Journal of General Virology (1992), 73, 2147-2149. Printed in Great Britain
2147
Haemagglutination by parvovirus B19 K. E. Brown*~ and B. J. Cohen Virus Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London N W 9 5HT, U.K.
H u m a n parvovirus B 19 is a m e m b e r o f the a u t o n o m o u s parvoviridae but in contrast to other members o f the genus has not been shown to agglutinate red blood cells. We now report that the virus agglutinates red cells of primate origin, though with plasma-derived
virus this activity is masked by the presence o f an IgMlike inhibitor. This observation is consistent with the presence on the erythroid precursor target cell o f a specific receptor for parvovirus B19.
Parvovirus B19, the causative agent of erythema infectiosum and viral aplastic crisis, was first described by Cossart et al. in 1975. It was subsequently shown to be a member of the autonomous parvoviridae but in contrast to other members of the genus has not been shown previously to agglutinate red blood cells (Cossart et al., 1975). It is also known that the target cell of B19 virus in vivo is an early erythrocyte precursor (Mortimer et al., 1983). We now report that the virus agglutinates red cells of primate origin, though with plasma-derived virus this activity is masked. Haemagglutination (HA) was carried out using a microtitre method in V-bottomed plates. Serial twofold dilutions of antigen were prepared in dextrose gelatin albumin buffer ( D G A ; 5 g/1 dextrose, 0.3 g/l gelatin, 0-2~ bovine plasma albumin, in 0-05 M-PBS pH 6-3). A further volume of D G A was added to each well before indicator cells [0.5 ~ v/v baboon (Papio sp.) erythrocytes in saline] were added and the plates were incubated for 2 h at 4 °C. The endpoint was 50?/o H A (1 HA5o unit). In contrast to previous reports, we found that the majority of samples of plasma and serum that contained B19 virus agglutinated red cells (baboon), although only at low titre. When sera were frozen and thawed repeatedly the titre of haemagglutinin was significantly increased (e.g. serum Sig from < 1:10 to 1:5120, Table 1). One serum (47432/90, Table 1) which had been subjected to repeated cycles of freezing and thawing showed an H A titre of 1:20000. Recombinant B19 virus proteins are known to form capsids (Kajigaya et al., 1989, 1991 ; Brown et al., 1990) and these also were examined for haemagglutinating activity. COS-7 cells were transfected with a B19 virus-
simian virus 40 hybrid plasmid (a gift from C. AsteU, University of British Columbia, Canada) as described previously (Beard et al., 1989) and lysed. The lysate was centrifuged through 20 ~ (w/v) sucrose (80000 g for 5 h); the resuspended pellet, which contained capsids, agglutinated baboon red cells to a titre of 1:1600 (Table 1). H u m a n O cells were also agglutinated (titre 1:800) but not tamarin (Saguinus sp.), horse, sheep, rabbit, guineapig, chicken or turkey erythrocytes. Agglutination was stable at both room temperature and 4 °C, but the titre was higher at 4 °C. Baculovirus-produced B19 capsids containing VP2 alone or VP1 and VP2 in combination (Kajigaya et al., 1991 ; a gift from S. Kajigaya, N H L B I , NIH, Bethesda,
t Present address: Cell Biology Section, Clinical Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, U.S.A. 0001-0834 © 1992 SGM
Table 1. HA titres of different parvovirus B19 antigens with 0.5%0 (v/v) baboon cells
Antigen Recombinant antigen COS-7 VP2 alone VPI + 2 Serum-derived antigen Sig 47432/90
VP2 (relative No. of concentration)* capsids* 2-2 12 20 100 90
4-4 × 109 2-6 x 10l0 4.0 × 101°
HA titre (reciprocal) 1600 6400 25000
2-0 x 1011 < 10t-5120~: 1.8 × 1011 20000~
* Antigen preparations were denatured in Laemmli sample buffer, electrophoresedon an SDS polyacrylamidegel (8% acrylamide,0.15~ bisacrylamide), stained with Coomassie blue, and destained for 48 h. VP2 bands were cut out, eluted with 25~ (v/v) pyridine and the absorbance was measured at 605 nm. The concentrationwas estimated by comparison with absorbances given by serial dilutions of a serum (Sig)containing 10 tag/mlB19 DNA, equivalent to 2 x 1011capsids/ml (Anderson et al., 1985). t Native. ~;After freezing and thawing.
