production of subgroup-specific monoclonal antibodies against human rotaviruses and their...

Journal of Medical Virology 14:115-125 (1984)

Production of Subgroup-Specific Monoclonal Antibodies Against Human Rotaviruses and Their Application to an Enzyme-Linked Immunosorbent Assay for Subgroup Determination Koki Taniguchi, Tomoko Urasawa, Shozo Urasawa, and Toshio Yasuhara

Department of Hygiene (K. T., S. U., T. Y ) and School of Allied Health Professions (7: U.), Sapporo Medical College, Sapporo, Japan

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid.

Using the three monoclonal antibodies (S2-37, YO-5, and Y0-156), an enzyme- linked irnmunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.

Key words: human rotavirus, monoclonal antibody, subgroup antigen, ELISA

INTRODUCTION

Recent extensive serological studies indicate that rotaviruses have two distinct antigenic specificities: subgroup and serotype specificities [Kapikian et al, 19811. Subgroup specificity has been detected by the enzyme-linked immunosorbent assay (ELISA) [Zissis and Lambert, 19801, complement fixation test [Zissis and Lambert, 19781, immune adherence hemagglutination assay (IAHA) [Kapikian et al, 19811, and immune electron microscopy (IEM) [Zissis and Lambert, 19781, and is associated with a major component of inner capsid (42,000-dalton protein; 42K protein), which is coded by the sixth gene segment [Mason et al, 1980; Smith et al, 1980; Kalica et al, 1981; Greenberg et al, 1983a1, while serotype specificity is defined by the virus neutralization test [Kapikian et al, 19811 and is associated with outer capsid protein

Accepted for publication January 16, 1984.

Address reprint requests to Dr. Koki Taniguchi, Department of Hygiene, Sapporo Medical College, Sapporo 060, Japan.

0 1984 Alan R. Liss, Inc.

116 Taniguchi et a1

(34K to 37K protein) which the eighth or ninth gene segment codes for [Kalica et al, 1981; McCrae and McCorquodale, 1982; Greenberg et al, 1983bl.

To date, two subgroups of human rotaviruses (HRVs) and four serotypes have been found [Kapikian et al, 1981; Urasawa et al, 1982; Wyatt et al, 19831. Until 1981, the only HRV, with the exception of rescued viruses obtained by genetic reassortment between fastidious HRVs and ts mutants of cultivable bovine rotavirus [Greenberg et al, 1981, 19821, that could grow in cell culture was the Wa strain obtained by multiple serial passage in gnotobiotic piglets [Wyatt et al, 19801. However, since the successful direct propagation of HRVs in cell culture [Sato et al, 1981; Urasawa et al, 19811, it has become possible to employ numerous cultivable HRVs for studying the serological properties of viruses. Using viruses isolated in cell culture, we defined three distinct serotypes of HRVs by plaque reduction assay and also suggested the antigenic complexity of HRVs [Urasawa et al, 19821. In this respect, it was considered that further antigenic characterization of HRVs, especially with the aid of strain-, subgroup-, and serotype-specific monoclonal antibodies, would yield useful informa- tion for epidemiological studies, diagnosis, and development of vaccines.

Recently, we prepared subgroup- and serotype-specific monoclonal antibodies against HRVs. In this paper, we describe the characterization of commonly reactive and subgroup-specific nonneutralizing monoclonal antibodies and their application for detecting and subgrouping HRV isolates by ELISA tests.

MATERIALS AND METHODS Viruses

The cultivable HRVs used in this study included S2, DS-1, and HN-126 strains (subgroup I, serotype 2); KU, K8, and Wa strains (subgroup 11, serotype 1); and YO, S3, and P strains (subgroup 11, serotype 3). Serological characterizations of these strains have already been reported [Urasawa et al, 1982, 1984; Wyatt et al, 19831. S2, KU, K8, YO, and S3 strains were isolated in our laboratory, while DS-1, HN- 126, Wa, and P strains were kindly provided by R.G. Wyatt, National Institutes of Health, Bethesda, Maryland. As reference animal rotaviruses, simian rotavirus (SA- 11 strain) and calf rotavirus (Lincoln strain) were employed. The viruses were pretreated with trypsin (10 pg/ml), propagated in MA-104 cells in the presence of trypsin (1 pg/ml), and harvested 1 to 3 days after infection.

