Enterovirus IgM detection: specificity of μ-antibody-capture radioimmunoassays using virions and procapsids of Coxsackie B virus

191

Journal of ~iroiog~c~~ methods, 24 (1989) 191-202 Elsevier JVM 00873

Enterovirus IgM detection: specificity of pantibody-capture radioimmunoassays using virions and procapsids of Coxsackie B virus G. Frisk’,

E. Nilsson’,

A. Ehrnst*

and H. Diderholm’

‘Department of Medical Virology, Biomedical Centre, University of Uppsala, Uppsala, and 2Central Microbiological Laboratory of Stockholm County (A. E.), Stockholm, Sweden

(Accepted 11 January 1989)

-.-

-_-

Summary A predominantly type-specific F-capture radioimmunoassay (RIA) of IgM antibodies to Coxsackie Bl-B5 (CBl-CB5) viruses was previously described (Frisk et al., 1984). The present study is concerned with the specificity of this assay, using as antigen different strains of one serotype (CB5) and procapsids of two serotypes (CB3 and CB5). Eight strains of CB5 virions were tested against acute and/or convalescent sera from 10 patients from whom CB5 had been isolated. Seven patients’ sera were tested against their own strain. The frequency of IgM-positive patients varied from 9 of 10 (90%) to 5 of 10 (50%). In three cases the highest titres were obtained with the patients’ own strain. When sera from patients with other enterovirus infections were tested against the CB5 strains, heterotypic titres were obtained to a certain extent (O-15.6%). The strains giving a high frequency of homotypic titres varied concerning heterotypic reactions. It is concluded that the choice of strain is important if a high frequency of homotypic titres with no or only a few heterotypic reactions is to be obtained. When procapsids were used as antigen, both homotypic and heterotypic titres were seen to a large extent. All patients with homotypic IgM against CB3 or CB5 virions showed IgM against the CB3 or the CB5 procapsids, respectively. When sera from patients with other enterovirus infections were tested, IgM was found in 54 of 93 patients (58%) with use of the CB3 procapsid and in 52 of 87 patients (60%) with the CB5 procapsid. It was often not the same patients who showed

Correspondence ro: Hans Dider~olm, Department of Medical Virology. Biomedical Centre, Box 584, S-751 23 Uppsala, Sweden.

0166-0934/89/$03.50 @ 1989 Eisevier Science Publishers B.V. (Biomedical Division)

192

IgM against the two different procapsids. When both procapsids were used, IgM positivity was found in 62 of 81 patients (77%) with other enterovirus infections. It is concluded that the use of two or more procapsids in combination is of value for the diagnosis of a recent or current enterovirus infection. RIA; IgM; Coxsackie; Echo; Procapsid

Introduction The serological diagnosis of enterovirus infections has been based mainly on the demonstration of a significant rise in antibody titre. Another approach has been to search for antibodies of the IgM class. There are several reports describing the detection of Coxsackie B (CB)-virus-specific IgM by RIA (Dorries and Ter Meulen, 1980; Morgan-Capner and McSorley, 1983; Pugh, 1984; Torfason et al., 1984; Frisk et al., 1984) or ELISA (El-Hagrassy and Banatvala, 1980; Dorries and Ter Meulen, 1983; King et al., 1983; Hannington et al., 1986). The results described vary in both specificity and sensitivity. This is not unexpected, since both the assay procedures and the purity of the antigens used differed in most reported studies. We described previously a predominantly type-specific RIA for the detection of IgM antibodies to CBl-CB5, and echo- (E) 11 and 30 viruses (Torfason et al., 1984, Frisk et al., 1984). The viruses were purified by ultracentrifugations in CsCl and the y-capture technique was used. In the present study, using the same techniques, different strains of CB5 were tested in order to look for strain specificity. In addition, specificity was studied with procapsids derived from CB3 and CB5 as antigen.

