_Archives
Vi rology
Arch Virol (1992) 122:359-365
© Springer-Verlag 1992 Printed in Austria
Glycoprotein-specific immune response in canine herpesvirus infection
Brief R e p o r t X. Xuan, T. Horimoto, J. A. Limcumpao, Y. Tohya, E. Takahashi, and T. Mikami
Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Accepted August 26, 1991
Summary. Sera from dogs which were infected with canine herpesvirus (CHV) were analyzed for their serological reactivities against virus-specific glycoproteins (gps). By sequential immunoblot analysis using sera from experimentally infected dogs, it was found that the antibody response to gp 145/112 appeared first followed by responses to gp 47 and gp 80. In addition, all sera from naturally infected dogs which showed neutralizing activity to CHV reacted with gp 145/ 112, whereas 77% and 70% reacted with gp47 and gp 80, respectively. Furthermore, some of the sera also cross-neutralized feline herpesvirus type 1 (FHV1) and reacted with gp 143/108 of FHV-1, indicating that gp 145/112 of CHV induced cross-neutralizing antibody response to FHV-1.
Canine herpesvirus (CHV), family Herpesviridae, subfamily Alphaherpesvirinae, causes a fatal haemorrhagic disease in neonatal and infant puppies and an upper respiratory tract disease in adult dogs [2-4, 14]. CHV appears to have a worldwide distribution as it has been isolated from dogs in many countries [1, 7, 10, 17, 21]. At present, the detection of CHV infection is done mainly by the complement-dependent virus neutralization (VN) test [6, 11]. Recently, we developed an enzyme-linked immunosorbent assay (ELISA) [20] and a haemagglutination-inhibition (HI) test [16] for the serological diagnosis of CHV infection. Previously, with the use of monoclonal antibodies (MoAbs), 145/112kDa glycoproteins (gp 145/112), gp80, and gp47 were identified to be the major immunogenic gps of CHV [24]. Furthermore, these gps were purified and used in immunizing mice to determine their relative immunogenicity [13]. However, the immune responses in dogs against these gps have not been investigated yet.
360
x. Xuan et al.
The purpose of this study is to examine the temporal appearance of antibodies to CHV in experimentally and naturally infected dogs by immunoblot and other conventional methods. Additionally, cross-reactivities between gps of CHV and feline herpesvirus type 1 (FHV-1) are discussed. Y P l l [25] and YP8702 [16] strains of CHV and C7301 strain [15] of FHV-1, which were respectively propagated in Madin-Darby canine kidney (MDCK) and Crandell feline kidney (CRFK) cell cultures, were used throughout the present study. The procedures of VN test [8, 20], HI test [16], and ELISA [20] were described previously. Briefly, for detection of VN antibodies serial dilutions of sera were mixed with virus (50 plaque-forming units) and 15% fresh guinea pig serum as a source of complement and incubated for 1 h at 37 °C in the microplate-wells. Then, suspensions of M D C K or C R F K cells were added to the wells and incubated for 3 days. VN titers were expressed as the reciprocal of the highest serum dilution showing 50% inhibition of the appearance of cytopathic effects. For detection of HI antibodies, the mixture of diluted sera and haemagglutinin-antigen (4 haemagglutination units) were incubated for 1 h at 37 °C. Then dog red blood cell suspensions (0.4%) were added and incubated for an additional 2 h before reading. HI titers were expressed as the reciprocal of the highest serum dilution resulting in complete haemagglutination-inhibition. For detection of ELISA antibodies, virus- or mock-infected cell lysates prepared by solubilizing with 0.5% sodium deoxycholate were used as ELISA antigens [20]. ELISA titers were expressed as the reciprocal of the highest serum dilution giving the absorbance difference at 405 nm of 0.1 or larger between the virus- and mock-infected cell lysate antigens. Immunoblot assay was carried out according to the previous method [24]. Virus- or mock-infected cell lysates which were separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% running gel under nonreducing conditions were electrophoretically transferred to polyvinyliden difluoride papers (Immobilon, Japan Millipore, Tokyo) by the Towbin's method [22]. The blotting paper was used for immunodetection. First, we examined the development of immune responses to CHV in experimentally infected dogs. The manners of experimental infection of a dog using CHV YP 11 strain were described elsewhere [20]. No clinical symptoms were manifested after the CHV inoculation. Sequential serum samples from the dog were analyzed by VN test, HI test, ELISA, and immunoblot assay (Fig. 1). No antibodies were detected in the sera collected 1 and 2 weeks post-inoculation (WPI). ELISA and VN antibodies were first detected at 3 and 4 WPI, respectively, while HI antibodies were at 10 WPI. On the other hand, the serum at 3 WPI reacted weakly with gp 145/112 but not with the other proteins in immunoblot analysis. Antibodies to gp 47 and gp 80 were first detected in sera at 9 and 10 WPI. Similar results were obtained with the series of sera collected from another dog experimentally infected with CHV YP 8702 strain (data not shown). These results suggest that antibodies to gp 145/112 appeared in the early stages of the infection and those to gp 47 and gp 80 appeared later, although
Immune responses to CHV glycoproteins
361
512 256 128
.~ 64 "-
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4
Vi rology
Arch Virol (1992) 122:359-365
© Springer-Verlag 1992 Printed in Austria
Glycoprotein-specific immune response in canine herpesvirus infection
Brief R e p o r t X. Xuan, T. Horimoto, J. A. Limcumpao, Y. Tohya, E. Takahashi, and T. Mikami
Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Accepted August 26, 1991
Summary. Sera from dogs which were infected with canine herpesvirus (CHV) were analyzed for their serological reactivities against virus-specific glycoproteins (gps). By sequential immunoblot analysis using sera from experimentally infected dogs, it was found that the antibody response to gp 145/112 appeared first followed by responses to gp 47 and gp 80. In addition, all sera from naturally infected dogs which showed neutralizing activity to CHV reacted with gp 145/ 112, whereas 77% and 70% reacted with gp47 and gp 80, respectively. Furthermore, some of the sera also cross-neutralized feline herpesvirus type 1 (FHV1) and reacted with gp 143/108 of FHV-1, indicating that gp 145/112 of CHV induced cross-neutralizing antibody response to FHV-1.
