-Sepharose columns and biotinylated for Western blot and enzyme-linked immunosorbent assays (ELISA) as described previously (Ey et a/., 1978; Simkins et a/., 1989; Ojeh et a/., 1992). Production
of MAbs
Mabs to the Cowden and Ah strains of porcine gp C rotavirus were generated as previously described (Ojeh et a/., 1991, 1992). Briefly spleen cells from mice immunized with Ah gp C rotaviruses were fused with SP2/0 myeloma cells using procedures similarto those described previously (Welch and Saif, 1988; Kang et a/., 1989; Simkins et a/., 1989). Hybridoma supernatants were screened for antibody production against Cowden and Shintoku gp C rotaviruses and NCDV gp A rotavirus by cell culture IF (CCIF) tests. Hybridomas which were positive against both rotavirus serogroups in CCIF tests were cloned five times by limiting dilution to ensure that they were products of single cell clones.
REACTIVE MAbs
273
The lg isotypes of the MAbs was determined by Ouchterlony immunodiffusion assays with monospecific anti-mouse immunoglobulin sera (ICN ImmunoBiologicals, Lisle, IL). Ascitic fluids were produced in pristane-primed BALB/c mice (Welch and Saif, 1988). The ascitic fluids were heat inactivated (56” for 30 min) and used in CCIF and fluorescent focus neutralization (FFN) tests (Kang ef al., 1989). The IgG MAbs were purified from ascitic fluids with protein A-Sepharose columns and used in ELISA (Ey eta/., 1978; Ojeh eta/., 1992). For the Western blot assays, purified MAbs were biotinylated as described previously (Simkins et a/., 1989; Ojeh, 1992). CCIF test Confluent monolayers of MA1 04 cells in 96-well microplates were infected with Cowden porcine gp C rotavirus, Shintoku bovine gp C rotavirus, NCDV bovine gp A rotavirus, and OSU or Gottfried porcine gp A rotaviruses at a multiplicity of infection of 0.2 to 0.4 fluorescence focus units (FFU) per cell as described previously (Terrett and Saif, 1987; Saif et al., 1988; Kang et a/., 1989; Tsunemitsu et al., 1991). After incubation at 37” for 20 hr, the infected cells were washed with phosphate-buffered saline (PBS; pH 7.4) and fixed with 80% acetone. To screen the hybridomas, the infected cells were reacted with 50 ~1 of hybridoma culture fluids at 37” for 1 hr. After washing with PBS, the cells were stained with fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG, IgA, and IgM (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The fixed, infected cells were examined for IF by using a fluorescent microscope. To determine the antibody titers of the MAbs, serial twofold dilutions of ascitic fluids (starting at 1: 10) were added to gp C or gp A rotavirus-infected fixed cells, and the same procedure as described above was followed. The antibody titer of each MAb was expressed as the reciprocal of the highest dilution at which fluorescent cells were detected. FFN test The neutralizing activity of the MAbs against the Cowden and Shintoku strains of gp C rotaviruses and the NCDV strain of gp A rotavirus was determined by a FFN test as described previously (Ojeh et al., 1991). Briefly, equal volumes of ascitic fluids (serial twofold dilutions starting at 1: 10) and viruses containing 200 FFU per 0.05 ml were mixed and reacted at 37” for 1 hr and then at 4” overnight. The mixtures (0.05 ml) were inoculated onto monolayers of MA104 cells grown in 96-well plates. After adsorption at 37” for 1 hr, the cells were washed once with Eagle’s minimum essential me-
274
TSUNEMITSU
dium, received the same medium containing 25 pg of pancreatin per milliliter and were incubated at 37” for 20 hr. The cells were washed with PBS, fixed with 80% acetone, and stained with FITC-conjugated hyperimmune gnotobiotic pig anti-gp A or gp C rotavirus serum. Neutralizing antibody titers were expressed as the reciprocal of the highest ascites dilution that resulted in an 80% or more reduction of FFU. Western
blot assay