Materials and Methods

JJH cells, a strain of HeLa cells (Beatrice et al., 1980), were cultivated in monolayers. GMK cells, a continuous line of green monkey kidney cells, were grown in 96-well tissue culture plates. The growth medium was Eagle’s minimum essential medium (MEM) supplemented with 10% bovine serum (fetal for JJH cells, newborn calf for GMK cells) and antibiotics. Medium without serum was used after infection. Viruses

The viruses used were CB3, strain Nancy, and in-house strains of CBl, CB2, CB4 and CB.5 designated CBl-E, CB2-E, CB4-E and CBS-E, respectively. The viruses used for testing strain specificity were eight isolates of CB5 from different patients collected over a period of over two decades. These strains were calfed V136, V2042, V4429, V4710, V5784, V9699, V9869 and CBS-E. The viruses used

193

for the preparation of procapsids were the Nancy strain of CB3 and a strain of CB.5, designated F84552. [3sS]methionine-labelled viruses were propagated in JJH cells in monolayers (120 cm2). After infection at a multiplicity of 2%30 TCD,, per cell and adsorption for 30 min, the cells were washed and incubated further with methionine-free MEM. After an additional 120 min, 1 mCi [35S]methionine (Amersham) was added and the cultures were incubated for 14-25 h, depending on the grade of CPE. Only extracellular virus was harvested. Virio~ and ~~oca~~id ~ur~~cation

Medium from infected monolayers was layered on 2.1 mi cushions of CsCi with a density of 1.4 g/ml in 5.5 ml polycar~nate tubes and centrifuged for 3 h at 124000

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Fig. 1. (A) Distribution of radioactivity and infectivity of [%]methionine-labelled CB3 virions after CsCl density gradient centrifugation. (B) Autoradiogram of the two peaks seen in (A) after separation by SDS-PAGE. Radioactivity, w-.-. 0; infectivity, o-o; density, A-A.

194

x g in a swing-out rotor. Fifteen-drop fractions were collected through the bottom of the tubes. The six to eight fractions with the highest infectivity were pooled and dialysed against a phosphate buffer devoid of calcium and magnesium (PBS-A). The dialysed fractions were mixed with 2.1 g of solid CsCl. The volume was then adjusted to 5.0 ml and centrifuged for 18-20 h at I240~ x g. Thirteen-drop fractions were collected. The peak of highest infectivity and the first peak of radioactivity coincided. The second peak of radioactivity contained the procapsids (Fig. 1). From both peaks three to five fractions were collected and pooled. The pools were dialysed against PBS-A before use in RIA. Serum specimens We used 161 serum specimens collected from 99 patients with various CB, Coxsackie A (CA), or E virus infections verified by virus isolation (Table 1). There were 62 serum pairs and 37 single specimens. The sera were collected over a period of several years. Two groups of controls were included. One group consisted of serum pairs from 30 patients with significant rises in complement-fixation (CF) titre against viruses other than enteroviruses. These were influenza A virus (6 pairs}, influenza B virus (4), rubella virus (S), herpes simplex virus (l), varicella-zoster virus (2), morbillivirus (l), respiratory syncytial virus (3), parainfluenza 1 and 3 viruses (l)? adenovirus (5) and mumps virus (2). In addition, five serum pairs with significant rises in CF titre against Mycoplasma pneumoniae and three pairs against Chlamydia psittaci were tested. The second control group comprised serum specimens from 61 medical students without any signs of infection. RIA procedure The RIA procedure used was described elsewhere (Torfason et al., 1984). Briefly,

polystyrene

beads were coated overnight at room temperature

with 9.4 pgiml (1.0

TABLE 1 Specification of serum specimens from patients from whom virus was isolated Virus isolated

Number of serum pairs

Total number of sera

Single sera A

c

Cl31 CB2 CB3 CB4 CB5 CA9 CA2,6,7,14 E6 El1 E30 E4,9,15,18,24

5 2 3 6 5 2 10 16 7 6

3 4 3 1 4 1 3 0 2 1 0

0 0 1

2 2 0 1 1 2 2 d

3 14 8 9 18 11 8 21 36 17 16

Total

62

22

15

161

0

A, acute sera; C, convalescent sera.

