JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 2051-2055 0095-1137/91/092051-05$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 29, No. 9
Reactivity of Monoclonal Antibodies to the 41-Kilodalton Protein of Porcine Group C Rotavirus with Homologous and Heterologous Rotavirus Serogroups in Immunofluorescence Testst C. K. OJEH,1 B. M. JIANG,2 H. TSUNEMITSU,2 S. Y. KANG,3 P. A. WEILNAU,2 AND L. J. SAIF2* Department of Veterinary Microbiology and Parasitology, University of Ibadan, Ibadan, Nigeria'; Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 446912; and Department of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea3 Received 21 February 1991/Accepted 29 February 1991
Three monoclonal antibodies (MAbs) to porcine group C rotavirus immunoprecipitated the major inner capsid protein (41 kDa) but failed to precipitate group A rotavirus proteins. In immunofluorescence tests of rotavirus-infected cell cultures or pig intestines, the MAbs recognized porcine and bovine group C rotaviruses but not group A or B rotaviruses. These MAbs may recognize the group C rotavirus counterpart to VP6 of group A rotaviruses and may be useful as diagnostic reagents.
Rotaviruses have been established as a leading etiological
making it easier to further study this group of viruses and to develop the diagnostic reagents necessary for virus detection and serogroup analysis. Consequently, the objective of this study was to produce monoclonal antibodies (MAbs) to the 41-kDa protein of porcine gp C rotavirus reactive in immunofluorescence (IF) tests. Virus strains and intestinal mucosal smears. The gp C rotaviruses used in this study included two strains adapted to serial propagation in MA104 cells: the Cowden porcine gp C rotavirus (28, 32) and the Shintoku bovine gp C rotavirus (36). Three non-cell-culture-adapted field strains of porcine gp C rotavirus, each having a distinct double-stranded RNA electropherotype and designated NB, SB, and FS, respectively, were obtained from 4- to 27-day-old pigs with diarrhea (16). Other rotavirus serogroups used in this study included the porcine (OSU and Gottfried) and bovine (NCDV) gp A rotaviruses and the porcine (Ohio) and bovine (ATI) gp B rotaviruses (26, 29, 35). Each rotavirus strain (except the three field strains) was inoculated onto MA104 cell monolayers for use in IF tests as described previously (2, 28, 29, 33, 34). Gnotobiotic pigs inoculated with porcine gp C rotaviruses, including the Cowden and the three field strains, Gottfried gp A rotavirus, Ohio gp B rotavirus, or the porcine coronavirus, the transmissible gastroenteritis virus (TGEV), were euthanized at the onset of diarrhea (approximately 20 to 48 h postexposure). Small intestinal mucosal smears were prepared as described previously (1, 2, 35, 38). Smears were first reacted with the MAbs (ascitic fluids diluted 1:50) followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG), IgA, and IgM (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.). As controls, smears were reacted directly with the polyclonal pig anti-gp A, B, or C sera conjugated to FITC. Sections were examined for IF by using a fluorescence microscope (Olympus IM, Tokyo, Japan). Production of MAbs. Female BALB/c mice (age, 8 to 10 weeks) were immunized intraperitoneally at weekly intervals with the Cowden strain of gp C rotavirus, which was semipurified from the contents of the large intestines of infected gnotobiotic piglets (1, 17, 38). Spleen cells from these mice were fused with SP2/0 mouse myeloma cells by following the procedures of Welch and Saif (38) and Kang et
agent of gastroenteritis in both human infants and neonatal