Group C (Cowden) and gp A (NCDV) rotavirus were purified from infected MA1 04 cells on CsCl gradients as described previously (Jiang eta/., 1990). The purified viruses were heated at 100” for 3 min in sample buffer [0.05 M Tris (pH 6.8) 100/o glycerol, 2% SDS, 5% 2mercaptoethanol, 0.002% bromophenol blue] and then electrophoresed in 10% polyacrylamide separating gels and 4% stacking gels with Laemmli discontinuous buffer systems (Laemmli, 1970). The gels were electroblotted onto nitrocellulose membranes in Trisglycine buffer containing 20% methanol for 4 hr at 70 V. The blotted membranes were cut into 5-mm-wide strips and washed three times with PBS containing 0.1% Tween 20 (PBS-T). The strips containing molecular weight standards (Bio-Rad Laboratories, Richmond, CA) were stained with 0.1% Amido black 1O-B. Other strips were incubated at 4” overnight with 0.5% NP-40 in PBS, washed three times with PBS-T, and then blocked by incubation with 3% bovine serum albumin (BSA) in PBS at room temperature for 2 hr. Biotinylated purified MAbs or hyperimmune polyclonal antibodies diluted to 1 pg per milliliter with 39/o BSA in PBS-T or ascitic fluids containing IgM MAb diluted 1: 1000 with the same buffer were reacted with the strips at room temperature for 2 hr. Bound antibodies were detected with horseradish peroxidase-conjugated streptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) or horseradish peroxidase-conjugated antimouse lg (A, G, and M; Zymed Laboratories, San Francisco, CA) diluted 1: 1000 in 3% BSA in PBS-T. After washing with PBS-T, the strips were developed with 0.06% diaminobenzidine in 0.1 M Tris-HCI (pH 7.6) containing 0.03% CaCI, and 0.03% H,O,. ELISA An ELISA for determining the reactivities of MAbs against gp A and gp C rotaviruses was performed by using a MAb to capture viral antigens (Burns et al., 1989; Ojeh et a/., 1992). Microtiter plates (MaxiSorp; Nunc, Inc., Naperville, IL) were coated with 0.5 to 10 pg of purified MAbs per milliliter or a 1:400 dilution of ascitic fluids (IgM MAb) in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4’. A purified
ET AL.
MAb to transmissible gastroenteritis virus (25E4; IgG2a) (Simkins et a/., 1989) a gp C rotavirus-specific MAb (RC8B4; IgGl) (Ojeh eta/., 1991, 1992) and a gp A rotavirus-specific MAb (RG25AlO; IgGl) (Kang et a/., 1989) were included as controls. The plates were washed with PBS-T and blocked with 2% BSA in PBS at room temperature for 1 hr. After washing with PBS-T, serial twofold dilutions of cell culture preparations at initial concentrations of 10’ FFU/ml of NCDV gp A rotavirus or 3 X lo5 FFU/ml of Cowden gp C rotavirus or 1:40 dilutions of fecal suspensions from gp A or gp C rotavirus-infected gnotobiotic pigs in PBS-T containing 0.5% BSA and 29/o fetal calf serum were added. The plates were incubated at 37” for 2 hr. Mock-infected cell culture fluids and fecal suspensions from uninoculated gnotobiotic pigs were used for virus-negative controls. After washing with PBS-T, gnotobiotic pig anti-gp A or gp C rotavirus serum diluted 1:20 in PBS-T was added to one of two duplicate wells to ensure the specificity of the reactions by a blocking test. The plates were incubated at 37” for 1 hr and washed with PBS-T. Biotinylated anti-gp A or C rotavirus IgG at a concentration of 1 pg/ml in 0.1 M bicarbonate buffer containing 0.01 M NaCl and 0.002 M KCI (pH 8.2) were added and incubated at 37” for 1 hr. After washing with PBS-T, horseradish peroxidase-conjugated streptavidin diluted 1:4000 in PBS-T was added. The plates were incubated at room temperature for 30 min and washed with PBS-T. ABTS [2,2’-azino-bis(3-ethylbenthizoline-6 sulfonic acid)] in 0.1 l\/I sodium citrate buffer (pH 4.2) containing 0.03% H,O, was added to each well as substrate. The plates were developed at room temperature for 30 min and the reaction was terminated by the addition of 5% SDS. The absorbance of the samples was measured at 414 nm with a microELISA reader (Flow Laboratories, Inc., Reston, VA). Epitope mapping Epitopes were mapped by a competition MAb-capture ELISA as described previously (Hendry et al., 1985; Burns et al., 1988, 1989; Ojeh et al., 1992). Microplates were coated with 1 to 10 pg per milliliter of purified MAbs and blocked with 2% BSA in PBS. Competing MAbs at initial concentrations of 10 pg/ml were serially diluted in PBS-T containing 0.5% BSA and 2% fetal calf serum, mixed with the same volumes of Cowden gp C rotavirus and incubated at 37” for 1 hr and then 4” overnight. The virus-MAb mixtures were added to the above plates and incubated at 37” for 2 hr. The plates were processed as described above, with biotinylated anti-gp C rotavirus IgG as the detector antibody. The percentage of competition was calculated using the formula [lOO(A-B)/A], where A is the
ISOLATION
OF GROUP A AND C ROTAVIRUS
REACTIVE MAbs
275
FIG. 1. lmmunofluorescence in MA1 04 cell monolayers infected with (a) Cowden porcine group C rotavirus and (b) NCDV bovine group A rotavirus and reacted with MAb RCA1 1Al prepared against AH gp C rotavirus (hybridoma cell culture fluid) and then with FITC-conjugated anti-mouse IgG, IgA, and IgM. Magnification, X400.