Preparation of Hybridomas

The 8-azaguanine-resistant (HGPRT) myeloma cell line P3-X63-Ag8.653 was grown in Dulbecco’s modified Eagle medium (DME) with 15% fetal calf serum. The production of hybridomas was carried out as described by Nowinski et a1 [ 19791 with several modifications. BALB/c mice were inoculated intraperitoneally with purified S2 or YO strain of HRV, and then were boosted via the same route on Day 30 or 45. The mice were sacrificed 3 days later, the spleen was removed, and spleen cells were fused with 4 X lo7 P3-X63-Ag8.653 cells using polyethylene glycol 1540 at a final concentration of 50%. The cell mixture was then suspended in HAT (hypoxanthine- aminopterin-thymidine) medium and was plated into microplates at a concentration of 3 X lo5 cells/well. About 2 weeks after the fusion, hybridomas producing anti-HRV antibodies were identified by assaying the medium from each well by indirect solid- phase radioimmunoassay (RIA) as described below. Hybridomas producing anti-

ELISA for Subgrouping Human Rotaviruses 117

HRV antibodies with high binding activity in RIA were selected and cloned by limiting dilution. The isotypes of the immunoglobulins produced by the hybridomas were determined by Ouchterlony double-immunodiffusion tests using sheep antisera for mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 (Serotec Ltd., Bincester, England). To obtain ascitic fluid, lo7 hybridoma cells were inoculated intraperitoneally into pristane-primed BALB/c mice.

Radioirnrnunoassay

For screening the anti-HRV activity of culture fluid and examining the reactivity patterns of monoclonal antibodies to various rotavirus strains, indirect radioimmunoassay (RIA) was employed. The wells of 96-well polyvinyl microtiter plates were precoated with purified rotavirus in 100 pl of 10 mM phosphate-buffered saline (PBS; pH 7.4) for 18 hr at 4°C. After the plates were washed with PBS, 1 % bovine serum albumin (BSA) in PBS was added to each well, after which the plates were kept overnight at 4°C. Next, they were washed twice with PBS, and 50 p1 of the test sample was added and allowed to react for 1 hr at room temperature. After washing three times with PBS containing 0.05% Tween 20 (PBS-Tween), the plates were incubated with 40 p1 of '251-labeled goat anti-mouse immunoglobulins (4 x lo5 cpm/ well) for 1 hr at room temperature. After a final additional washing three times with PBS-Tween, the wells were separated from the plates and counted in a gamma counter.

lrnrnunoprecipitation Analysis

Monolayer cultures of MA-104 cells were infected with S2 or YO strain pretreated with trypsin (10 pg/ml) at a multiplicity of 5 pfu/cell. After 1 hr adsorption at 37"C, the inocula were removed and the cells were washed. At 9 hr after infection, the cells were labeled for 1 hr with 100 pCi of [35S]methionine (800 to 1200 Ci/ mmol; Amersham Corp.)/ml in methionine-free Eagle's minimum essential medium (MEM). After another 1 hr incubation in Eagle's MEM, the cells were washed with cold Tris-HCI buffered saline (pH 7.5) and were then mixed for 1 hr at 4°C in extraction buffer consisting of 0.1 M Tris-HC1 (pH 7 . 3 , 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. After centrifugation at 40,000 rpm (Hitachi rotor RP40) for 1 hr, the supernatants were employed for immunoprecipitation, which was carried out as described by Lee et al [1981]. The immunoprecipitated proteins were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in 12.5% slab gels with 4 % stacking gels using the Laemmli's discontinuous Tris- glycine buffer system [Laemmli, 19701.

IEM Purified HRV particles (S2 or YO strain) were mixed with culture fluid (100 pI)

from hybridomas and incubated for 1 hr at room temperature. The mixture was then processed for EM, as described previously [Taniguchi et al, 19811.

ELISA

The buffer used for the dilution of ascitic fluid was 10 mM PBS (pH 7.4). PBS- Tween was used for washing the microplates and for diluting stool suspensions, rabbit anti-HRV serum, and goat anti-rabbit IgG alkaline phosphatase conjugate.