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Table 2. H A inhibition titres of parvovirus B19 MAbs MAb* A JC (negative control) C VRL/B19-3 I VRL/B19-7 C VRL/B19-11 A A A E A L A N H R96-B6 A R92-F6 A 162-2B A 521-5D A BEll
Source laboratoryt V V V V O O O O B B C1 C2 F
Antibody subclass NK§ NK IgG2a NK IgG2a IgG1 IgG2a IgG2b NK NK IgM IgG NK
Reactivityin Westernblot:~ Neg Neg VP2 Neg Neg VP2 VP1 + 2 VP2 Neg VP1 + 2 Neg Neg VP1 + 2
HAI titre (reciprocal) < 100 < 100 < 100 < 100 < 100 > 1000 < 100 < 100 < 100 < 100 200 > 100000 >100
* A, ascites fluid, unpurified; C, ascites fluid purified by caprylic acid precipitation; I, ascites fluid purified by ion-exchange chromatography; H, hybridoma fluid. t Source laboratories (and references to MAbs) V, VRL, CPHL, London, U.K. (Cohen et al., 1983); O, LCDC, Ottawa, Canada (Yoshimoto etal., 1991); B, RVL, Belfast, U.K. (Dr H. O'Neill); C1, CDC, Atlanta, Ga., U.S.A. (Anderson et aL, 1986); C2, CDC, Atlanta, Ga., U.S.A. (Kajigaya et al., 1991); F, Kyushu University, Fukuoka, Japan (Sato et al., 1991). :~ Transfected COS-7 cell lysates were denatured in Laemmli sample buffer, electrophoresed on an SDS-polyacrylamide gel ( 8 ~ acrylamide, 0.15~ bisacrylamide), and electrophoretically transferred to nitrocellulose membranes. MAbs (1:1000 dilution) were incubated overnight with membranes, and detected by goat anti-mouse-horseradish peroxidase and diaminobenzidine enhanced with 0.3~ cobalt chloride. Neg, Negative. § NK, Not known.
Md., U.S.A.) both showed haemagglutinating activity (Table 1); this suggests that agglutination was a function of the VP2 alone. An HA inhibition (HAI) test to detect antibody to BI9 virus was set up using the haemagglutinin prepared from transfected COS-7 cells. Serial twofold dilutions of sera or monoclonal antibody (MAb) in DGA were incubated with an equal volume of haemagglutinin suspension containing 4 HAso units. The plates were incubated for 1 h at room temperature, a suspension of 0.5~ baboon erythrocytes was added and, after incubation for 2 h at 4 °C, the endpoint was read as 50~ HA. HA could be inhibited by three MAb preparations (Table 2). One of these preparations, MAb E, has been shown to bind to a neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein (Yoshimoto et al., 1991). This region corresponds to amino acids 71 to 91 of canine parvovirus (CPV) VP2 and contributes to the spike region on the 'threefold' axis of the capsid (Tsao et al., 1991). The HA site for CPV is not yet known. Sequence analysis has indicated that two residues (Arg 373, Val 558) are associated with the ability of CPV to agglutinate red cells (Parrish et al., 1988), but neither maps on the surface of the CPV VP2. Human sera with or without parvovirus B19-specific IgG and/or IgM detectedby radioimmunoassay (Cohen et al., 1983) were tested for HAl. All serum specimens,
regardless of their anti-B19 status, inhibited HA (titres > 1 : 80) which suggests that non-specific inhibitor(s) of HA are consistently present in human serum. Attempts to remove inhibitors by standard methods such as absorption with kaolin, manganous chloride/heparin, periodate, acetone and receptor-destroying enzyme (Lennette & Schmidt, 1979) were all unsuccessful. The inhibitory activity was not removed by heating at 60 °C for l h, but it was reduced if serum has been frozen and thawed and left at room temperature for several hours. It was not removed by overnight treatment with phospholipase A2 (10 units/ml) or phospholipase C (1 unit/I.d) although it was partially destroyed by proteinase K (2 mg/ml), trypsin (8 mg/ml), 2-mercaptoethanol (0.2 M) or two extractions with n-butanol. Overnight