animals (11, 18). Subsequent studies have revealed that there are rotaviruses that are morphologically indistinguishable but that are antigenically and electropherotypically distinct from each other (4, 8-10, 13, 14, 20-29, 34-37). As a result of these distinctive characteristics, the rotaviruses were subdivided into at least seven different serogroups, namely, groups A to G (23, 24, 26, 29). Group (gp) A rotaviruses have been extensively studied and characterized, whereas the other serogroups (B to G), often referred to as atypical or non-group A rotaviruses, remain poorly characterized. The gp C rotaviruses, or pararotaviruses, were first reported in swine (27) and later in humans (25). Recent findings also suggest their occurrence in cattle with diarrhea (36). Studies on the viral structural polypeptides of porcine gp C rotavirus have revealed six or seven polypeptides (3, 15), with the 41-kDa inner shell protein being the most abundant (15). Similarities in several properties (molecular mass, inner shell protein, and greatest abundance) suggest that the 41-kDa protein may be the counterpart to VP6 of gp A rotavirus, which carries the common group antigen (11, 18). Like gp A rotaviruses, porcine gp C rotaviruses experimentally induce diarrhea in gnotobiotic piglets (1, 2); preliminary serological surveys have shown that they are prevalent in swine herds in the United States, Europe, and Australia (5, 8, 22, 26, 33), although their global distribution and epidemiological significance have yet to be determined. Only a few sporadic cases of diarrhea associated with gp C rotaviruses in children were reported initially (9, 10, 25), but
more recently, gp C rotaviruses have been associated with outbreaks of diarrhea in children and adults in the United Kingdom (7) and Japan (20, 37). The non-group A rotaviruses are fastidious in their in vitro growth requirements, and this has greatly limited serological studies and further characterization of these viruses. Recently, however, a porcine gp C rotavirus has been successfully propagated in vitro in both primary porcine kidney cells and a monkey kidney (MA104) cell line (28, 32), thereby
Corresponding author. t This is journal article 24-91 of the Ohio Agricultural Research and Development Center, Ohio State University. *
2051
2052
J. CLIN. MICROBIOL.
NOTES
supernatant __ ascites polyclonal U GpA (.PC U GpA GPC U GpA CGpC
TABLE 1. Ig isotypes of MAbs to the 41-kDa protein of porcine gp C rotavirus and their antibody titers with homologous and heterologous porcine and bovine rotavirus serogroups in CCIF tests MAb Ig (ascites) isotype
RC8B4 RC9E5 RC1SB7
IgGl IgG3 IgGl
205
Antibody titers of MAbs against the following rotavirus serogroups with the indicated hosta: C A B Porcine Bovine
51,200 25,600 12,800 6,400 51,200 51,200
M
116 97
Porcine Bovine Porcine Bovine
Vol. 29, No. 9
Reactivity of Monoclonal Antibodies to the 41-Kilodalton Protein of Porcine Group C Rotavirus with Homologous and Heterologous Rotavirus Serogroups in Immunofluorescence Testst C. K. OJEH,1 B. M. JIANG,2 H. TSUNEMITSU,2 S. Y. KANG,3 P. A. WEILNAU,2 AND L. J. SAIF2* Department of Veterinary Microbiology and Parasitology, University of Ibadan, Ibadan, Nigeria'; Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 446912; and Department of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea3 Received 21 February 1991/Accepted 29 February 1991
Three monoclonal antibodies (MAbs) to porcine group C rotavirus immunoprecipitated the major inner capsid protein (41 kDa) but failed to precipitate group A rotavirus proteins. In immunofluorescence tests of rotavirus-infected cell cultures or pig intestines, the MAbs recognized porcine and bovine group C rotaviruses but not group A or B rotaviruses. These MAbs may recognize the group C rotavirus counterpart to VP6 of group A rotaviruses and may be useful as diagnostic reagents.
