Immunology and Cell Biology (1991) 69, 47-49
Monoclonal antibodies to Kunjin and Kokobera viruses ROY A. HALL1.2, GRAHAM W. BURGESS2, BRIAN H. KAYi AND PAULA CLANCY2 ^ Queensland Institute of Medical Research, Bramston Terrace, Herston, Queensland 4006, and the'^GraduateSchool of Tropical Veterinary Science, Townsville, Qld 4811, Australia (Submitted 9 October 1990. Accepted for publication 6 February 1991.) Summary Five monoclonal antibodies (MoAb) were produced to the envelope (E) protein of Kunjin (KUN) virus. Three of these were specific for the KUNAVest Nile complex, and two reacted with epitopes that were common to theflavivirusfamily. These MoAb defined at least four distinct epitopes on the E protein of KUN virus. Eight MoAb were also produced to unidentified proteins of Kokobera (KOK) virus. Seven were specific for KOK, while the remaining antibody cross-reacted with Stratford virus and an unregisteredflavivirusCS946. The application of these MoAb to arboviral surveillance is discussed. INTRODUCTION Kunjin (KUN) and Kokobera (KOK) viruses are mosquito-bome flaviviruses that are native to Australia and possibly Papua New Guinea and Indonesia (1). KUN virus is one ofthe causative agents of Australian encephalitis which occurs as isolated cases, sporadic outbreaks and occasionally as major epidemics (2,3). KOK virus infection has been associated with an acute polyarticular disease in humans (4). Until recently, virus neutralization has been the established method for identifying new flavivirus isolates in the surveillance of arbovirus prevalence and distribution. However, this procedure is relatively slow and labour intensive. A more convenient and specific identification system involves the use of type-specific monoclonal antibodies (MoAb) in rapid immunoassays such as immunofluorescence or ELISA (5). The characterization of MoAb produced to proteins of KUN and KOK viruses is reported and, briefly, their potential for use in arbovirus surveillance is discussed. MATERIALS AND M E T H O D S The following strains offlaviviruseswere used in this study: Murray Valley encephalitis (MVE) —
strain MVE/3/51; KUN — MRM 16; Alfuy (ALF) — MRM 3929; KOK — MRM 32; Stratford (STR) — C338; Edge Hill (EH) — C281; Japanese encephalitis (JBE) — Nakayama; West Nile (WN) — Sarafend; dengue type 1 (DEN 1) — Hawaii; dengue type 2 (DEN 2) — New Guinea C; dengue type 3 (DEN 3) — H87; Saint Louis encephalitis (SLE) — Hubbard; yellow fever (YF) — 17D 204 vaccine; Sepik (SEP) — MK 7148. A new flavivirus isolate, CS946, was obtained from the CSIRO Division of Tropical Animal Production, Indooroopilly, Queensland. To produce hybridomas to KUN and KOK viruses, 6-10 week old BALB/c mice were immunized with live virus (6). Spleen cells from immunized mice were then fused with NS-1 myeloma cells according to the method of Galfre et al. (7). Cloning of the resulting hybridomas and characterization of the secreted MoAb was performed using previously described methods (5,6). RESULTS AND D I S C U S S I O N Supematants from 14 hybridoma cell lines produced to KUN virus were tested by ELISA for reactivity to 15 different flaviviruses. Antibody in each supematant reacted equally well with
Correspondence: Dr R. A. Hall, Department of Microbiology, QE II Medical Centre, University of Western Australia, Nedlands, WA 6009, Australia. Abbreviations used in this paper. ELISA, enzyme-linked immunosorbent assay: HI, haemagglutination inhibition; KOK, Kokobera virus; KUN, Kunjin virus; MoAb, monoclonal antibody; WN, West Nile virus.
48
R. A. HALL ETAL.
Table 1. Characteristics of five MoAb to KUN virus.
Clone
HI
KUN2-1D4 KUN2-3B4 KUN2-2B2 KUN2-3F2 KUN2-1B2
-f
Neutralization
Reactive protein
Viral specificity
-1-
E E E E E
KUN, WN KUN, WN KUN, WN Flavivirus Flavivirus
NT
NT = not tested.