treatment with trypsin followed by inactivation of the enzyme by heating at 60 °C for 1 h and two n-butanol extractions did, however, remove all inhibitory activity. To identify the nature of the inhibitor(s), parvovirus B19 IgG-/IgM-negative sera (I00 ~tl) were centrifuged through 10 ~ to 50 ~ (w/v) sucrose in PBS at 135 000 g for 16 h. Fractions were collected by upward displacement and tested in the HA1 assay. Inhibitory activity was found in the lower fractions indicating an inhibitor with a sedimentation coefficient of approximately 19S. Flotation centrifugation (Haukenes & Blom, 1975) showed inhibitory activity at a density > 1-25 g/ml,
Short communication
2
r
l
I
[ T I I I I
I I [ I I I
I I 1 I I I' I [
I ]
1.8 1.6 ,-, 1.4
~ =
12 1
"~ 0-8 0-6 0-4 0.2 0
i ~ m m m ~ m ~ m ~ 18 19 20 21 22 2g 24 25 26 27 28 29 3~ 31 32 33 34 ~5 36 37 38 x9 40 41 42 43
Fraction no. Fig. 1. Fractionation of human sera containing an inhibitor of parvovirus BI9 HA. Bl9-specific antibody-negative serum was separated through a 35 x 1 cm Sephacryl S-300 column at 5 ml/h and 0-5 ml fractions were collected. Total protein ( I ) was estimated by absorbance (280 nm), and total IgM ([:]) and IgG (*) by enzyme immunoassay. The HAl titre is shown (ll).
indicating that a lipoprotein was not involved in the inhibition reaction. Gel filtration chromatography of B 19 IgG-/IgM-negative sera confirmed the presence of a large molecule inhibitor which co-purified with IgM (Fig. 1). Purified human r IgM myeloma protein (50 pg/ml; obtained from The Binding Site, Birmingham University, U.K.) inhibited the HA. Pre-incubation of serum with a rabbit anti-human IgM ~ chain-specific; Dako) antibody preparation blocked inhibitory activity. The finding that B19 virus agglutinates primate erythrocytes is important for studies of its pathogenesis and for diagnosis. Mature red cells and their precursors probably share the same surface receptor for B 19 virus. The interaction of the virus with an IgM-like molecule in plasma may allow it to evade the immune response, and it would prevent in vivo agglutination of erythrocytes. In vitro HA by parvovirus B19 may prove to be a useful property for clinical laboratory diagnosis. We thank Drs D. Kennedy, H. O'Neill, L. Anderson and K. Okochi for the supply of MAb, J. Norcott for technical assistance and Drs P. P. Mortimer and D. W. G. Brown for helpful comments.
2149
References ANDERSON, L. J., TSOU, C., PARKER, R. A., CHORBA,T. U, WULFF, H., TATTERSALL, P. & MORTIMER, P. P. (1986). Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 24, 522-526. ANDERSON, M. J., J O N E S , S . E . & M I N S O N , A . C . (1985). Diagnosis of human parvovirus infection by dot-blot hybridisation using cloned viral DNA. Journal of Medical Virology 15, 163-172. B E A R D , C . , S T A M A N D , J . & A S T E L L , C . R . (1989). Transient expression of B 19 parvovirus gene products in COS-7 cells transfected with B 19SV40 hybrid vectors. Virology 172, 659-664. BROWN, C. S., SALIMANS,M. M. M., NOTEBORN, M. H. M. & WE1LAND, H. T. (1990). Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system. Virus Research 15, 197-211. COHEN, B. J., MORTIMER, P. P. & PEREIRA, i. S . (1983). Diagnostic assays with monoclonal antibodies for the human serum parvoviruslike virus (SPLV). Journal of Hygiene 91, 113-130. COSSART, Y. E., F I E L D , A . M . , C A N T , B . & W I D D O W S , D . (1975). Parvovirus-like particles in human sera. Lancet i, 72-73. HAUKENES, G. & BLOM, M. (1975). False positive rubella virus haemagglutination inhibition reactions: occurrence and disclosure. Medical Microbiology and Immunology 161, 