_Archives
Vi rology
Arch Virol (1992) 124:355-361
© Springer-Verlag 1992 Printed in Austria
Detection of anti-gag antibodies of feline immunodeficieney virus in cat sera by enzyme-linked immunosorbent assay Brief R e p o r t
T. Furuyat, A. Hasegawa2, T. Miyazawa1, K. Miki 2, and T. Mikami~ Department of Veterinary Microbiology,Faculty of Agriculture,The Universityof Tokyo, Bunkyo-ku, Tokyo 2Fundamental Research Laboratory, Tohnen Corporation, Ohimachi, Saitama, Japan Accepted November 11, 1991
Summary. Using gag protein of feline immunodeficiency virus (FIV) expressed in Escherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIV gag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIV gag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9 %) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gpl30 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.
Feline immunodeficiency virus (FIV) is a lentivirus which was first isolated from a cat with an immunodeficiency-like syndrome in the United States in 1986 [17]. This virus displays typical lentivirus morphology and has Mg 2+dependent reverse transcriptase activity like human immunodeficiency virus (HIV), the causative agent of an acquired immune deficiency syndrome (AIDS) in humans. It is known that FIV causes cytolytic infections in feline T-lymphocytes in vitro. The virus persists in infected cats and occasionally causes chronic diseases [24]. The similar virus was also isolated in the United Kingdom [6-[, Japan [7, 12], and other countries [2, 14]. Serological studies revealed the
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prevalence of FIV infections in the United Kingdom [9], the United States [23], Canada [23], Japan [4, 7, 12], Europe [5], Australia [19], and New Zealand [21]. Besides, molecular clones of FIV were obtained [11, 15], and the nucleotide sequences of several distinct FIVs were determined [16, 18, 10, 22], indicating that FIV genome has three major open reading frames (ORFs); gag, pol, and env, as seen in other lentiviruses. Among these ORFs, gag encodes the viral core and capsid proteins. Recently, we developed an enzyme-linked immunosorbent assay (ELISA) system using the expressed gag proteins in Escheriehia coli to detect anti-gag antibodies, and the degree of ELISA value (EV) found to correspond to the intensity of p26 band of the major core protein detected by Western blotting analysis [3]. In this report, we investigated serological responses to the gag protein in cats experimentally and naturally infected with FIV using the ELISA and a radioimmunoprecipitation assay (RIPA). Sera from 6 specific pathogen free (SPF) cats infected with several strains of FIV were used. Two female cats, cat 103(TM 1) of 4.5 months and cat 104(TM 2) of 5 months old, were inoculated intraperitoneally (IP) with 0.5 ml of peripheral blood taken from cats infected with FIV TM 1 and TM 2 strains, respectively, and a male cat of 5 months old [cat 105(Petaluma)] was inoculated IP with primary feline peripheral blood mononuclear cells infected with FIV Petaluma strain as described previously [12]. Six-month-old female and male cats [cat 106(IC) and cat 108(KYO-1)] were inoculated IP with 6 x 106 cells of MYA-1 [13] infected with an infectious molecular clone (IC) of TM 1 strain [11] and those infected with KYO-1 strain which was isolated in Tokyo area, respectively. A female cat of the same age [cat 107(CRFK/Petaluma)] was inoculated IP with 6 x 106 cells of MYA-1 cocultured for 7 days with Crandell feline kidney (CRFK) cells which were persistently infected with the Petaluma strain [24]. We also used a total of 415 field cat sera. These included 363 sera used in a previous report [4], and 52 sera from