195

*g/bead) of human IgM-Fc-specific antibodies raised in rabbits (Jackson Immunoresearch Laboratories, Inc, USA) and diluted in carbonate buffer, pH 9.6. The serum specimens, diluted in PBS-A containing 0.5% bovine serum albumin, 0.5% Tween 20 and 0.02% NaN,, were incubated with the coated beads for 2 h at 37°C. After washing, the 35S-labelled virions or the procapsids, diluted in the serum dilution buffer with 20 or 10% newborn calf serum, respectively, were added. After incubation overnight at room temperature the beads were washed, The cut-off point used in the end-point calculation was three times the CPM value of the buffer blank and a negative serum (both gave the same cpm value), with the proviso that the cut-off value should be at least 150 CPM. The results were expressed as reciprocal serum titres. The amount of labelled antigen added was essential for specificity and sensitivity. Standardization of the labelled virions was described previously (Frisk et al., 1984). Briefly, the antigen concentration was chosen by testing each new batch against 3-5 sera with a known homotypic IgM titre and about 15 sera from patients with heterotypic enterovirus infections. The concentration giving an IgM titre only with the homotypic specimens was used. In the study on strain specificity, each CB5 strain was standardized against 17 homotypic and 44 heterotypic sera on the same occasion. The antigen concentration for the procapsids was chosen by assaying 161 acute and/or convalescent sera from 99 patients with enterovirus infections and all the control sera mentioned above. The amount of labelled procapsids which gave the greatest discrepancy between the group of patients with enterovirus infections and the two control groups was then used (Fig. 2). Each new batch of labelied procapsids was standardized against 10-15 sera with known titres. 00000

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Iglll-titres in sera of enterovirus infected and non-enterovirus infected patients and healthy controls using CB5 procapsids as antigen. A, acute sera (0); C, convalescent sera (0).

196

SDS-polyacrylamide

gel electrophoresis

(SDS-PAGE)

Samples were prepared in 0.8% Tris, 10% glycerol, 2% SDS and 5% 2-mercaptoethanol and heated in a boiling water bath for 3 min and electrophoresed on SDS-polyacrylamide gels with a 15 cm resolving gel with 11% acrylamide and a 2 cm stacking gel with 4.0% acrylamide. After the electrophoresis the gels were in-

TABLE 2 Frequency of IgM responses in patients with enterovirus infections studied by RIA using CBI-CBS virions and procapsids of CB3 and CB5 as antigen Virus isolated

Acute (A) or convalescent (C) sera

Antigen Virion Homotypic

CBl CB2 CB3 CB4 CB5 CA9 CA2,6,7,14 E6 El1 E30 E4,9,15,18,24

Total number of positive assays/total number of assays Total number of positive serum pairsb/total number of serum pair assays

A A C A C A c A C A C A C A c A C A C A c

213

Procapsid Heterotypic

CB3

CB5

CB3 and/or CB5

315 313 414 415 l/l0 818 NT NT NT NT NT NT NT NT NT NT NT NT

1112 O/36 3120 o/20 0112 0116 0120 1140 3132 0130 0125 0125 o/15 5!50 4155 6190 6190 1140 8145 2130 4i50

213 519 215 415 313 214 215 4110 318 216 415 l/.5 113 4110 7111 13118 12118 418 619 016 4110

6110 6111 12118 11/18 318 519 116 6110

313 R/l0 lOill 15118 15118 518 719 116 8i10

37152 (71%)

441753 (5.8%)

851161 (53%)

931161 (58%)

1241161” (77%)

29/36 (81%)

24145I (5.3%)

59199 (60%)

64199 (65%)

79199’ (80%)

719 51.5

313 419 315 315 113 414 215 6110 818 316 215 315 113

313 519 415 415 313 414 315 7110 8/S 416 415 315

“Total number of positive serakotal number of sera tested. bAcute and/or convalescent sera positive. From some patients only acute or convalescent sera were obtained. Total number of positive serum pairs/total number of serum pairs tested. NT, Not tested.

197

cubated for 20 min with a fluorographic reagent (Amplifyer, and layered on a Hyperfilm-B-max (Amersham).

Amersham),

dried

Results IgM RZA wing virions us anrigen

Table 2 (columns 3 and 4) shows the IgM results obtained with virions as antigen. Seventy-one percent of the homotypic sera had IgM against CBl-CBS, whereas 5.8% of the heterotypic assays were positive. Both homotypic and heterotypic reactions were predominantly found in the convalescent sera. The percentage of positive serum pairs (acute and/or convalescent sera positive) was 81 when homotypic virions were used, whereas 5.3% of the heterotypic serum pair assays were positive. Titres of up to 32000 were noted, the median titre being 2000. There were no differences in magnitude between homotypic and heterotypic titres. Different strains of CB5 gave homotypic IgM titres of different levels (Table 3). The strains V9699, V9869 and CBS-E were not so broad in antigenicity as V136, V4429 and V4710. With use of the former strains the IgM positivity was 40-47%, whereas 73--80% of the sera showed IgM against the latter strains. No IgM was found in five patients when V9699 was used and in four when V9869 was used. The CBS-E strain failed to detect IgM in three patients, and the strains V136, V2042 and V5784 in two patients. V136, V4429 and V4710 were the three strains against which most of the patients had IgM. From four patients only acute serum was obtained and in one of these all the strains failed to show IgM. The titer levels were not correlated in such a way that the sera from one patient had the highest titre when the isolated strain from the same patient was used as antigen, except for the CBS-strains V136, V4429 and V9699. As shown in Table 4, when tested against sera from patients with other enterovirus infections all strains of CB5 except CBS-E gave heterotypic titres to different extent. The highest frequency of heterotypic reactions (15.6%) was obtained with the V5784 strain. There were four strains (V136, V4429, V9699 and V9869) with low heterotypic IgM reactivity (4.4%) but with homotypic IgM positivity varying from 40 to 73%. The highest ratio (16.6) between the frequency of homotypic and that of heterotypic responses was obtained with two strains, V136 and V4429. IgM RiA using procapsids as antigen