absorbance in the absence of competitor and B is the absorbance in the presence of competing MAb. Strong competition was considered to be greater than 67% inhibition; intermediate competition, between 33% and 67% inhibition; and weak or no competition, less than 339/o (Simkins et al., 1989). RESULTS Production
of cross-reactive
MAbs
Five MAbs were generated that reacted with both gp A and C rotaviruses in the CCIF tests (Table 1). One MAb (RC8G3) was derived from a mouse immunized
with the Cowden gp C rotavirus and the other four (RCA3D1, RCA5E4, RCA4D10, and RCA1 1Al) were produced from mice immunized with the Ah gp C rotavirus. From three fusions of spleen cells of mice immunized with Ah gp C rotavirus, the cross-reactive hybridomas represented O-2% of the total hybridomas which reacted with Cowden gp C rotavirus in the CCIF tests. All were of the IgG isotype except for RCA4D10, which was IgM. All MAbs showed strong intracytoplasmic fluorescence by CCIF in MA1 04 cells infected with gp A or gp C rotaviruses (Fig. 1). None of the MAbs reacted with mock-infected control cells. The antimouse Ig-FITC conjugate and preinoculation sera from
276
TSUNEMITSU
ET AL.
TABLE 1 IMMUNOGLOBULIN(lg) ISOWPESOF MAbs PREPAREDTO Group C ROTAVIRUSREACTIVEWITH GROUPA AND GROUPC ROTAVIRUSES AND THEIR ANTIBODY TITERSAGAINSTBOTH ROTAVIRUSSEROGROUPSIN A CCIF TEST Indirect CCIF titef
against rotavirus (Host/virus
Group C MAb (ascites)
lg isotype
RC8G3 RCA3Dl RCA5E3 RCA4DlO RCA1 1Al RC8134c
IgGl IgG2a IgGl IgM IgG2a IgGl
Po/Cowden/ND
Group A Bo/Shintoku/ND
102,400 204,800 204,800 25,600 409,600 51,200
6,400 1,600 6,400 25,600 102,400 25,600
Bo/NCDV/l 6,400 12,800 102,400 102,400 409,600 RCA3D1, RCA4DlO ti RCA8G3. Their relative affinity for the NCDV strain of gp A rotavirus was RCA1 1Al > RG25AlO > RCA8G3 > RCA5E3, RCA3Dl > RCA4DlO. The relatively high affinity of RCA1 1Al for both virus strains permitted its use as a capture antibody in assays for detecting rotavirus in fecal samples from ani-
Po/osu/1 12,800 3,200 12,800 25,600 102,400
191, 272-281
(1992)
Production and Characterization of Monoclonal Antibodies to Porcine Group C Rotaviruses Cross-Reactive with Group A Rotaviruses HIROSHI TSUNEMITSU,**t CLEM K. OJEH,t+ BAOMING JIANG,?’ RON A. SIMKINS,t PEGGY A. WEILNAU,t AND LINDA J. SAIFt*’ ‘Hokkaido
Prefectural Shintoku Animal Husbandry Experiment Station, Shintoku, Hokkaido 08 1, Japan; tFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 4469 1; and *Department of Veterinary Microbiology and Parasitology, University of Ibadan, lbadan, Nigeria Received May 26, 1992; accepted July 21, 1992
Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41 -kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is 0 1992 Academic Press, Inc. recognized by certain MAbs in various serologic assays.