cats brought to veterinary hospitals for some medical treatments in Tokyo area. All the samples were tested for antibodies to FIV by an indirect immunofluorescence assay (IFA) in the manner which was reported previously [12]. In the ELISA, we used gag antigen-coated and control antigen-coated plates for each sample. The gag antigen was prepared from E. coli HB 101 expressing FIV gag protein as described previously [3], and control antigen was obtained from intact E. coli by the same manner as prepared the gag antigen. The methods of the immune and enzymatic reactions were described previously [3]. After enzymatic reaction, optical density at 415 nm was read for the both types of plates. The EV of each sample was obtained by subtracting the optical density of the control antigen-coated well from that of the gag antigen-coated well. The cut-off value of the ELISA was determined to be 0.6 as described below. At first, we performed the ELISA using serum samples which were sequentially collected from cats experimentally infected with FIVs. As shown in Fig. 1, the EV increased remarkably in all cats after virus inoculation. The results
Detection of anti-FIV gag antibodies E
A 2.0
2,0
357
B
r1.0 J~ 0 el
1.0 i
0.0
i 10
~ . i ~ i • , , i¸ . 20 30 120 130 140
J
0.0
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I0
150
Weeks
After
n
2O
Inoculation
Fig. l. Results of ELISA with serum samples sequentially obtained from the cats which
were experimentally infected with several strains and an infectious molecular clone (IC) of FIV. The serum samples were diluted 1 : 80. A Cat 103(TM 1) (Z]), cat 104(TM 2) (ll), and cat 105(Petaluma) (A). B Cat 106(TM 1 IC) (©), cat 107(CRFK/Petaluma) (O), cat 108(KYO-1) (A)
indicated that the antigenicity of gag proteins is conserved among FIVs used, and anti-gag antibodies can be detected in cats infected with various strains of FIV by the ELISA. These results might be due to the low variability of the gag gene, as compared with other genes of FI¥, which was predicted by the comparison of two FIV isolates by the amino acid sequence data [10]. The peak EVs in cat 105(Petaluma) and cat 108(KYO-1) were lower than those in the other cats. The reason for this diversity may be due to the differences in growth kinetics of these viruses in vivo. Judging from the results of the following experiments, the serum samples whose EV exceeded 0.6 were considered to be positive for FIV infection. The EV of the sera collected from cat 103(TM 1), cat 104(TM 2), and cat 105(Petaluma) exceeded 0.6 at 4, 7, and 8 weeks after inoculation (WAI), respectively. All other cats could be judged as positive for FIV infection at least 10 WAI. The results indicated that, when anti-gag antibodies is not detected in a cat serum at early stage of infection, we should reexamine the cat al least 10 weeks after the first examination for confirmatory diagnosis. The EV decreased gradually after the peak point, and especially in the cat 105(Petaluma), and the EV was below 0.6 at 120 WAI. There is a possibility that the decreases of EV are followed by feline AIDS, because all three cats had oral cavity diseases since 150 WAI. Further examination of these cats will give us some information for the confirmation of this possibility. The frequency distributions for IFA positive and negative populations of the field cats are shown in Fig. 2. In IFA negative sera, 342 (99.1%) out of 345 samples had EV below 0.6. Three IFA negative samples had EV above 0.6, but it seemed to be due to non-specific reactions because we could not find any specific bands for the gag or other proteins of FIV in these samples by the Western blotting analysis (data not shown). In IFA positive sera, 61 (87.1%)