99-106. KAJIGAYA, S., SHIMADA, T., FUJITA, S. & YOUNG, N. S. (1989). A genetically engineered cell line that produced empty capsids of B I 9 (human) parvovirus. Proceedingsof the NationalAcademy of Sciences, U.S.A. 86, 7601-7605. KAJIGAYA, S., FUJII, H., FIELD, A., ANDERSON, S., ROSENFIELD, S., ANDERSON, L. J., SHIMADA, T. & YOUNG, N. S. (1991). Self assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunologically similar to native virus. Proceedings of the National Academy of Sciences, U.S.A. 88, 4646-4650. LENNETTE, E. M. & SCHMIDT, N. (editors)(1979). Viral, Rickettsial and Chlamydial Infections. Washington, D.C. American Public Health Association. MORTIMER, P. P., HUMPHRIES, R. K., MOORE, J. G., PURCELL, R. H. & YOUNG, N. S. (1983). A human parvovirus-like virus inhibits haematopoietic colony formation in vitro. Nature, London 302, 426M29. PARRISH, C. R., BURTONBOY, G. & CARMICHAEL, L. E. (1988). Characterization of a non-hemagglutinating mutant of canine parvovirus. Virology 163, 230-232. SATO, H., HlgmA, J., FURUKAWA, M., KURODA, N., SHIRAKI, H., MAEDA, Y. & OKOCHI, K. (1991). Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvov i rus B 19. Journalof Virology 65, 1667-1672. TSAO, J., CHAPMAN, M. S., AGBANDIE, M., KELLER, W., SMITH, K., WA, H., Luo, M., SMITH,T. J., ROSSMANN,M. G., COMPANS,R. W. & PARRISn, C. R. (1991). The three-dimensional structure of canine parvovirus and its functional implications. Science 251, 1456-1463. YOSHIMOTO, K., ROSENFELD, S., FRICKHOFEN, N., KENNEDY, D., HILLS, R., KAJIGAYA, S. & YOUNG, N. S. (1991). A second neutralizing epitope of Bt 9 parvovirus implicates the spike region in the immune response. Journal of Virology 65, 7056-7060.
(Received 7 January 1992; Accepted 7 April 1992)
2147
Haemagglutination by parvovirus B19 K. E. Brown*~ and B. J. Cohen Virus Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London N W 9 5HT, U.K.
H u m a n parvovirus B 19 is a m e m b e r o f the a u t o n o m o u s parvoviridae but in contrast to other members o f the genus has not been shown to agglutinate red blood cells. We now report that the virus agglutinates red cells of primate origin, though with plasma-derived
virus this activity is masked by the presence o f an IgMlike inhibitor. This observation is consistent with the presence on the erythroid precursor target cell o f a specific receptor for parvovirus B19.
Parvovirus B19, the causative agent of erythema infectiosum and viral aplastic crisis, was first described by Cossart et al. in 1975. It was subsequently shown to be a member of the autonomous parvoviridae but in contrast to other members of the genus has not been shown previously to agglutinate red blood cells (Cossart et al., 1975). It is also known that the target cell of B19 virus in vivo is an early erythrocyte precursor (Mortimer et al., 1983). We now report that the virus agglutinates red cells of primate origin, though with plasma-derived virus this activity is masked. Haemagglutination (HA) was carried out using a microtitre method in V-bottomed plates. Serial twofold dilutions of antigen were prepared in dextrose gelatin albumin buffer ( D G A ; 5 g/1 dextrose, 0.3 g/l gelatin, 0-2~ bovine plasma albumin, in 0-05 M-PBS pH 6-3). A further volume of D G A was added to each well before indicator cells [0.5 ~ v/v baboon (Papio sp.) erythrocytes in saline] were added and the plates were incubated for 2 h at 4 °C. The endpoint was 50?/o H A (1 HA5o unit). In contrast to previous reports, we found that the majority of samples of plasma and serum that contained B19 virus agglutinated red cells (baboon), although only at low titre. When sera were frozen and thawed repeatedly the titre of haemagglutinin was significantly increased (e.g. serum Sig from < 1:10 to 1:5120, Table 1). One serum (47432/90, Table 1) which had been subjected to repeated cycles of freezing and thawing showed an H A titre of 1:20000. Recombinant B19 virus proteins are known to form capsids (Kajigaya et al., 1989, 1991 ; Brown et al., 1990) and these also were examined for haemagglutinating activity. COS-7 cells were transfected with a B19 virus-