118 Taniguchi et a1

The wells of polyvinyl microtiter plates were coated for 18 hr at 4°C with S2- 37, YO-5, or YO-156 ascitic fluid diluted 1:10,000. After the plates were washed three times, 1% BSA was added and the plates were kept overnight at 4°C. The plates were washed three times, and 75 pl of the test samples was reacted for 1 hr at 37°C. After washing, rabbit anti-HRV serum diluted 1: 1,OOO to 1: 10,OOO was added and the plates were incubated for 1 hr at 37°C. After additional washing, 100 pl of alkaline-phosphatase-conjugated goat IgG antibody to rabbit IgG was added. The plates were incubated for 1 hr at 37"C, and 100 pl of p-nitrophenylphosphoric acid disodium salt solution (1 mg/ml) in 0.1 M sodium carbonate buffer (pH 9.6) was added. Absorbance was then measured at 400 nm with a micro ELISA reader (Model MTP-12, Corona Electric, Katsuta, Japan).

Polyacrylamide Gel Electrophoresis of RNA of HRV HRV in stool specimens or culture fluids was concentrated by ultracentrifuga-

tion through 45% sucrose. The RNA patterns of HRV were analyzed as described previously [Taniguchi et al, 19821 exept that 10% (w/v) polyacrylamide gel was used in this study.

RESULTS Derivation of Six Monoclonal Antibodies to HRV

Six separate monoclonal antibodies were selected on the basis of high binding activity in RIA using S2 or YO strain as coating antigen. S2-37 and S2-65 hybridomas were derived from fusion using a mouse immunized with S2 strain (subgroup I, serotype 2). The remaining clones (YO-5, YO-71, YO-89, and YO-156) were obtained in fusion experiments using YO strain (subgroup 11, serotype 3) as the immunizing antigen. The isotype of S2-37, YO-5, and YO-89 antibodies was IgG1. YO-156 hybridoma secreted the IgG2a subclass of immunoglobulin, and S2-65 and YO-71 hybridomas produced IgG2b. They were all nonneutralizing when examined using a 1: 10 dilution of the culture fluids in fluorescent-focus neutralization tests.

Immunoprecipitation studies were carried out to determine the viral protein against which these monoclonal antibodies are directed. As shown in Figure I , all of the six monoclonal antibodies precipitated 42K protein; a major component of inner capsid. Since the 42K protein has been shown to be a component of single-shelled particles [Estes et al, 19811, these monoclonal antibodies directed to the 42K protein were expected to bind to single-shelled particles but not to double-shelled particles. IEM using these monoclonal antibodies and the S2 or YO strain revealed that all six monoclonal antibodies agglutinated only single-shelled particles. Figure 2 shows the reaction between YO-I56 antibody and the single-shelled particles of YO strain.

Reactivity Patterns of Monoclonal Antibodies to HRV in RIA Reactivity patterns in RIA between the monoclonal antibodies and various strains

of HRV and simian and calf rotaviruses are shown in Table I. S2-65, YO-71, YO-89, and YO-156 antibodies reacted commonly to all rotaviruses examined, although their reactivity patterns to the viruses varied to some extent. In contrast, S2-37 antibody reacted only with the subgroup I rotaviruses (S2, HN-126, and DS-1 strains of HRV and SA-11 and Lincoln strains of animal rotaviruses), while YO-5 antibody bound to only the subgroup I1 rotaviruses (KU, K8, Wa, YO, and S3 strains of HRV). From


Fig. 1. Immunoprecipitation of [35S]methionine-labeled YO or S2 strain proteins by monoclonal antibody. (1) YO lysate only; (2) to (6) reactions between YO lysate and YO-5 antibody (2), YO-89 antibody (3), YO-71 antibody (4 ) , YO-156 antibody (3, and anti-YO mouse serum (6); (7) to (9) reactions between S2 lysate and anti-S2 mouse serum (7), S2-37 antibody (8), and S2-65 antibody (9); (10) S2 lysate only. Anti-YO mouse serum and antiLS2 mouse serum were obtained from the mice, on the day of sacrifice, whose spleens were employed for the cell fusions. Numbers on the left indicate molecular weight x

Fig. 2. IEM between YO-156 antibody and YO strain of HRV. Bar represents 100 nm.

these results, it was concluded that S2-37 and YO-5 antibodies had antibody specificities identical to previously characterized subgroup I and subgroup I1 specificities, respectively.

ELSA With Monoclonal Antibodies for Detecting and Subgrouping HRV Isolates

Based on the reactivity patterns of the monoclonal antibodies, we selected three monoclonal antibodies for ELISA: commonly reactive YO- 156 antibody (ELISA-

TABL

E I.