358
T. Furuya et al.
IFA Positive Samples 6¸
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IFA Negative Samples 200
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ELISA Value
Fig.2 Fig. 2. The frequency distributions of the ELISA values for the IFA positive and negative populations of the field cats. The serum samples were diluted 1:80
Fig. 3. Reactivity of cat sera for antibody to FIV proteins as determined by RIPA. Cell lysates labeled with [35S]methionine from FIV-infected MYA-1 cells (a) and uninfected MYA-1 cells (b) were reacted with serum samples from the following cats; 1 IFA positive cat 103(TM 1) with a high EV (1.88), 2 IFA positive field cat with a low EV (0.00), 3 normal SPF cat. Protein bands of 97 and 44 k Mr in 1 a, 2 a and 3 a are caused by nonspecific reactions to the cellular proteins. Mr of FIV specific proteins (gp130, p50, p26, and p15) and molecular mass standards are shown in the left and fight column, respectively
out of 70 samples showed EV above 0.6, which indicated the remaining 9 (12.9%) I F A positive samples did n o t have antibodies to the gag proteins. To examine a possible involvement of antibodies against other t h a n the gag protein in the 9 I F A positive samples, we selected 3 serum samples and carried out the RIPA. FIV-infected a n d -uninfected cells were labeled for 16 h with 50 gCi of a mixture of [3SS]L-methionine and [35S]L-cysteine (1198 Ci/mmol; New England Nuclear, Boston, Mass.) per ml in methionine-free R P M I 1640
Detection of anti-FIV gag antibodies
359
medium containing 5% fetal calf serum and lysed with lysis buffer (0.5% Nonidet P-40, sodium deoxycholate, 0.05 M Tris-HC1 (pH 7.2), 0.1 M NaC1, 1 mM EDTA, t m M phenylmethylsulfonyl fluoride). The celt lysates were immunoprecipitated with the sera and the immune complexes were analyzed by SDS-PAGE (12.5% gel) by the method of Laemmli [8]. As shown in Fig. 3, p26 and p15 were precipitated by a serum from cat 103(TM 1) which had a high EV (EV 1.88, IFA positive) (Fig. 3, 1 a) but not by one of the 3 serum samples, which had a low EV (EV 0, IFA positive) (Fig. 3, 2 a), whereas gpl30 was precipitated by both of the sera (Fig. 3, 1 a and 2a). Two other serum samples with a low EV (0) had also antibodies to gpl30 but not to p26 (data not shown). No FIV-specific protein was detected in a serum from a normal SPF cat (Fig. 3, 3 a). These cat sera reacted strongly with normal components of uninfected MYA- 1 cells but not with the same components of infected cells. The reason is not clear at present. The gpl30 was identified as an envelope protein, and p26 and p15 were as major core proteins of FIV [20]. The result indicated that there are some IFA positive samples which do not have antibodies to the major core proteins but have those to the envelope protein. Further, a protein band of 50 k Mr, which was reported to be a putative precursor of p26 [20], was also observed in both of the samples though p26 was only observed in one of the 3 serum samples tested (Fig. 3, 1 a and 2 a). The reasons for this inconsistency might be due to the difference of protein concentration in the cell lysates or the difference of antigenicity between p26 and p50. In HIV infection, it was reported that among the patients who had antibodies to HIV envelope protein, less than half of the AIDS patients had antibodies against HIV major core protein, although most samples from AIDS-related complex patients also reacted with both of the proteins [1]. In this study, we found that there are some FIV-positive samples without anti-p26 antibodies in field cat sera. Since health conditions of the cats were unknown, we could not find whether the cats had clinical status related to the feline AIDS. However, the ELISA in this study will be valuable for further studies about clinical stages of FIV infections when we use it together with other physical examinations.
Acknowledgements We are grateful to Dr. Hayami (Kyoto University, Kyoto, Japan) for providing FIV KYO1 strain. We also thank Dr. J. K. Yamamoto (University of California, Davis, California, U.S.A.) and H. Koyama (Kitasato University, Towada, Japan) for providing the FIV Petaluma-infected CRFK cells.This study was supported in part by grants from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare of Japan.
References 1. Barin F. Mclane MF, Allan JS, Lee TH, Groopman JE, Essex M (1985) Virus envelope protein of HTLV-III represents major target antigen for antibodies in AIDS patiens. Science 228:1094-1096 2. Egberink H, Borst M, Niphuis H, Balzarini J, Neu H, Schellekens H, Clercq ED, Horzinek M, Koolen M (1990) Suppression of feline immunodeficiencyvirus infection
360
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4. 5.
6.
7. 8. 9.
10.
11.
12.
13.
14. 15.
16. 17.
18. 19. 20.