Table 2. Characteristics of eight MoAb to KOK virus. Clone
HI
Neutralization
Reactive protein
Viral specificity
K0K1-1C5 K0K1-1E3 K0K1-1E9 KOKl-lAl KOKl-lCl KOK1-10C9 KOK2-3D6 KOK1-2E5
— — — — — — — —
— — — — NT — NT NT
43-45 kDa 43-45 kDa 43-45 kDa
KOK KOK KOK KOK KOK KOK KOK KOK, STR, CS946
7* ? ? ?
18 kDa
'Unreactive in Western blot. NT = not tested.
both KUN and WN viruses. This antigenic similarity is consistent with the high degree of homology (93%) between the complete amino acid sequences of these two viruses (8). While three hybridomas were specific for these two viruses, two were flavivirus group-reactive. The remainder reacted with various flavivirus subgroups, predominantly within the JBE virus antigenic complex. Five hybridomas specific for the envelope (E) protein by immunoblot were cloned and further characterized (Table 1). These MoAb defined at least four distinct epitopes on the E protein of KUN virus: (i) KUN/WN specific with haemagglutination inhibition (HI) activity only; (ii) KUN/WN specific, with neutralizing activity only; (iii) KUN/WN specific, with HI and neutralizing activity; and (iv) flavivirus specific, with HI and neutralizing activity. Thirty-four uncloned hybridoma lines produced to KOK virus were also tested by ELISA. Fourteen were specific for KOK, seven were specific for a KOK/STR/CS946 complex while the remainder were cross-reactive with various subgroups within the flavivirus family. None reacted with group-specific epitopes, or with antigens of ALF, SEP or EH viruses. Seven KOK
type-specific hybridomas and one that crossreacted with STR and CS946 viruses were cloned for further characterization (Table 2). These antibodies failed to neutralize ICOK virus or inhibit haemagglutination of gander red cells. In immunoblots three MoAb reacted with a doublet of 43-45 kDa, one reacted with a protein of approximately 18 kDa, while four were unreactive. The identity ofthe 18 kDa protein is uncertain. However, the approximate molecular weight of the 43-45 kDa doublet and the fact that MoAb to this protein have no HI or neutralizing activity, imply it is the non-structural protein NSl of KOK virus. The MoAb described in this report complement the battery of MoAb previously produced to Murray Valley encephalitis (6). Selected MoAb from this collection are now routinely used to specifically identify new strains of MVE, KUN and KOK isolated from mosquitoes captured in northwestern Australia (unpubl. data). These reagents are also being assessed for use in rapid immunoassays to detect viral antigen directly in mosquitoes, and for use in specific, epitope-blocking serological assays. These MoAb are available to other laboratories as reference or research reagents.
MoAb TO KUNJIN AND KOKOBERA VIRUSES
ACKNOWLEDGEMENTS We would like to thank Dr A. Broom, Mr M. Lindsay, Mr M. Poidinger and Mr D. Segal for helpful advice, manuscript review and technical assistance. This work was funded by the National Health and Medical Research Council and the Australian Water Resources Advisory Council, Canberra.
REFERENCES 1. French, E. L. 1973. A review of arthropod-borne infections affecting man and animals in Australia. Aust. J. Exp. Biol. Med Sci. 51: 131-158. 2. Stanley, N. F. 1982. Human arbovirus infections in Australia. Arbovirus Res. Aust. 3: 216-226. 3. Muller, D., McDonald, M., Stallman, N. and King, J. 1986. Kunjin virus encephalomyelitis. Med J. Aust. 144: 41-42.
49
4. Boughton, C. R., Hawkes, R. and Naim, H. M. 1986. Illness caused by Kokobera-like virus in south-eastern Australia. Med. J. Aust. 145: 9092. 5. Hall, R. A., Kay, B. H. and Burgess, G. W. 1987. An enzyme immunoassay to detect Australian flaviviruses and identify the encephalitic subgroup using monoclonal antibodies. Immunol. Cell Biol. 65: 103-110. 6. Hall, R. A., Kay, B. H., Burgess, G. W., Clancy, P. and Fanning, I. D. 1990. Epitope analysis ofthe envelope and non-structural glycoproteins of Murray Valley encephalitis virus. J. Gen. Virol. 71: 2923-2930. 7. Galfre, G., Hove, C. S., Milstein, C , Butcher, G. W. and Howard, J. W. 1977. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266: 550-552. 8. Coia, G., Parker, M. D., Speight, G., Byrne, M. E. and Westaway, E. G. 1988. Nucleotide and complete amino sequences of Kunjin virus: Definitive gene order and characteristics of the virus-specified proteins. J. Gen. Virol. 69: 1-21.