Rea

ctiv

ity P

atte

rns

of A

nti-H

RV

Mon

oclo

nal A

ntib

odie

s as

Det

erm

ined

by

RIA

P/N

rat

io"

Mon

oclo

nal

Subg

roup

I Su

bgro

up I1

an

tibod

y su

bcla

ss

S2

DS-

I H

N-1

26

Linc

oln

SA-I

1 K

U

K8

wa

YO

s3

S2-3

1 1

30.8

23

.3

29.7

25

.0

39.3

1.

6 1.

5 2.

0 1.

3 1.

5 S2

-65

2b

25.8

19

.8

24.8

29

.9

34. I

12

.1

27.6

16

.6

16.0

7.

1 Y

O-5

I

1.8

1.2

1.3

0.8

2.1

23.5

30

.8

24.4

25

.7

24.6

Y

O-I

I 2b

22

.8

21.1

20

.3

29.0

29

.5

23.4

28

.0

30.3

29

.0

17.0

Y

O-8

9 1

17.7

15

.0

15.8

25

.6

18.1

16

.6

46.5

14

.8

24.2

10

.7

YO

-I56

2a

41

.2

38.2

21

.9

45.5

40

.1

37.3

57

.5

29.5

47

.7

39.3

"The

PIN

rat

io is

the

cpr

n of

an

indi

vidu

al m

onoc

lona

l ant

ibod

y bo

und

to th

e in

dica

ted

viru

s di

vide

d by

the

cou

nts

per

min

ute

of c

ultu

re

fluid

of

P3-X

63-A

g8.6

53 ce

lls b

ound

to th

e in

dica

ted

viru

s. A

rat

io o

f >

3.0

was

con

side

red

as a

sig

nific

ant r

eact

ion.


156), subgroup I-specific S2-37 antibody (ELISA-37), and subgroup 11-specific YO- 5 antibody (ELISA-5) (Table II). The sensitivity of ELISA-156 was evaluated by the quantitation of two HRV strains, S2 and YO, and it was found that ELISA-156 was about 10 times as sensitive as direct EM examination. The specificity of ELISA-156 was verified by the use of culture fluids from HRV-infected cells and HRV-positive or negative stool suspensions (Table III). Thus, the utility of ELISA-156 for detecting HRV was confirmed.

For subgrouping HRV isolates, subgroup-specific monoclonal antibodies (S2- 37 and YO-5) were employed for ELISA as the coating antibody. The specificity of the ELISA subgrouping was examined by testing 10 rotavirus strains which had been previously subgrouped by conventional ELISA or IAHA. As shown in Table LI, our ELISA subgrouping results were in good agreement with earlier subgrouping results. Finally, using the ELISA developed here, we examined the subgroup specificity of HRV in stool suspensions and in culture fluids of HRV-infected cells. In 11 out of 13 stool specimens and 14 out of 15 culture fluids, viral subgroups were successfully determined (Table 111). The HRV in three samples did not react in ELISA-37 or ELISA-5 despite their positive reaction in ELISA-156. These results might imply the possible presence of a third subgroup of HRV.

Correlation Between Subgroup Specificity and RNA Patterns of HRV The RNA patterns of the HRV isolates employed for ELISA were examined by

polyacrylamide gel electrophoresis (Tables I1 and 111). All of the isolates defined as subgroup I showed “short” RNA patterns that 10th and 11th RNA segments migrate

TABLE 11. Subgrouping of Previously Characterized HRV and Animal Rotavirus Strains by ELISA With Monoclonal Antibodies

Previously ELISA testa determined ELISA- ELISA-5/ Ascribed RNA

Strain subgroup ELISA-37 ELISA-5 156 ELISA-37b subgroup pattern

HRV s2 I 1033 142 1081 0.14 I Short DS-1 I 1278 203 1231 0.16 I Short HN-126 I 1300 123 1224 0.09 I Short KU I1 20 1 I128 1078 5.6 11 Long K8 I1 63 1083 1118 17.2 II Long Wa I1 123 1055 1053 8.6 11 Long YO I1 55 1041 1059 18.9 11 Long P I1 107 700 899 6.5 11 Long Animal rotavirus Lincoln I 1088 223 986 0.20 I Long SA-I 1 I 1298 89 1298 0.07 I Lone

”ELISA-37, ELISA-5, and ELISA-156 indicate the ELISA tests with S2-37, YO-5, and YO-156 antibodies, respectively. The numbers in the table show the sum of OD400 x loo0 in two wells. Values over 300 in each ELISA test were considered to show positive reactions. bThe ELISA-YELISA-37 ratio is the OD,, obtained in ELISA-5 divided by that in ELISA-37. Samples with a ratio of < 0.4 were considered to belong to subgroup I, while samples with a ratio of > 2.5 were placed in subgroup 11.