T. Furuya et al. in vivo by 9-(2-phosphonomethoxyethyl)adenine. Proc Natl Acad Sci USA 87: 30873091 Furuya T, Hasegawa A, Saitoh M, Miyazawa T, Tohya Y, Hayami M, Takahashi E, Miki K, Mikami T (1992) Expression of feline immunodeficiency virus gag gene in Escherichia coli. Arch Virol 122:383-390 Furuya T, Kawaguchi Y, Miyazawa T, Fujikawa Y, Tohya Y, Azetaka M, Takahashi E, Mikami T (1990) Existence of feline immunodeficiency virus infection in Japanese cat population since 1968. Jpn J Vet Sci 52:891-893 Gruffydd-Jones TJ, Hopper CD, Harvour DA, Lutz H (1988) Serological evidence of feline immunodeficiency virus infection in UK cats from 1975-76. Vet Rec 123: 569570 Harbour DA, Williams PD, Gruffydd-Jones TJ, Burbridge J, Pearson GR (1988) Isolation of a T-lymphotropic tentivirus from a persistently leucopenic domestic cat. Vet Rec 122:84-86 Ishida T, Washizu T, Toriyabe K, Motoyoshi S (1988) Detection of feline T-lymphotropic lentivirus (FTLV) infection in Japanese domestic cats. Jpn J Vet Sci 50:39-44 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227:680-685 Lutz H, Arnold P, Hiibscher U, Egberink H, Pedersen N, Horzinek MC (1988) Specificity assessment of feline T-lymphotropic lentivirus serology. J Vet Med B 35: 773778 Maki N, Miyazawa T, Fukasawa M, Hasegawa A, Hayami M, Miki K, Mikami T (1992) Molecular characterization and heterogeneity of feline immunodeficiencyvirus isolates. Arch Virol 123:29--45 Miyazawa T, Fukasawa M, Hasegawa A, Maki N, Ikuta K, Takahashi E, Hayami M, Mikami T (1991) Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U.S. isolate. J Virol 65: 15721577 Miyazawa T, Furuya T, Itagaki S, Tohaya Y, Nakano K, Takahashi E, Mikami T (1989) Preliminary comparisons of the biological properties of two strains of feline immunodeficiency virus (FIV) isolated in Japan with FIV Petatuma strain isolated in the United States. Arch Virol 108:59-68 Miyazawa T, Furuya T, Itagaki S, Tohya Y, Takahashi E, Mikami T (1989) Establishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus. Arch Virol 108:131-135 Morikawa S, Booth TF, Bishop DHL (1991) Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors. Virology 183:288-297 Olmsted RA, Barnes AK, Yamamoto JK, Hirsch VM, Purcell RH, Johnson PR (1989) Molecular cloning of feline immunodeficiency virus. Proc Natl Acad Sci USA 86: 24482452 Olmsted RA, Hirsch VM, Purcell RH, Johnson PR (t989) Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentiviruses. Proc Natl Acad Sci USA 86:8088-8092 Pedersen NC, HoEW, BrownML, Yamamoto JK (1987) Isolation ofaT-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235: 790794 Phillips TR, Talbott RL, Lamont C, Muir S, Lovelace K, Elder JH (1990) Comparison of two host cell variants of feline immunodeficiency virus. J Virol 64:4605-4613 Sabine M, Michelsen J, Thomas F, Zheng M (1988) Feline AIDS. Aust Vet Practit 18:105-107 Steinman R, Dombrowski J, O'Connor T, Montelaro RC, Tonelli Q, Lawrence K,
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24.