INTRODUCTION
sumoto et a/., 1989; Penaranda et al., 1989; Ushijima et al., 1989; Caul et a/., 1990), and more recently in cattle (Tsunemitsu et al., 1991) with diarrhea. Preliminary serological surveys indicated that gp C rotaviruses were prevalent in swine in North America, Australia, and Europe (Bridger et al., 1985; Nagesha et a/., 1988; Saif, 1990). Recently, a large-scale outbreak of diarrhea caused by gp C rotavirus in children in Japan has been reported (Matsumoto eta/., 1989). Studies of the viral structural proteins of porcine gp C rotavirus have revealed that the 41 -kDa inner shell protein is the counterpart to VP6 of gp A rotavirus and contains common group antigens (Bremont et al., 1988; Jiang et a/., 1990; Ojeh et al., 1991, 1992). At present only porcine and bovine gp C rotavirus have been successfully propagated in a monkey kidney cell line (MA104) (Terrett and Saif, 1987; Saif et a/., 1988; Tsunemitsu et a/., 1991). Although the failure of in vitro propagation of other non-gp A rotaviruses and limited quantities of samples containing non-gp A rotaviruses have hampered antigenic comparisons, most reports suggest that no common antigens exist between rotaviruses from different serogroups (Pedley et al., 1983, 1986; Snodgrass et al., 1984; Saif and Theil, 1985). A prior report from our laboratory confirmed that three monoclonal antibodies (MAbs) produced to the 41 -kDa protein of porcine gp C rotavirus failed to react with several gp A and B rotaviruses in CCIF and ELISA tests (Ojeh et al., 199 1, 1992). However, we previously
Rotaviruses are classified as a genus within the family Reoviridae, based on a characteristic morphology and a genome containing 11 segments of doublestranded (ds) RNA within a double-shelled capsid. At present, rotaviruses are assigned to seven groups (A to G), on the basis of serologic and genome analysis (Pedley et al., 1983, 1986; McNulty et a/., 1984; Snodgrass et al., 1984; Saif and Theil, 1985; Saif, 1990). Each group (gp) of rotaviruses shares its own common group antigen and showssimilarelectrophoretic mobilities of the ds RNA segments. The gp A rotaviruses are widespread, are a major cause of diarrhea in infants and neonatal animals, and compared with non-gp A rotaviruses, are well studied and characterized (Flewett and Woode, 1978; McNulty, 1978). A common group antigen located on the VP6 protein of the inner capsid provides the basis for immunoassays used to detect gp A rotaviruses from different species (Estes and Cohen, 1989; Kapikian and Chanock, 1990). The gp C rotaviruses have been found in swine (Saif et al., 1980; Bohl et a/., 1982; Janke et al., 1990), humans (Bridger et a/., 1986; Mat-
’ Present address: Viral Gastroenteritis Unit, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, GA 30333. ’ To whom reprint requests should be addressed. 0042-6822/92
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction I” any form reserved.