122 Taniguchi et al

TABLE 111. Subgrouping of HRV in Culture Fluids and Stool Suspensions by ELISA With Monoclonal Antibodies

ELISA testa ELISA-5/ Ascribed RNA Specimen ELISA-37 ELISA-5 ELISA-156 ELISA-37b subgroup pattern

Culture fluid

AK-10 AK- 12 AK-13 AK- 14 AK-15 AK-2 1 AK-27 AK-35 s4 s5 YMI M-24 P2 FU s 12

Stool suspension

(EM positive)

AK- 119 AK-190 AK-247 AK-386 ID OG IK HM KB MK KN MD KU

(EM negative)

ST HN AG 11

81 752 126 180 48

1432 164 146 92

174 1254 200

48 68

104

1130 112 24 1 440 41 13 12 73 22 83 30 18 25

26 10 86

127

147 48

5 86 6 16 493 112 820 850 733 5 14 218

1086 782 452 618

60 7 80

1178 37

590 68

736 920 962 753 135 760 810

74 72

124

1025 926

1006 832

1128 1234 934

1272 I170 646 892

1336 928 786 758

1176 1126 1140 1019 94 I 994 904 998

1056 989 929 838

1074

45 24 70 58

-

0.06 4.7 3.4

10.3 0.08 5 .O 5.8 8.0 3.0 0.17 5.4

16.3 6.6 5.9

0.05 7.0 4.9 0.08

14.4

61.3 12.6 43.7 9.1

42.2 32.4

-

-

- -

-

-

- I I1 I1 I1 I I1 I1 I1 I1 I I1 I1 I1 I1

I I1 I1 I I1

I1 I1 I1 I1

I1 I1

-

-

- - - -

Long Short Long Long Long Short Long Long Long Long Short Long Long Long Long

Short Long Long Short Long Long Long Long Long Long Long Long Long

-

~

- -

"sbSee footnotes in Table 11.

slowly. In contrast, the RNA patterns of the isolates determined as subgroup I1 and those of the undetermined subgroup were found to show "long" patterns.

DISCUSSION

Although the etiological importance of HRV in acute nonbacterial gastroenteritis has been acknowledged since its first visualization by EM by Bishop et a1 in 1973, virological, clinical, and epidemiological studies of HRV have been hampered be-


cause of the difficulty in propagating HRV in cultured cells. However, in 1980, Wyatt et a1 successfully established a cultivable HRV, the Wa strain, after multiple serial passages in gnotobiotic piglets. Subsequently, Greenberg et a1 [ 198 1, 19821 established large numbers of rescued viruses by genetic reassortment between fastidious HRV and ts mutants of cultivable bovine rotaviruses. At present, the cultivation of numerous HRVs in cell cultures has become possible by employing roller-tube cultures with trypsin-containing medium [Sato et al, 1981; Urasawa et al, 19811. In our laboratory, more than 40 HRVs have been isolated in MA-104 cells and is being employed for further serological characterizations of HRV.

In HRVs, two independent antigenic specificities, subgroup and serotype specificities, have been revealed [Kapikian et al, 19811. For epidemiological studies and diagnosis in particular, rapid methods for determining the subgroup and serotype of HRV have been described [Yolken et al, 1978; Thouless et al, 19821. In these studies, however, sera absorbed with heterologous viruses to reduce cross-reactions were used. In contrast, production of subgroup- and serotype-specific monoclonal antibodies to HRV provide an unlimited supply of extremely specific reagents. In recent studies, a number of monoclonal antibodies to animal rotaviruses and human Wa strain have been prepared and characterized [Greenberg et al, 1983a,c; Roseto et al, 1983; Soma et al, 19831. Furthermore, Greenberg et a1 [1983a,c] have successfully applied subgroup-specific and serotype 3-specific monoclonal antibodies for serological characterizations of animal and human rotaviruses.