In the CsCl gradient the first peak of radioactivity contained the bulk of the virions. A second peak which banded at a density of 1.31 g/cm3 was also recorded (Fig. 1). SDS-PAGE revealed that this peak consisted of particles which were dissociated into mainly VPO, 1 and 3 (lAB, 1D and lC, respectively, according to the new L 434 nomenclature of Rueckert and Wimmer, 1984). There was also a weak band coinciding with VP2 (Fig. 1). These results showed that the bulk of particles in the second peak consisted of procapsids and are in agreement with the results of Putnack and Philips (1981). All sera with IgM against CB3 or CB.5 virions were also positive with CB3 and

62.5 2000

2 000

S5784-A S5784-c

S9699-A S9699-C

500

-

12/15 (80%) 9110 (90%)

11115 (73%) 9110 (90%)

9115 (60%)

8110

(80%)

250

2 000

2000

4000 16000

1000

250

8 000

4 000

500 2 000

500

500

1000

500

4 000

4 000

1000 4 000

2.50 16000

2000

125 4000

v4710

16000

2 000

2000 1000

v4429

“Strains and sera from the same patient are indicated by the same number. sAcute and/or convalescent sera positive.

11115 Total number of positive serakotal (73%) number of sera 8110 Total number of (80%) positive patie&‘/total number of patients

59864-C

S6233-A

S6610-A

s9869-c

2 000

1000

500

S4710-A

S9869-A

16000

62.5 4000

S4429-A S4429-C

500

4 000

S2042-A

2000

v2042

500 4000

V136”

Strain of CB5

S136-A S136-C

Sera acute (A) or convalescent (C)

5110

(50%)

a/i0

6115 (40%)

1000

8 000

62.5 1000

62.5

1000

V9699

(80%)

10115 (67%)

1000

-

4 000

4000

62.5 16000

62.5

32 000

1000

1000 4 000

V5784

Homotypic IgM RIA titres using different strains of CB5 virions; in seven cases the patient’s own strain was tested

TABLE 3

6110 (60%)

7115 (47%)

62.5

-

_

1000

1000

_

-

_ 2000

125

62.5 125

V9869

7110 (70%)

6115 (40%)

62.5

1000

_ 1000

_ 125

_ 1000

500

500

CBS-E

5

Ratio between frequencies of homotypic and heterotypic resDonses

16.6

(4?J%)

9.0

(6?7%)

16.6

(4.24%)

9.0

(8:0/o)

4.3

(1:6%)

45

El8

Heterotypic (CB2, CBJ, CB4, Eli, and E30) 9.1

(4?4%)

10.7

(42%)

(4;%)

(4G%)

(4i%).

15

Homotypic (CB5)

(::%)

CBS-E

The number and percentages of homotypic and heterotypic IgM responses to different strains of CB5 virions as antigen in the RIAs .~ Sera No. of sera Strain of CB5 tested V136 v2042 V4429 v4710 VS784 V9699 V9869