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Seymour C, Goodness J, Pedersen NC, Andersen PR (1990) Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus. J Gen Virol 71:701-706 Swinney GR, Pauli JV, Jones BR, Wilks CR (1989) Feline T-lymphotropic virus (FTLV) (feline immunodeficiency virus infection) in cats in New Zealand. N Z Vet J 37: 4143 Talbott RL, Sparger EE, Lovelace KM, Fitch WM, Pedersen NC, Luciw PA, Elder JH (1989) Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc Natl Acad Sci USA 86:5743-5747 Yamamoto JK, Hansen H, Ho EW, Morishita TY, Okuda T, Sawa TR, Nakamura RM, Pedersen NC (1989) Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. J Am Vet Med Assoc 194:213-220 Yamamoto JK, Sparger E, Ho EW, Andersen PR, O'Conner TP, Mandell CP, Lowenstine L, Munn R, Pedersen NC (1988) Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am J Vet Res 49:1246-1258
Authors' address: Dr. T. Mikami, Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. Received August 26, 1991
Vi rology
Arch Virol (1992) 124:355-361
© Springer-Verlag 1992 Printed in Austria
Detection of anti-gag antibodies of feline immunodeficieney virus in cat sera by enzyme-linked immunosorbent assay Brief R e p o r t
T. Furuyat, A. Hasegawa2, T. Miyazawa1, K. Miki 2, and T. Mikami~ Department of Veterinary Microbiology,Faculty of Agriculture,The Universityof Tokyo, Bunkyo-ku, Tokyo 2Fundamental Research Laboratory, Tohnen Corporation, Ohimachi, Saitama, Japan Accepted November 11, 1991
Summary. Using gag protein of feline immunodeficiency virus (FIV) expressed in Escherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIV gag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIV gag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9 %) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gpl30 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.
Feline immunodeficiency virus (FIV) is a lentivirus which was first isolated from a cat with an immunodeficiency-like syndrome in the United States in 1986 [17]. This virus displays typical lentivirus morphology and has Mg 2+dependent reverse transcriptase activity like human immunodeficiency virus (HIV), the causative agent of an acquired immune deficiency syndrome (AIDS) in humans. It is known that FIV causes cytolytic infections in feline T-lymphocytes in vitro. The virus persists in infected cats and occasionally causes chronic diseases [24]. The similar virus was also isolated in the United Kingdom [6-[, Japan [7, 12], and other countries [2, 14]. Serological studies revealed the
356
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prevalence of FIV infections in the United Kingdom [9], the United States [23], Canada [23], Japan [4, 7, 12], Europe [5], Australia [19], and New Zealand [21]. Besides, molecular clones of FIV were obtained [11, 15], and the nucleotide sequences of several distinct FIVs were determined [16, 18, 10, 22], indicating that FIV genome has three major open reading frames (ORFs); gag, pol, and env, as seen in other lentiviruses. Among these ORFs, gag encodes the viral core and capsid proteins. Recently, we developed an enzyme-linked immunosorbent assay (ELISA) system using the expressed gag proteins in Escheriehia coli to detect anti-gag antibodies, and the degree of ELISA value (EV) found to correspond to the intensity of p26 band of the major core protein detected by Western blotting analysis [3]. In this report, we investigated serological responses to the gag protein in cats experimentally and naturally infected with FIV using the ELISA and a radioimmunoprecipitation assay (RIPA). Sera from 6 specific pathogen free (SPF) cats infected with several strains of FIV were used. Two female cats, cat 103(TM 1) of 4.5 months and cat 104(TM 2) of 5 months old, were inoculated intraperitoneally (IP) with 0.5 ml of peripheral blood taken from cats infected with FIV TM 1 and TM 2 strains, respectively, and a male cat of 5 months old [cat 105(Petaluma)] was inoculated IP with primary feline peripheral blood mononuclear cells infected with FIV Petaluma strain as described previously [12]. Six-month-old female and male cats [cat 106(IC) and cat 108(KYO-1)] were inoculated IP with 6 x 106 cells of MYA-1 [13] infected with an infectious molecular clone (IC) of TM 1 strain [11] and those infected with KYO-1 strain which was isolated in Tokyo