In this study, using the HRV strains isolated in cell culture, we prepared six monoclonal antibodies to HRV. They were, however, all nonneutralizing. For this result, two major reasons are conceivable. First, in this study mice were immunized with HRV cultured in the presence of trypsin, which was found to be unstable during the purification process. Second, the selection of monoclonal antibodies with high binding activity by means of RIA may have resulted in the preferential selection of nonneutralizing antibodies. The latter phenomenon was suggested also in a recent report by Greenberg et a1 [ 1983al. Taking these points into consideration, in our latest fusion experiments in which HRV cultured in trypsin-free medium and a fluorescent focus neutralization test as screening assay were employed, we obtained numerous neutralizing monoclonal antibodies to HRV (manuscript in preparation).

Even in the commonly reactive monoclonal antibodies, the reaction strengths to various HRV strains differed to some extent. These results may reflect the slight differences in the avidities of the antibodies for HRVs, which are probably caused by slight differences in the common antigenic determinants among various HRV strains [Goldstein et al, 19821. Further, these results raise the possibility that some HRV strains might escape recognition in a serological assay in which the monoclonal antibodies are used for detecting HRVs [Richman et al, 19821. Therefore, we chose YO-156 antibody in ELISA for detecting HRVs since this antibody reacted almost evenly to various HRV strains examined. To date, we have tested 53 samples in which HRV particles were observed electron-microscopically, and none escaped recognition by ELISA-156 tests.

The specificity of ELISA-37 and ELISA-5 employing subgroup I- and subgroup 11-specific monoclonal antibodies was confirmed by testing the previously characterized HRV strains. ELISA subgrouping with the monoclonal antibodies was in excellent agreement with earlier subgrouping results. In further tests, 25 HRV samples out of 28 examined were clearly determined for their subgroup specificity; 5

124 Taniguchi et a1

HRV samples for subgroup I and 20 samples for subgroup 11. In contrast, 3 samples reacted in neither ELISA-37 nor ELISA-5, although they reacted in ELISA- 156. This may imply that these samples might belong to a third subgroup specificity as suggested in recent reports [Lambert et al, 1983; Tufvesson, 19831. However, we cannot yet exclude the possibility that these samples contained HRV subgroup I1 variants reacting very weakly to YO-5 antibody, because the sensitivity of ELISA-5 was slightly lower than those of ELISA-37 and ELISA-156. In this respect, production and characterization of monoclonal antibodies to one of those strains not determined for their subgroup specificity will be useful for further characterization of the subgroup antigens of HRV.

In addition to subgrouping, rapid and simple serotyping of HRV is urgently needed for serological studies of the virus. Recently, using KU (serotype l), S2 (serotype 2), and YO (serotype 3) strains, we obtained serotypes 1-, 2-, and 3-specific monoclonal anibodies. Studies on the application of these serotype-specific monoclonal antibodies to ELISA or other serological tests for serotyping HRV isolates are currently underway.

ACKNOWLEDGMENTS

We wish to thank Dr. Y. Chiba and Dr. Y. Saeki for providing stool specimens. This work was supported in part by Grants 57770349 and 585702 13 from the Ministry of Education, Science, and Culture of Japan.

REFERENCES

Bishop RF, Davidson GP, Holmes IH, Ruck BJ (1973): Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 11: 1281-1283.

Estes MK, Graham DY, Mason BB (1981): Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. Journal of Virology 39:879-888.

Goldstein LC, McDougall J, Hackman R, Meyers JD, Thomas ED, Nowinski RC (1982): Monoclonal antibodies to cytomegalovirus: Rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia. Infection and Immunity 38:273-281.

Greenberg HB, Kalica AR, Wyatt RG, Jones RW, Kapikian AZ, Chanock RM (1981): Rescue of noncultivatable human rotavirus by gene reassortment during mixed infection ts mutant of a cultivable bovine rotavirus. Proceedings of the National Academy of Science, USA 78:420-424.

Greenberg HB, Wyatt RG, Kapikian AZ, Kalica AR, Flores I, Jones R (1982): Rcscue and serotype characterization of noncultivatable human rotavirus by gene reassortment. Infection and Immunity 37: 104-109.