Monoclonal antibodies to Kunjin and Kokobera viruses ROY A. HALL1.2, GRAHAM W. BURGESS2, BRIAN H. KAYi AND PAULA CLANCY2 ^ Queensland Institute of Medical Research, Bramston Terrace, Herston, Queensland 4006, and the'^GraduateSchool of Tropical Veterinary Science, Townsville, Qld 4811, Australia (Submitted 9 October 1990. Accepted for publication 6 February 1991.) Summary Five monoclonal antibodies (MoAb) were produced to the envelope (E) protein of Kunjin (KUN) virus. Three of these were specific for the KUNAVest Nile complex, and two reacted with epitopes that were common to theflavivirusfamily. These MoAb defined at least four distinct epitopes on the E protein of KUN virus. Eight MoAb were also produced to unidentified proteins of Kokobera (KOK) virus. Seven were specific for KOK, while the remaining antibody cross-reacted with Stratford virus and an unregisteredflavivirusCS946. The application of these MoAb to arboviral surveillance is discussed. INTRODUCTION Kunjin (KUN) and Kokobera (KOK) viruses are mosquito-bome flaviviruses that are native to Australia and possibly Papua New Guinea and Indonesia (1). KUN virus is one ofthe causative agents of Australian encephalitis which occurs as isolated cases, sporadic outbreaks and occasionally as major epidemics (2,3). KOK virus infection has been associated with an acute polyarticular disease in humans (4). Until recently, virus neutralization has been the established method for identifying new flavivirus isolates in the surveillance of arbovirus prevalence and distribution. However, this procedure is relatively slow and labour intensive. A more convenient and specific identification system involves the use of type-specific monoclonal antibodies (MoAb) in rapid immunoassays such as immunofluorescence or ELISA (5). The characterization of MoAb produced to proteins of KUN and KOK viruses is reported and, briefly, their potential for use in arbovirus surveillance is discussed. MATERIALS AND M E T H O D S The following strains offlaviviruseswere used in this study: Murray Valley encephalitis (MVE) —
strain MVE/3/51; KUN — MRM 16; Alfuy (ALF) — MRM 3929; KOK — MRM 32; Stratford (STR) — C338; Edge Hill (EH) — C281; Japanese encephalitis (JBE) — Nakayama; West Nile (WN) — Sarafend; dengue type 1 (DEN 1) — Hawaii; dengue type 2 (DEN 2) — New Guinea C; dengue type 3 (DEN 3) — H87; Saint Louis encephalitis (SLE) — Hubbard; yellow fever (YF) — 17D 204 vaccine; Sepik (SEP) — MK 7148. A new flavivirus isolate, CS946, was obtained from the CSIRO Division of Tropical Animal Production, Indooroopilly, Queensland. To produce hybridomas to KUN and KOK viruses, 6-10 week old BALB/c mice were immunized with live virus (6). Spleen cells from immunized mice were then fused with NS-1 myeloma cells according to the method of Galfre et al. (7). Cloning of the resulting hybridomas and characterization of the secreted MoAb was performed using previously described methods (5,6). RESULTS AND D I S C U S S I O N Supematants from 14 hybridoma cell lines produced to KUN virus were tested by ELISA for reactivity to 15 different flaviviruses. Antibody in each supematant reacted equally well with
Correspondence: Dr R. A. Hall, Department of Microbiology, QE II Medical Centre, University of Western Australia, Nedlands, WA 6009, Australia. Abbreviations used in this paper. ELISA, enzyme-linked immunosorbent assay: HI, haemagglutination inhibition; KOK, Kokobera virus; KUN, Kunjin virus; MoAb, monoclonal antibody; WN, West Nile virus.
48
R. A. HALL ETAL.
Table 1. Characteristics of five MoAb to KUN virus.
Clone
HI
KUN2-1D4 KUN2-3B4 KUN2-2B2 KUN2-3F2 KUN2-1B2
-f
Neutralization
Reactive protein
Viral specificity
-1-
E E E E E
KUN, WN KUN, WN KUN, WN Flavivirus Flavivirus
NT
NT = not tested.
Table 2. Characteristics of eight MoAb to KOK virus. Clone
HI
Neutralization
Reactive protein
Viral specificity
K0K1-1C5 K0K1-1E3 K0K1-1E9 KOKl-lAl KOKl-lCl KOK1-10C9 KOK2-3D6 KOK1-2E5
— — — — — — — —
— — — — NT — NT NT
43-45 kDa 43-45 kDa 43-45 kDa
KOK KOK KOK KOK KOK KOK KOK KOK, STR, CS946
7* ? ? ?