area, respectively. A female cat of the same age [cat 107(CRFK/Petaluma)] was inoculated IP with 6 x 106 cells of MYA-1 cocultured for 7 days with Crandell feline kidney (CRFK) cells which were persistently infected with the Petaluma strain [24]. We also used a total of 415 field cat sera. These included 363 sera used in a previous report [4], and 52 sera from cats brought to veterinary hospitals for some medical treatments in Tokyo area. All the samples were tested for antibodies to FIV by an indirect immunofluorescence assay (IFA) in the manner which was reported previously [12]. In the ELISA, we used gag antigen-coated and control antigen-coated plates for each sample. The gag antigen was prepared from E. coli HB 101 expressing FIV gag protein as described previously [3], and control antigen was obtained from intact E. coli by the same manner as prepared the gag antigen. The methods of the immune and enzymatic reactions were described previously [3]. After enzymatic reaction, optical density at 415 nm was read for the both types of plates. The EV of each sample was obtained by subtracting the optical density of the control antigen-coated well from that of the gag antigen-coated well. The cut-off value of the ELISA was determined to be 0.6 as described below. At first, we performed the ELISA using serum samples which were sequentially collected from cats experimentally infected with FIVs. As shown in Fig. 1, the EV increased remarkably in all cats after virus inoculation. The results
Detection of anti-FIV gag antibodies E
A 2.0
2,0
357
B
r1.0 J~ 0 el
1.0 i
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Weeks
After
n
2O
Inoculation
Fig. l. Results of ELISA with serum samples sequentially obtained from the cats which
were experimentally infected with several strains and an infectious molecular clone (IC) of FIV. The serum samples were diluted 1 : 80. A Cat 103(TM 1) (Z]), cat 104(TM 2) (ll), and cat 105(Petaluma) (A). B Cat 106(TM 1 IC) (©), cat 107(CRFK/Petaluma) (O), cat 108(KYO-1) (A)
indicated that the antigenicity of gag proteins is conserved among FIVs used, and anti-gag antibodies can be detected in cats infected with various strains of FIV by the ELISA. These results might be due to the low variability of the gag gene, as compared with other genes of FI¥, which was predicted by the comparison of two FIV isolates by the amino acid sequence data [10]. The peak EVs in cat 105(Petaluma) and cat 108(KYO-1) were lower than those in the other cats. The reason for this diversity may be due to the differences in growth kinetics of these viruses in vivo. Judging from the results of the following experiments, the serum samples whose EV exceeded 0.6 were considered to be positive for FIV infection. The EV of the sera collected from cat 103(TM 1), cat 104(TM 2), and cat 105(Petaluma) exceeded 0.6 at 4, 7, and 8 weeks after inoculation (WAI), respectively. All other cats could be judged as positive for FIV infection at least 10 WAI. The results indicated that, when anti-gag antibodies is not detected in a cat serum at early stage of infection, we should reexamine the cat al least 10 weeks after the first examination for confirmatory diagnosis. The EV decreased gradually after the peak point, and especially in the cat 105(Petaluma), and the EV was below 0.6 at 120 WAI. There is a possibility that the decreases of EV are followed by feline AIDS, because all three cats had oral cavity diseases since 150 WAI. Further examination of these cats will give us some information for the confirmation of this possibility. The frequency distributions for IFA positive and negative populations of the field cats are shown in Fig. 2. In IFA negative sera, 342 (99.1%) out of 345 samples had EV below 0.6. Three IFA negative samples had EV above 0.6, but it seemed to be due to non-specific reactions because we could not find any specific bands for the gag or other proteins of FIV in these samples by the Western blotting analysis (data not shown). In IFA positive sera, 61 (87.1%)
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Fig.2 Fig. 2. The frequency distributions of the ELISA values for the IFA positive and negative populations of the field cats. The serum samples were diluted 1:80