simian virus 40 hybrid plasmid (a gift from C. AsteU, University of British Columbia, Canada) as described previously (Beard et al., 1989) and lysed. The lysate was centrifuged through 20 ~ (w/v) sucrose (80000 g for 5 h); the resuspended pellet, which contained capsids, agglutinated baboon red cells to a titre of 1:1600 (Table 1). H u m a n O cells were also agglutinated (titre 1:800) but not tamarin (Saguinus sp.), horse, sheep, rabbit, guineapig, chicken or turkey erythrocytes. Agglutination was stable at both room temperature and 4 °C, but the titre was higher at 4 °C. Baculovirus-produced B19 capsids containing VP2 alone or VP1 and VP2 in combination (Kajigaya et al., 1991 ; a gift from S. Kajigaya, N H L B I , NIH, Bethesda,
t Present address: Cell Biology Section, Clinical Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, U.S.A. 0001-0834 © 1992 SGM
Table 1. HA titres of different parvovirus B19 antigens with 0.5%0 (v/v) baboon cells
Antigen Recombinant antigen COS-7 VP2 alone VPI + 2 Serum-derived antigen Sig 47432/90
VP2 (relative No. of concentration)* capsids* 2-2 12 20 100 90
4-4 × 109 2-6 x 10l0 4.0 × 101°
HA titre (reciprocal) 1600 6400 25000
2-0 x 1011 < 10t-5120~: 1.8 × 1011 20000~
* Antigen preparations were denatured in Laemmli sample buffer, electrophoresedon an SDS polyacrylamidegel (8% acrylamide,0.15~ bisacrylamide), stained with Coomassie blue, and destained for 48 h. VP2 bands were cut out, eluted with 25~ (v/v) pyridine and the absorbance was measured at 605 nm. The concentrationwas estimated by comparison with absorbances given by serial dilutions of a serum (Sig)containing 10 tag/mlB19 DNA, equivalent to 2 x 1011capsids/ml (Anderson et al., 1985). t Native. ~;After freezing and thawing.
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Table 2. H A inhibition titres of parvovirus B19 MAbs MAb* A JC (negative control) C VRL/B19-3 I VRL/B19-7 C VRL/B19-11 A A A E A L A N H R96-B6 A R92-F6 A 162-2B A 521-5D A BEll
Source laboratoryt V V V V O O O O B B C1 C2 F
Antibody subclass NK§ NK IgG2a NK IgG2a IgG1 IgG2a IgG2b NK NK IgM IgG NK
Reactivityin Westernblot:~ Neg Neg VP2 Neg Neg VP2 VP1 + 2 VP2 Neg VP1 + 2 Neg Neg VP1 + 2
HAI titre (reciprocal) < 100 < 100 < 100 < 100 < 100 > 1000 < 100 < 100 < 100 < 100 200 > 100000 >100
* A, ascites fluid, unpurified; C, ascites fluid purified by caprylic acid precipitation; I, ascites fluid purified by ion-exchange chromatography; H, hybridoma fluid. t Source laboratories (and references to MAbs) V, VRL, CPHL, London, U.K. (Cohen et al., 1983); O, LCDC, Ottawa, Canada (Yoshimoto etal., 1991); B, RVL, Belfast, U.K. (Dr H. O'Neill); C1, CDC, Atlanta, Ga., U.S.A. (Anderson et aL, 1986); C2, CDC, Atlanta, Ga., U.S.A. (Kajigaya et al., 1991); F, Kyushu University, Fukuoka, Japan (Sato et al., 1991). :~ Transfected COS-7 cell lysates were denatured in Laemmli sample buffer, electrophoresed on an SDS-polyacrylamide gel ( 8 ~ acrylamide, 0.15~ bisacrylamide), and electrophoretically transferred to nitrocellulose membranes. MAbs (1:1000 dilution) were incubated overnight with membranes, and detected by goat anti-mouse-horseradish peroxidase and diaminobenzidine enhanced with 0.3~ cobalt chloride. Neg, Negative. § NK, Not known.