Rotaviruses have been established as a leading etiological
making it easier to further study this group of viruses and to develop the diagnostic reagents necessary for virus detection and serogroup analysis. Consequently, the objective of this study was to produce monoclonal antibodies (MAbs) to the 41-kDa protein of porcine gp C rotavirus reactive in immunofluorescence (IF) tests. Virus strains and intestinal mucosal smears. The gp C rotaviruses used in this study included two strains adapted to serial propagation in MA104 cells: the Cowden porcine gp C rotavirus (28, 32) and the Shintoku bovine gp C rotavirus (36). Three non-cell-culture-adapted field strains of porcine gp C rotavirus, each having a distinct double-stranded RNA electropherotype and designated NB, SB, and FS, respectively, were obtained from 4- to 27-day-old pigs with diarrhea (16). Other rotavirus serogroups used in this study included the porcine (OSU and Gottfried) and bovine (NCDV) gp A rotaviruses and the porcine (Ohio) and bovine (ATI) gp B rotaviruses (26, 29, 35). Each rotavirus strain (except the three field strains) was inoculated onto MA104 cell monolayers for use in IF tests as described previously (2, 28, 29, 33, 34). Gnotobiotic pigs inoculated with porcine gp C rotaviruses, including the Cowden and the three field strains, Gottfried gp A rotavirus, Ohio gp B rotavirus, or the porcine coronavirus, the transmissible gastroenteritis virus (TGEV), were euthanized at the onset of diarrhea (approximately 20 to 48 h postexposure). Small intestinal mucosal smears were prepared as described previously (1, 2, 35, 38). Smears were first reacted with the MAbs (ascitic fluids diluted 1:50) followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG), IgA, and IgM (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.). As controls, smears were reacted directly with the polyclonal pig anti-gp A, B, or C sera conjugated to FITC. Sections were examined for IF by using a fluorescence microscope (Olympus IM, Tokyo, Japan). Production of MAbs. Female BALB/c mice (age, 8 to 10 weeks) were immunized intraperitoneally at weekly intervals with the Cowden strain of gp C rotavirus, which was semipurified from the contents of the large intestines of infected gnotobiotic piglets (1, 17, 38). Spleen cells from these mice were fused with SP2/0 mouse myeloma cells by following the procedures of Welch and Saif (38) and Kang et
agent of gastroenteritis in both human infants and neonatal
animals (11, 18). Subsequent studies have revealed that there are rotaviruses that are morphologically indistinguishable but that are antigenically and electropherotypically distinct from each other (4, 8-10, 13, 14, 20-29, 34-37). As a result of these distinctive characteristics, the rotaviruses were subdivided into at least seven different serogroups, namely, groups A to G (23, 24, 26, 29). Group (gp) A rotaviruses have been extensively studied and characterized, whereas the other serogroups (B to G), often referred to as atypical or non-group A rotaviruses, remain poorly characterized. The gp C rotaviruses, or pararotaviruses, were first reported in swine (27) and later in humans (25). Recent findings also suggest their occurrence in cattle with diarrhea (36). Studies on the viral structural polypeptides of porcine gp C rotavirus have revealed six or seven polypeptides (3, 15), with the 41-kDa inner shell protein being the most abundant (15). Similarities in several properties (molecular mass, inner shell protein, and greatest abundance) suggest that the 41-kDa protein may be the counterpart to VP6 of gp A rotavirus, which carries the common group antigen (11, 18). Like gp A rotaviruses, porcine gp C rotaviruses experimentally induce diarrhea in gnotobiotic piglets (1, 2); preliminary serological surveys have shown that they are prevalent in swine herds in the United States, Europe, and Australia (5, 8, 22, 26, 33), although their global distribution and epidemiological significance have yet to be determined. Only a few sporadic cases of diarrhea associated with gp C rotaviruses in children were reported initially (9, 10, 25), but
more recently, gp C rotaviruses have been associated with outbreaks of diarrhea in children and adults in the United Kingdom (7) and Japan (20, 37). The non-group A rotaviruses are fastidious in their in vitro growth requirements, and this has greatly limited serological studies and further characterization of these viruses. Recently, however, a porcine gp C rotavirus has been successfully propagated in vitro in both primary porcine kidney cells and a monkey kidney (MA104) cell line (28, 32), thereby
Corresponding author. t This is journal article 24-91 of the Ohio Agricultural Research and Development Center, Ohio State University. *
2051
2052
J. CLIN. MICROBIOL.
NOTES
supernatant __ ascites polyclonal U GpA (.PC U GpA GPC U GpA CGpC
TABLE 1. Ig isotypes of MAbs to the 41-kDa protein of porcine gp C rotavirus and their antibody titers with homologous and heterologous porcine and bovine rotavirus serogroups in CCIF tests MAb Ig (ascites) isotype
RC8B4 RC9E5 RC1SB7
IgGl IgG3 IgGl
205
Antibody titers of MAbs against the following rotavirus serogroups with the indicated hosta: C A B Porcine Bovine
51,200 25,600 12,800 6,400 51,200 51,200
M
116 97
Porcine Bovine Porcine Bovine