TABLE 4

200

CB5 procapsids, respectively. In addition, heterotypic IgM titres were obtained to a large extent with both procapsids, as seen in Table 2 (columns 5-7). Using the CB3 procapsids, 53% of all sera were positive, whereas the corresponding figure for CB5 was 58%. The corresponding proportions of positive serum pairs out of the total number of serum pairs (acute and/or convalescent sera positive) were 60 and 65% (58 and 60% of heterotypic pairs), respectively, showing that the sensitivity was about the same. However, some patients were negative with one procapsid but positive with the other. When both were used the results revealed IgM positivity to CB3 and/or CB.5 procapsids in 77% of the sera and in 80% of the patients (77% of patients with heterotypic enterovirus infections) (Table 2, last column). No definite differences between homotypic and heterotypic titre levels were seen. It was not found that either of the procapsids had any kind of subgroup (CA, CB or E) specificity. The enterovirus-specificity of the assay was investigated by testing serum specimens from the two control groups, patients with non-enterovirus infections and healthy subjects. In the former group IgM positivity was displayed by 10.5 and 7.9% with use of the CB3 and CBS procapsids, respectively. The percentage using CB3 and CB5 procapsids was 15.8. Among the healthy subjects 4.9 and 1.6% showed IgM positivity with the use of the CB3 and CB.5 procapsids, respectively, and the percentage with both procapsids was 6.6. The frequencies of IgM-positive patients with enterovirus infections, differed significantly (P
Discussion We described previously a predominantly type-specific b-capture RIA for the detection of IgM against CBl-CBS viruses (Torfason et al., 1984; Frisk et al., 1984). In the present study the specificity of the RIA was investigated using different strains of CB5 virions and procapsids of CB3 and CB5 as antigen. In addition, tests with CBl-CB4 virions against homotypic and heterotypic sera were performed. In tests of the CBl-CB5 virions, the frequency of Ig~-positivity among homotypic serum pairs (acute and/or convalescent sera positive) was 81%, whereas heterotypic reactions were noted in 5.3% of the assays (Table 2). In the earlier study (Frisk et al., 1984), in which El1 and 30 were also included, the corresponding figures were 97% (29 of 30 cases) and 2.1% (5 of 237 assays), respectively. The lower frequency of homotypic titres in the present study might be due to the fewer convalescent serum specimens. In both studies IgM titres were detected to a greater extent in the convalescent specimens than in the acute ones. It must be emphasized that ‘heterotypic’ titres may in fact be homotypic reactions due to double infections or to anamnestic responses. The RIAs using different strains of CB5 virions revealed differences both regarding homotypic and heterotypic reactions (Tables 3 and 4). The percentages of the former varied between 40 and 80 (50 and 90 of the patients), whereas the cor-

201

responding figures for heterotypic sera were 0 and 15.6 (0 and 16.6% of the patients). It is evident that the choice of strain is important for obtaining high specificity and sensitivity. The strains V136 and V4429 yielded highest ratio (16.6) between homotypic and heterotypic titres, while V5784 showed the lowest ratio (4.3). In the RIAs of Table 2 the CB.5 virion was represented by the CBS-E strain. The outcome would not have been the same with one of the other strains. Our results are not surprising. Intratypic variations have been reported for a number of enteroviruses. Using the RNA-hybridization technique, Brown and Wild (1974) found considerable differences between the Faulkner strain (CB5 prototype) and several isolates of CB5. With the aid of monoclonal antibodies to CB4, Prabhakar et al. (1982) identified over 13 variants of CB4 and showed that there were major antigenic differences among naturally occurring isolates. The RIAs using CB3 and CB5 procapsids revealed a number of heterotypic responses in addition to homotypic reactions. The two procapsids differed in antigenicity, since several sera were positive with only one of them. With the use of these procapsids IgM positivity was observed in 62 of 81 patients (77%) with heterotypic enterovirus infections. It is impossible to establish whether the heterotypic reactivity might be due to double infections or to anamnestic responses. The few positive reactions noted among the controls may be due to enterovirus infections or anamnestic responses. The possibility that there are cross-reacting antibodies is unlikely. Schmidt et al. (1963) and Forsgren (1968, 1969) purified enteroviruses by CsCl density gradients and found type-specific reactions by immunodiffusion in the fraction coinciding with the highest infectivity and group-specific reactions at a lower density. The findings of these studies are in line with our results. We found no reactions in our RIAs when heated virions were used (results not presented). Procapsids of poliovirus have been reported to express N,, N,, and H antigenic epitopes at pH 7.2 (Rombaut et al., 1982). We do not know which epitope(s) is reacting in our procapsid RIAs. Apparently the two procapsids show different epitopes, since some sera were positive with only one procapsid. Furthermore, the epitope(s) is probably neither the same one as appears after heating of the virions, nor N, or N, on the native virions. It is not unexpected that enterovirus-IgM assays vary in specificity and sensitivity (for references, see Introduction). Some investigators have used crude antigens which contain procapsids. In addition, the strains chosen might have been too broad or too narrow in antigenicity. The IgM-RIA using purified virions is predominantly type-specific. However, as shown with CB5, the choice of an appropriate strain is important. The procapsids of CB3 and CB5 show both homotypic and heterotypic reactions. Thus, using procapsids of at least two types a, for diagnostic purposes, relatively satisfactory broadly reacting IgM response may be detected. When a type-specific IgM response is required, an IgM-RIA based on purified virions may be selected.