Md., U.S.A.) both showed haemagglutinating activity (Table 1); this suggests that agglutination was a function of the VP2 alone. An HA inhibition (HAI) test to detect antibody to BI9 virus was set up using the haemagglutinin prepared from transfected COS-7 cells. Serial twofold dilutions of sera or monoclonal antibody (MAb) in DGA were incubated with an equal volume of haemagglutinin suspension containing 4 HAso units. The plates were incubated for 1 h at room temperature, a suspension of 0.5~ baboon erythrocytes was added and, after incubation for 2 h at 4 °C, the endpoint was read as 50~ HA. HA could be inhibited by three MAb preparations (Table 2). One of these preparations, MAb E, has been shown to bind to a neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein (Yoshimoto et al., 1991). This region corresponds to amino acids 71 to 91 of canine parvovirus (CPV) VP2 and contributes to the spike region on the 'threefold' axis of the capsid (Tsao et al., 1991). The HA site for CPV is not yet known. Sequence analysis has indicated that two residues (Arg 373, Val 558) are associated with the ability of CPV to agglutinate red cells (Parrish et al., 1988), but neither maps on the surface of the CPV VP2. Human sera with or without parvovirus B19-specific IgG and/or IgM detectedby radioimmunoassay (Cohen et al., 1983) were tested for HAl. All serum specimens,
regardless of their anti-B19 status, inhibited HA (titres > 1 : 80) which suggests that non-specific inhibitor(s) of HA are consistently present in human serum. Attempts to remove inhibitors by standard methods such as absorption with kaolin, manganous chloride/heparin, periodate, acetone and receptor-destroying enzyme (Lennette & Schmidt, 1979) were all unsuccessful. The inhibitory activity was not removed by heating at 60 °C for l h, but it was reduced if serum has been frozen and thawed and left at room temperature for several hours. It was not removed by overnight treatment with phospholipase A2 (10 units/ml) or phospholipase C (1 unit/I.d) although it was partially destroyed by proteinase K (2 mg/ml), trypsin (8 mg/ml), 2-mercaptoethanol (0.2 M) or two extractions with n-butanol. Overnight treatment with trypsin followed by inactivation of the enzyme by heating at 60 °C for 1 h and two n-butanol extractions did, however, remove all inhibitory activity. To identify the nature of the inhibitor(s), parvovirus B19 IgG-/IgM-negative sera (I00 ~tl) were centrifuged through 10 ~ to 50 ~ (w/v) sucrose in PBS at 135 000 g for 16 h. Fractions were collected by upward displacement and tested in the HA1 assay. Inhibitory activity was found in the lower fractions indicating an inhibitor with a sedimentation coefficient of approximately 19S. Flotation centrifugation (Haukenes & Blom, 1975) showed inhibitory activity at a density > 1-25 g/ml,
Short communication
2
r
l
I
[ T I I I I
I I [ I I I
I I 1 I I I' I [
I ]
1.8 1.6 ,-, 1.4
~ =
12 1
"~ 0-8 0-6 0-4 0.2 0
i ~ m m m ~ m ~ m ~ 18 19 20 21 22 2g 24 25 26 27 28 29 3~ 31 32 33 34 ~5 36 37 38 x9 40 41 42 43
Fraction no. Fig. 1. Fractionation of human sera containing an inhibitor of parvovirus BI9 HA. Bl9-specific antibody-negative serum was separated through a 35 x 1 cm Sephacryl S-300 column at 5 ml/h and 0-5 ml fractions were collected. Total protein ( I ) was estimated by absorbance (280 nm), and total IgM ([:]) and IgG (*) by enzyme immunoassay. The HAl titre is shown (ll).
indicating that a lipoprotein was not involved in the inhibition reaction. Gel filtration chromatography of B 19 IgG-/IgM-negative sera confirmed the presence of a large molecule inhibitor which co-purified with IgM (Fig. 1). Purified human r IgM myeloma protein (50 pg/ml; obtained from The Binding Site, Birmingham University, U.K.) inhibited the HA. Pre-incubation of serum with a rabbit anti-human IgM ~ chain-specific; Dako) antibody preparation blocked inhibitory activity. The finding that B19 virus agglutinates primate erythrocytes is important for studies of its pathogenesis and for diagnosis. Mature red cells and their precursors probably share the same surface receptor for B 19 virus. The interaction of the virus with an IgM-like molecule in plasma may allow it to evade the immune response, and it would prevent in vivo agglutination of erythrocytes. In vitro HA by parvovirus B19 may prove to be a useful property for clinical laboratory diagnosis. We thank Drs D. Kennedy, H. O'Neill, L. Anderson and K. Okochi for the supply of MAb, J. Norcott for technical assistance and Drs P. P. Mortimer and D. W. G. Brown for helpful comments.