18 kDa
'Unreactive in Western blot. NT = not tested.
both KUN and WN viruses. This antigenic similarity is consistent with the high degree of homology (93%) between the complete amino acid sequences of these two viruses (8). While three hybridomas were specific for these two viruses, two were flavivirus group-reactive. The remainder reacted with various flavivirus subgroups, predominantly within the JBE virus antigenic complex. Five hybridomas specific for the envelope (E) protein by immunoblot were cloned and further characterized (Table 1). These MoAb defined at least four distinct epitopes on the E protein of KUN virus: (i) KUN/WN specific with haemagglutination inhibition (HI) activity only; (ii) KUN/WN specific, with neutralizing activity only; (iii) KUN/WN specific, with HI and neutralizing activity; and (iv) flavivirus specific, with HI and neutralizing activity. Thirty-four uncloned hybridoma lines produced to KOK virus were also tested by ELISA. Fourteen were specific for KOK, seven were specific for a KOK/STR/CS946 complex while the remainder were cross-reactive with various subgroups within the flavivirus family. None reacted with group-specific epitopes, or with antigens of ALF, SEP or EH viruses. Seven KOK
type-specific hybridomas and one that crossreacted with STR and CS946 viruses were cloned for further characterization (Table 2). These antibodies failed to neutralize ICOK virus or inhibit haemagglutination of gander red cells. In immunoblots three MoAb reacted with a doublet of 43-45 kDa, one reacted with a protein of approximately 18 kDa, while four were unreactive. The identity ofthe 18 kDa protein is uncertain. However, the approximate molecular weight of the 43-45 kDa doublet and the fact that MoAb to this protein have no HI or neutralizing activity, imply it is the non-structural protein NSl of KOK virus. The MoAb described in this report complement the battery of MoAb previously produced to Murray Valley encephalitis (6). Selected MoAb from this collection are now routinely used to specifically identify new strains of MVE, KUN and KOK isolated from mosquitoes captured in northwestern Australia (unpubl. data). These reagents are also being assessed for use in rapid immunoassays to detect viral antigen directly in mosquitoes, and for use in specific, epitope-blocking serological assays. These MoAb are available to other laboratories as reference or research reagents.
MoAb TO KUNJIN AND KOKOBERA VIRUSES
ACKNOWLEDGEMENTS We would like to thank Dr A. Broom, Mr M. Lindsay, Mr M. Poidinger and Mr D. Segal for helpful advice, manuscript review and technical assistance. This work was funded by the National Health and Medical Research Council and the Australian Water Resources Advisory Council, Canberra.
REFERENCES 1. French, E. L. 1973. A review of arthropod-borne infections affecting man and animals in Australia. Aust. J. Exp. Biol. Med Sci. 51: 131-158. 2. Stanley, N. F. 1982. Human arbovirus infections in Australia. Arbovirus Res. Aust. 3: 216-226. 3. Muller, D., McDonald, M., Stallman, N. and King, J. 1986. Kunjin virus encephalomyelitis. Med J. Aust. 144: 41-42.
49
4. Boughton, C. R., Hawkes, R. and Naim, H. M. 1986. Illness caused by Kokobera-like virus in south-eastern Australia. Med. J. Aust. 145: 9092. 5. Hall, R. A., Kay, B. H. and Burgess, G. W. 1987. An enzyme immunoassay to detect Australian flaviviruses and identify the encephalitic subgroup using monoclonal antibodies. Immunol. Cell Biol. 65: 103-110. 6. Hall, R. A., Kay, B. H., Burgess, G. W., Clancy, P. and Fanning, I. D. 1990. Epitope analysis ofthe envelope and non-structural glycoproteins of Murray Valley encephalitis virus. J. Gen. Virol. 71: 2923-2930. 7. Galfre, G., Hove, C. S., Milstein, C , Butcher, G. W. and Howard, J. W. 1977. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266: 550-552. 8. Coia, G., Parker, M. D., Speight, G., Byrne, M. E. and Westaway, E. G. 1988. Nucleotide and complete amino sequences of Kunjin virus: Definitive gene order and characteristics of the virus-specified proteins. J. Gen. Virol. 69: 1-21.