272
ISOLATION
OF GROUP A AND C ROTAVIRUS
reported minor cross-reactivity between gp A and C rotaviruses in indirect immunofluorescent (IF) tests by using hyperimmune polyclonal antisera and gp C rotaviruses propagated in MA1 04 cells (Tsunemitsu e[ al., 1991). In the present study, we produced and characterized MAbs reactive to both gp A and C rotaviruses, confirming the presence of a common antigen shared by both rotavirus serogroups. MATERIALS Rotaviruses
AND METHODS
and hyperimmune
antisera
The Cowden strain of porcine gp C rotavirus was propagated in MA1 04 cells or gnotobiotic pigs (Bohl et a/., 1982; Terrett and Saif, 1987; Saif et al., 1988). Six non-cell-culture-adapted field strains of porcine gp C rotaviruses, each having a distinct ds RNA electropherotype and designated NB, WH, Ah, HF, KH, and Wi, were propagated in gnotobiotic pigs (Jiang et al., 199 1). The Shintoku strain of bovine gp C rotavirus was propagated in MA1 04 cells or colostrum-deprived calves (Tsunemitsu eta/., 1991). Human gp C rotavirus (88-261 strain) was provided as a stool specimen collected from a child with diarrhea by M. Oseto and Y. Yamashita, Ehime Prefectural Institute of Public Health, Ehime, Japan. The porcine (OSU and Gottfried strains), bovine (NCDV strain), and human (Wa strain) gp A rotaviruses were propagated in MA1 04 cells and gnotobiotic pigs (Kang et al., 1989). Hyperimmune antisera to bovine gp A rotavirus (NCDV) and porcine gp C rotavirus (Cowden) were prepared in gnotobiotic pigs (Bohl eT al., 1982; Saif et a/., 1988). lmmunoglobulin (lg) G was purified from these antisera on protein A-Sepharose columns and biotinylated for Western blot and enzyme-linked immunosorbent assays (ELISA) as described previously (Ey et a/., 1978; Simkins et a/., 1989; Ojeh et a/., 1992). Production
of MAbs
Mabs to the Cowden and Ah strains of porcine gp C rotavirus were generated as previously described (Ojeh et a/., 1991, 1992). Briefly spleen cells from mice immunized with Ah gp C rotaviruses were fused with SP2/0 myeloma cells using procedures similarto those described previously (Welch and Saif, 1988; Kang et a/., 1989; Simkins et a/., 1989). Hybridoma supernatants were screened for antibody production against Cowden and Shintoku gp C rotaviruses and NCDV gp A rotavirus by cell culture IF (CCIF) tests. Hybridomas which were positive against both rotavirus serogroups in CCIF tests were cloned five times by limiting dilution to ensure that they were products of single cell clones.
REACTIVE MAbs
273
The lg isotypes of the MAbs was determined by Ouchterlony immunodiffusion assays with monospecific anti-mouse immunoglobulin sera (ICN ImmunoBiologicals, Lisle, IL). Ascitic fluids were produced in pristane-primed BALB/c mice (Welch and Saif, 1988). The ascitic fluids were heat inactivated (56” for 30 min) and used in CCIF and fluorescent focus neutralization (FFN) tests (Kang ef al., 1989). The IgG MAbs were purified from ascitic fluids with protein A-Sepharose columns and used in ELISA (Ey eta/., 1978; Ojeh eta/., 1992). For the Western blot assays, purified MAbs were biotinylated as described previously (Simkins et a/., 1989; Ojeh, 1992). CCIF test Confluent monolayers of MA1 04 cells in 96-well microplates were infected with Cowden porcine gp C rotavirus, Shintoku bovine gp C rotavirus, NCDV bovine gp A rotavirus, and OSU or Gottfried porcine gp A rotaviruses at a multiplicity of infection of 0.2 to 0.4 fluorescence focus units (FFU) per cell as described previously (Terrett and Saif, 1987; Saif et al., 1988; Kang et a/., 1989; Tsunemitsu et al., 1991). After incubation at 37” for 20 hr, the infected cells were washed with phosphate-buffered saline (PBS; pH 7.4) and fixed with 80% acetone. To screen the hybridomas, the infected cells were reacted with 50 ~1 of hybridoma culture fluids at 37” for 1 hr. After washing with PBS, the cells were stained with fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG, IgA, and IgM (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The fixed, infected cells were examined for IF by using a fluorescent microscope. To determine the antibody titers of the MAbs, serial twofold dilutions of ascitic fluids (starting at 1: 10) were added to gp C or gp A