202

Acknowledgement We wish to thank Hardyal Ahluwalia for excellent technical assistance and Dr Claes Kgllander for helpful suggestions concerning statistics. References Beatrice, S., Katze, M.G., Zajac, B.A. and Crowell, R.L. (1980) Induction of neutralizing antibodies by the coxsackie virus B3 virion polypeptide VP2 Virology 105, 426-438. Brown, F. and Wild, F. (1974) Variation in the Coxsackievirus type B5 and its possible role in the aetiology of swine vesicular disease. Inte~irology 3, 125-128. Dorries, R. and Ter Meulen, V. (1980) Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay. Med. Microbial. Immunol. 168, 159-171, Dbrries, R. and Ter Meulen, V. (1983) Specificity of IgM antibodies in acute human coxsackie B infections analysed by indirect solid phase enzyme immunoassay and immunoblot technique. J. Gen. Virol. 64, 159-167. El-Hagrassy, M.M.O., Banatvala, J.E. and Coltart, D.J. (1980) Coxsackie B-virus-specific-IgM-responses in patients with cardiac and other diseases. Lancet 29, 116t&1162. Forsgren, M. (1968) Studies of Echovirus type 6 antigens in immunodiffusion. 3. Antibody response against echovirus 6 antigens in human infections with homologous and heterologous enteroviruses. Acta Pathol. Microbial. Stand. 74, 611-623. Forsgren, M. (1969) Separation of Echovirus type 6 antigens by centrifugation in caesium chloride density gradients. 1. Infectivity and antigenic activities. Arch. Ges. Virusforsch. 28, 19-25. Frisk, G., Torfason, E. and Diderholm, H. (1984) Reverse radioimmunoassay of IgM and IgG antibodies to coxsackie B virus in patients with acute myopericarditis. J. Med. Virol. 14, 191-200. Hannington, G., Booth, J.C., Bowes, R.J. and Stern, H. (1986) Coxsackie B virus-specific IgM antibody and myocardial infarction, J. Med. Microbial. 21, 287-291. King, M.L., Shaikh, A., Bidwell, D., Voller, A. and Banatvala, J.E. (1983) Coxsackie-B-virus-specific IgM responses in children with insulin-dependent (juvenile-onset; Type 1) diabetes mellitus. Lancet 1, 1397-1399. Morgan-Capner, P. and McSorley, C. (1983) Antibody capture radioimmunoassay (MACRIA) for coxsackie virus B4 and CBS-specific IgM. 3. Hyg. 90, 333-349. Prabhakar. B.S., Haspel, M.V., McClintock. P.R. and Notkins, A.L. (1982) High frequency of antigenie variants among naturally human coxsackie B4 isolates identified by monoclonal antibodies. Nature (London) 300, 374-376. Pugh, S.F. (1984) Heterotypic reactions in a radioimmunoassay for coxsackie B virus specific IgM. J. Clin. Pathol. 37, 433-439. Putnak, J.R. and Philips, B.A. (1981) Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor. J. Virol. 40, 177-183. Rombaut, B., Vrijsen, R., Brioen, P. and Boeye, A. (1982) A pH dependent antigenic conversion of empty capsids of poliovirus studied with the aid of monoclonal antibodies to N and H antigen. Virology 122, 215-218. Rueckert, R. and Wimmer, E. (1984) Systematic nomenclature of picornavirus proteins. J. Virol. SO, 957-959. Schmidt, N.J., Dennis, J., Frommhagen, L.H. and Lennette, E.H. (1963) Serologic reactivity of certain antigens obtained by fractionation of coxsackie viruses in cesium chloride density gradients. J. Immunol. 90,654-662. Torfason, E., Frisk, G. and Diderholm, H. (1984) Indirect and reverse radioimmunoassays and their apparent specificities in the detection of antibodies to enteroviruses in human sera. J. Med. Virol. 13, 13-31.