Canine herpesvirus (CHV), family Herpesviridae, subfamily Alphaherpesvirinae, causes a fatal haemorrhagic disease in neonatal and infant puppies and an upper respiratory tract disease in adult dogs [2-4, 14]. CHV appears to have a worldwide distribution as it has been isolated from dogs in many countries [1, 7, 10, 17, 21]. At present, the detection of CHV infection is done mainly by the complement-dependent virus neutralization (VN) test [6, 11]. Recently, we developed an enzyme-linked immunosorbent assay (ELISA) [20] and a haemagglutination-inhibition (HI) test [16] for the serological diagnosis of CHV infection. Previously, with the use of monoclonal antibodies (MoAbs), 145/112kDa glycoproteins (gp 145/112), gp80, and gp47 were identified to be the major immunogenic gps of CHV [24]. Furthermore, these gps were purified and used in immunizing mice to determine their relative immunogenicity [13]. However, the immune responses in dogs against these gps have not been investigated yet.
360
x. Xuan et al.
The purpose of this study is to examine the temporal appearance of antibodies to CHV in experimentally and naturally infected dogs by immunoblot and other conventional methods. Additionally, cross-reactivities between gps of CHV and feline herpesvirus type 1 (FHV-1) are discussed. Y P l l [25] and YP8702 [16] strains of CHV and C7301 strain [15] of FHV-1, which were respectively propagated in Madin-Darby canine kidney (MDCK) and Crandell feline kidney (CRFK) cell cultures, were used throughout the present study. The procedures of VN test [8, 20], HI test [16], and ELISA [20] were described previously. Briefly, for detection of VN antibodies serial dilutions of sera were mixed with virus (50 plaque-forming units) and 15% fresh guinea pig serum as a source of complement and incubated for 1 h at 37 °C in the microplate-wells. Then, suspensions of M D C K or C R F K cells were added to the wells and incubated for 3 days. VN titers were expressed as the reciprocal of the highest serum dilution showing 50% inhibition of the appearance of cytopathic effects. For detection of HI antibodies, the mixture of diluted sera and haemagglutinin-antigen (4 haemagglutination units) were incubated for 1 h at 37 °C. Then dog red blood cell suspensions (0.4%) were added and incubated for an additional 2 h before reading. HI titers were expressed as the reciprocal of the highest serum dilution resulting in complete haemagglutination-inhibition. For detection of ELISA antibodies, virus- or mock-infected cell lysates prepared by solubilizing with 0.5% sodium deoxycholate were used as ELISA antigens [20]. ELISA titers were expressed as the reciprocal of the highest serum dilution giving the absorbance difference at 405 nm of 0.1 or larger between the virus- and mock-infected cell lysate antigens. Immunoblot assay was carried out according to the previous method [24]. Virus- or mock-infected cell lysates which were separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% running gel under nonreducing conditions were electrophoretically transferred to polyvinyliden difluoride papers (Immobilon, Japan Millipore, Tokyo) by the Towbin's method [22]. The blotting paper was used for immunodetection. First, we examined the development of immune responses to CHV in experimentally infected dogs. The manners of experimental infection of a dog using CHV YP 11 strain were described elsewhere [20]. No clinical symptoms were manifested after the CHV inoculation. Sequential serum samples from the dog were analyzed by VN test, HI test, ELISA, and immunoblot assay (Fig. 1). No antibodies were detected in the sera collected 1 and 2 weeks post-inoculation (WPI). ELISA and VN antibodies were first detected at 3 and 4 WPI, respectively, while HI antibodies were at 10 WPI. On the other hand, the serum at 3 WPI reacted weakly with gp 145/112 but not with the other proteins in immunoblot analysis. Antibodies to gp 47 and gp 80 were first detected in sera at 9 and 10 WPI. Similar results were obtained with the series of sera collected from another dog experimentally infected with CHV YP 8702 strain (data not shown). These results suggest that antibodies to gp 145/112 appeared in the early stages of the infection and those to gp 47 and gp 80 appeared later, although
Immune responses to CHV glycoproteins
361
512 256 128
.~ 64 "-
16
4