rotavirus-infected fixed cells, and the same procedure as described above was followed. The antibody titer of each MAb was expressed as the reciprocal of the highest dilution at which fluorescent cells were detected. FFN test The neutralizing activity of the MAbs against the Cowden and Shintoku strains of gp C rotaviruses and the NCDV strain of gp A rotavirus was determined by a FFN test as described previously (Ojeh et al., 1991). Briefly, equal volumes of ascitic fluids (serial twofold dilutions starting at 1: 10) and viruses containing 200 FFU per 0.05 ml were mixed and reacted at 37” for 1 hr and then at 4” overnight. The mixtures (0.05 ml) were inoculated onto monolayers of MA104 cells grown in 96-well plates. After adsorption at 37” for 1 hr, the cells were washed once with Eagle’s minimum essential me-
274
TSUNEMITSU
dium, received the same medium containing 25 pg of pancreatin per milliliter and were incubated at 37” for 20 hr. The cells were washed with PBS, fixed with 80% acetone, and stained with FITC-conjugated hyperimmune gnotobiotic pig anti-gp A or gp C rotavirus serum. Neutralizing antibody titers were expressed as the reciprocal of the highest ascites dilution that resulted in an 80% or more reduction of FFU. Western
blot assay
Group C (Cowden) and gp A (NCDV) rotavirus were purified from infected MA1 04 cells on CsCl gradients as described previously (Jiang eta/., 1990). The purified viruses were heated at 100” for 3 min in sample buffer [0.05 M Tris (pH 6.8) 100/o glycerol, 2% SDS, 5% 2mercaptoethanol, 0.002% bromophenol blue] and then electrophoresed in 10% polyacrylamide separating gels and 4% stacking gels with Laemmli discontinuous buffer systems (Laemmli, 1970). The gels were electroblotted onto nitrocellulose membranes in Trisglycine buffer containing 20% methanol for 4 hr at 70 V. The blotted membranes were cut into 5-mm-wide strips and washed three times with PBS containing 0.1% Tween 20 (PBS-T). The strips containing molecular weight standards (Bio-Rad Laboratories, Richmond, CA) were stained with 0.1% Amido black 1O-B. Other strips were incubated at 4” overnight with 0.5% NP-40 in PBS, washed three times with PBS-T, and then blocked by incubation with 3% bovine serum albumin (BSA) in PBS at room temperature for 2 hr. Biotinylated purified MAbs or hyperimmune polyclonal antibodies diluted to 1 pg per milliliter with 39/o BSA in PBS-T or ascitic fluids containing IgM MAb diluted 1: 1000 with the same buffer were reacted with the strips at room temperature for 2 hr. Bound antibodies were detected with horseradish peroxidase-conjugated streptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) or horseradish peroxidase-conjugated antimouse lg (A, G, and M; Zymed Laboratories, San Francisco, CA) diluted 1: 1000 in 3% BSA in PBS-T. After washing with PBS-T, the strips were developed with 0.06% diaminobenzidine in 0.1 M Tris-HCI (pH 7.6) containing 0.03% CaCI, and 0.03% H,O,. ELISA An ELISA for determining the reactivities of MAbs against gp A and gp C rotaviruses was performed by using a MAb to capture viral antigens (Burns et al., 1989; Ojeh et a/., 1992). Microtiter plates (MaxiSorp; Nunc, Inc., Naperville, IL) were coated with 0.5 to 10 pg of purified MAbs per milliliter or a 1:400 dilution of ascitic fluids (IgM MAb) in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4’. A purified
ET AL.
MAb to transmissible gastroenteritis virus (25E4; IgG2a) (Simkins et a/., 1989) a gp C rotavirus-specific MAb (RC8B4; IgGl) (Ojeh eta/., 1991, 1992) and a gp A rotavirus-specific MAb (RG25AlO; IgGl) (Kang et a/., 1989) were included as controls. The plates were washed with PBS-T and blocked with 2% BSA in PBS at room temperature for 1 hr. After washing with PBS-T, serial twofold dilutions of cell culture preparations at initial concentrations of 10’ FFU/ml of NCDV gp A rotavirus or 3 X lo5 FFU/ml of Cowden gp C rotavirus or 1:40 dilutions of fecal suspensions from gp A or gp C rotavirus-infected gnotobiotic pigs in PBS-T containing 0.5% BSA and 29/o fetal calf serum were added. The plates were incubated at 37” for 2 hr. Mock-infected cell culture fluids and fecal suspensions from uninoculated gnotobiotic pigs were used for virus-negative controls. After