Fig. 3. Reactivity of cat sera for antibody to FIV proteins as determined by RIPA. Cell lysates labeled with [35S]methionine from FIV-infected MYA-1 cells (a) and uninfected MYA-1 cells (b) were reacted with serum samples from the following cats; 1 IFA positive cat 103(TM 1) with a high EV (1.88), 2 IFA positive field cat with a low EV (0.00), 3 normal SPF cat. Protein bands of 97 and 44 k Mr in 1 a, 2 a and 3 a are caused by nonspecific reactions to the cellular proteins. Mr of FIV specific proteins (gp130, p50, p26, and p15) and molecular mass standards are shown in the left and fight column, respectively
out of 70 samples showed EV above 0.6, which indicated the remaining 9 (12.9%) I F A positive samples did n o t have antibodies to the gag proteins. To examine a possible involvement of antibodies against other t h a n the gag protein in the 9 I F A positive samples, we selected 3 serum samples and carried out the RIPA. FIV-infected a n d -uninfected cells were labeled for 16 h with 50 gCi of a mixture of [3SS]L-methionine and [35S]L-cysteine (1198 Ci/mmol; New England Nuclear, Boston, Mass.) per ml in methionine-free R P M I 1640
Detection of anti-FIV gag antibodies
359
medium containing 5% fetal calf serum and lysed with lysis buffer (0.5% Nonidet P-40, sodium deoxycholate, 0.05 M Tris-HC1 (pH 7.2), 0.1 M NaC1, 1 mM EDTA, t m M phenylmethylsulfonyl fluoride). The celt lysates were immunoprecipitated with the sera and the immune complexes were analyzed by SDS-PAGE (12.5% gel) by the method of Laemmli [8]. As shown in Fig. 3, p26 and p15 were precipitated by a serum from cat 103(TM 1) which had a high EV (EV 1.88, IFA positive) (Fig. 3, 1 a) but not by one of the 3 serum samples, which had a low EV (EV 0, IFA positive) (Fig. 3, 2 a), whereas gpl30 was precipitated by both of the sera (Fig. 3, 1 a and 2a). Two other serum samples with a low EV (0) had also antibodies to gpl30 but not to p26 (data not shown). No FIV-specific protein was detected in a serum from a normal SPF cat (Fig. 3, 3 a). These cat sera reacted strongly with normal components of uninfected MYA- 1 cells but not with the same components of infected cells. The reason is not clear at present. The gpl30 was identified as an envelope protein, and p26 and p15 were as major core proteins of FIV [20]. The result indicated that there are some IFA positive samples which do not have antibodies to the major core proteins but have those to the envelope protein. Further, a protein band of 50 k Mr, which was reported to be a putative precursor of p26 [20], was also observed in both of the samples though p26 was only observed in one of the 3 serum samples tested (Fig. 3, 1 a and 2 a). The reasons for this inconsistency might be due to the difference of protein concentration in the cell lysates or the difference of antigenicity between p26 and p50. In HIV infection, it was reported that among the patients who had antibodies to HIV envelope protein, less than half of the AIDS patients had antibodies against HIV major core protein, although most samples from AIDS-related complex patients also reacted with both of the proteins [1]. In this study, we found that there are some FIV-positive samples without anti-p26 antibodies in field cat sera. Since health conditions of the cats were unknown, we could not find whether the cats had clinical status related to the feline AIDS. However, the ELISA in this study will be valuable for further studies about clinical stages of FIV infections when we use it together with other physical examinations.
Acknowledgements We are grateful to Dr. Hayami (Kyoto University, Kyoto, Japan) for providing FIV KYO1 strain. We also thank Dr. J. K. Yamamoto (University of California, Davis, California, U.S.A.) and H. Koyama (Kitasato University, Towada, Japan) for providing the FIV Petaluma-infected CRFK cells.This study was supported in part by grants from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare of Japan.
References 1. Barin F. Mclane MF, Allan JS, Lee TH, Groopman JE, Essex M (1985) Virus envelope protein of HTLV-III represents major target antigen for antibodies in AIDS patiens. Science 228:1094-1096 2. Egberink H, Borst M, Niphuis H, Balzarini J, Neu H, Schellekens H, Clercq ED, Horzinek M, Koolen M (1990) Suppression of feline immunodeficiencyvirus infection
360
3.
4. 5.
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Authors' address: Dr. T. Mikami, Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. Received August 26, 1991