2149
References ANDERSON, L. J., TSOU, C., PARKER, R. A., CHORBA,T. U, WULFF, H., TATTERSALL, P. & MORTIMER, P. P. (1986). Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 24, 522-526. ANDERSON, M. J., J O N E S , S . E . & M I N S O N , A . C . (1985). Diagnosis of human parvovirus infection by dot-blot hybridisation using cloned viral DNA. Journal of Medical Virology 15, 163-172. B E A R D , C . , S T A M A N D , J . & A S T E L L , C . R . (1989). Transient expression of B 19 parvovirus gene products in COS-7 cells transfected with B 19SV40 hybrid vectors. Virology 172, 659-664. BROWN, C. S., SALIMANS,M. M. M., NOTEBORN, M. H. M. & WE1LAND, H. T. (1990). Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system. Virus Research 15, 197-211. COHEN, B. J., MORTIMER, P. P. & PEREIRA, i. S . (1983). Diagnostic assays with monoclonal antibodies for the human serum parvoviruslike virus (SPLV). Journal of Hygiene 91, 113-130. COSSART, Y. E., F I E L D , A . M . , C A N T , B . & W I D D O W S , D . (1975). Parvovirus-like particles in human sera. Lancet i, 72-73. HAUKENES, G. & BLOM, M. (1975). False positive rubella virus haemagglutination inhibition reactions: occurrence and disclosure. Medical Microbiology and Immunology 161, 99-106. KAJIGAYA, S., SHIMADA, T., FUJITA, S. & YOUNG, N. S. (1989). A genetically engineered cell line that produced empty capsids of B I 9 (human) parvovirus. Proceedingsof the NationalAcademy of Sciences, U.S.A. 86, 7601-7605. KAJIGAYA, S., FUJII, H., FIELD, A., ANDERSON, S., ROSENFIELD, S., ANDERSON, L. J., SHIMADA, T. & YOUNG, N. S. (1991). Self assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunologically similar to native virus. Proceedings of the National Academy of Sciences, U.S.A. 88, 4646-4650. LENNETTE, E. M. & SCHMIDT, N. (editors)(1979). Viral, Rickettsial and Chlamydial Infections. Washington, D.C. American Public Health Association. MORTIMER, P. P., HUMPHRIES, R. K., MOORE, J. G., PURCELL, R. H. & YOUNG, N. S. (1983). A human parvovirus-like virus inhibits haematopoietic colony formation in vitro. Nature, London 302, 426M29. PARRISH, C. R., BURTONBOY, G. & CARMICHAEL, L. E. (1988). Characterization of a non-hemagglutinating mutant of canine parvovirus. Virology 163, 230-232. SATO, H., HlgmA, J., FURUKAWA, M., KURODA, N., SHIRAKI, H., MAEDA, Y. & OKOCHI, K. (1991). Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvov i rus B 19. Journalof Virology 65, 1667-1672. TSAO, J., CHAPMAN, M. S., AGBANDIE, M., KELLER, W., SMITH, K., WA, H., Luo, M., SMITH,T. J., ROSSMANN,M. G., COMPANS,R. W. & PARRISn, C. R. (1991). The three-dimensional structure of canine parvovirus and its functional implications. Science 251, 1456-1463. YOSHIMOTO, K., ROSENFELD, S., FRICKHOFEN, N., KENNEDY, D., HILLS, R., KAJIGAYA, S. & YOUNG, N. S. (1991). A second neutralizing epitope of Bt 9 parvovirus implicates the spike region in the immune response. Journal of Virology 65, 7056-7060.
(Received 7 January 1992; Accepted 7 April 1992)