washing with PBS-T, gnotobiotic pig anti-gp A or gp C rotavirus serum diluted 1:20 in PBS-T was added to one of two duplicate wells to ensure the specificity of the reactions by a blocking test. The plates were incubated at 37” for 1 hr and washed with PBS-T. Biotinylated anti-gp A or C rotavirus IgG at a concentration of 1 pg/ml in 0.1 M bicarbonate buffer containing 0.01 M NaCl and 0.002 M KCI (pH 8.2) were added and incubated at 37” for 1 hr. After washing with PBS-T, horseradish peroxidase-conjugated streptavidin diluted 1:4000 in PBS-T was added. The plates were incubated at room temperature for 30 min and washed with PBS-T. ABTS [2,2’-azino-bis(3-ethylbenthizoline-6 sulfonic acid)] in 0.1 l\/I sodium citrate buffer (pH 4.2) containing 0.03% H,O, was added to each well as substrate. The plates were developed at room temperature for 30 min and the reaction was terminated by the addition of 5% SDS. The absorbance of the samples was measured at 414 nm with a microELISA reader (Flow Laboratories, Inc., Reston, VA). Epitope mapping Epitopes were mapped by a competition MAb-capture ELISA as described previously (Hendry et al., 1985; Burns et al., 1988, 1989; Ojeh et al., 1992). Microplates were coated with 1 to 10 pg per milliliter of purified MAbs and blocked with 2% BSA in PBS. Competing MAbs at initial concentrations of 10 pg/ml were serially diluted in PBS-T containing 0.5% BSA and 2% fetal calf serum, mixed with the same volumes of Cowden gp C rotavirus and incubated at 37” for 1 hr and then 4” overnight. The virus-MAb mixtures were added to the above plates and incubated at 37” for 2 hr. The plates were processed as described above, with biotinylated anti-gp C rotavirus IgG as the detector antibody. The percentage of competition was calculated using the formula [lOO(A-B)/A], where A is the
ISOLATION
OF GROUP A AND C ROTAVIRUS
REACTIVE MAbs
275
FIG. 1. lmmunofluorescence in MA1 04 cell monolayers infected with (a) Cowden porcine group C rotavirus and (b) NCDV bovine group A rotavirus and reacted with MAb RCA1 1Al prepared against AH gp C rotavirus (hybridoma cell culture fluid) and then with FITC-conjugated anti-mouse IgG, IgA, and IgM. Magnification, X400.
absorbance in the absence of competitor and B is the absorbance in the presence of competing MAb. Strong competition was considered to be greater than 67% inhibition; intermediate competition, between 33% and 67% inhibition; and weak or no competition, less than 339/o (Simkins et al., 1989). RESULTS Production
of cross-reactive
MAbs
Five MAbs were generated that reacted with both gp A and C rotaviruses in the CCIF tests (Table 1). One MAb (RC8G3) was derived from a mouse immunized
with the Cowden gp C rotavirus and the other four (RCA3D1, RCA5E4, RCA4D10, and RCA1 1Al) were produced from mice immunized with the Ah gp C rotavirus. From three fusions of spleen cells of mice immunized with Ah gp C rotavirus, the cross-reactive hybridomas represented O-2% of the total hybridomas which reacted with Cowden gp C rotavirus in the CCIF tests. All were of the IgG isotype except for RCA4D10, which was IgM. All MAbs showed strong intracytoplasmic fluorescence by CCIF in MA1 04 cells infected with gp A or gp C rotaviruses (Fig. 1). None of the MAbs reacted with mock-infected control cells. The antimouse Ig-FITC conjugate and preinoculation sera from
276
TSUNEMITSU
ET AL.
TABLE 1 IMMUNOGLOBULIN(lg) ISOWPESOF MAbs PREPAREDTO Group C ROTAVIRUSREACTIVEWITH GROUPA AND GROUPC ROTAVIRUSES AND THEIR ANTIBODY TITERSAGAINSTBOTH ROTAVIRUSSEROGROUPSIN A CCIF TEST Indirect CCIF titef
against rotavirus (Host/virus
Group C MAb (ascites)
lg isotype
RC8G3 RCA3Dl RCA5E3 RCA4DlO RCA1 1Al RC8134c
IgGl IgG2a IgGl IgM IgG2a IgGl
Po/Cowden/ND
Group A Bo/Shintoku/ND
102,400 204,800 204,800 25,600 409,600 51,200
6,400 1,600 6,400 25,600 102,400 25,600
Bo/NCDV/l 6,400 12,800 102,400 102,400 409,600 RCA3D1, RCA4DlO ti RCA8G3. Their relative affinity for the NCDV strain of gp A rotavirus was RCA1 1Al > RG25AlO > RCA8G3 > RCA5E3, RCA3Dl > RCA4DlO. The relatively high affinity of RCA1 1Al for both virus strains permitted its use as a capture antibody in assays for detecting rotavirus in fecal samples from ani-
Po/osu/1 12,800 3,200 12,800 25,600 102,400