Greenberg HB, McAuliffe V, Valdesuso J, Wyatt R, Flores J, Kalica A, Hoshino Y, Singh N (1983a): Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infection and Immunity 39:Yl-99.

Greenberg HB, Flores J, Kalica AR, Wyatt RG, Jones R (1983b): Gene coding assignments for growth restriction, neutralization and subgroup specificities of the W and DS-I strains of human rotavirus. Journal of General Virology 64:313-320.

Greenberg HB, Valdesuso J, van Wyke K, Midthun K, Walsh M, McAuliffe V, Wyatt RG, Kalica AR, Flores J, Hoshino Y (1983~): Production and preliminry characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus. Journal of Virology 47:267-275.

Kalica AR, Greenberg HB, Wyatt RG, Flores J, Sereno MM, Kapikian AZ, Chanock RM (1981): Genes of human (strain Wa) and bovine (strain UK) rotaviruses that code for neutralization and subgroup antigens. Virology 112:385-390.

Kapikian AZ, Cline WL, Greenberg HB, Wyatt RG, Kalica AR, Banks CE, Hames HD, Flores J, Chanock RM (1981): Antigenic characterization of human and animal rotaviruses by immune


adherence hemagglutination assay (IAHA): Evidence for distinctness of IAHA and neutralization antigens. Infection and Immunity 33:415-425.

Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

Lambert JP, Marissens D, Marbehant P, Zissis G (1983): Prevalence of subgroup 1,2, and 3 rotaviruses in Belgian children suffering from acute diarrhea (1978-1981). Journal of Medical Virology

Lee PWK, Hayes EC, Joklik WK (1981): Characterization of anti-reovirus immunoglobulins secreted by cloned hybridoma cell lines. Virology 108: 134-146.

Mason BB, Graham DY, Estes MK (1980): In vitro transcription and translation of simian rotavirus SA 11 gene products. Journal of Virology 33:1111-1121.

McCrae MA, McCorquodale JG (1982): The molecular biology of rotaviruses. 11. Identification of the protein-coding assignments of calf rotavirus genome RNA species. Virology 117:435-443.

Nowinski RC, Lostrom ME, Tam MR, Stone MR, Burnette WE (1979): The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses. Virology 93: 11 1- 126.

Richman DD, Cleveland PH, Oxman MN (1982): A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus. Journal of Medical Virology 9:299-305.

Roseto A, Sherrer R, Cohen J, Guillemin MC, Charpilienne A, Feynerol C, Peries J (1983): Isolation and characterization of anti-rotavirus immunoglobulins secreted by cloned hybridoma cell lines. Journal of General Virology 64:237-240.

Sat0 K, Inaba Y, Shinozaki T, Fujii R, Matsumoto M (1981): Isolation of human rotavirus in cell cultures. Archives of Virology 69: 155-160.

Smith ML, Lazdins I, Holmes IH (1980): Coding assignments of SA 11 rotavirus established by in vitro translation. Journal of Virology 33:976-982.

Sonza S, Breschkin AM, Holmes IH (1983): Derivation of neutralizing monoclonal antibodies against rotavirus. Journal of Virology 1143-1146.

Taniguchi K, Urasawa S, Urasawa T (1981): Further studies of 35-40 nm virus-like particles associated with outbreaks of acute gastroenteritis. Journal of Medical Microbiology 14: 107-1 18.

Taniguchi K, Urasawa S, Urasawa T (1982): Electrophoretic analysis of RNA segments of human rotaviruses cultivated in cell culture. Journal of General Virology 60: 171-175.

Thouless ME, Beards GM, Flewett TH (1982): Serotyping and subgrouping of rotavirus strains by the ELISA test. Archives of Virology 73:219-230.

Tufvesson B (1983): Detection of a human rotavirus strain different from types 1 and 2-a new subgroup? Epidemiology of subgroups in a Swedish and an Ethiopian community. Journal of Medical Virology 12:111-117.

Urasawa T, Urasawa S, Taniguchi K (1981): Sequential passages of human rotavirus in MA-104 cells. Microbiology and Immunology 25: 1025-1035.

Urasawa S, Urasawa T, Taniguchi K (1982): Three human rotavirus serotypes determined by plaque neutralization of isolated strains. Infection and Immunity 38:781-784.

Urasawa S, Urasawa T, Taniguchi K, Chiba S (1984): Serotype determination of human rotavirus isolates and antibody prevalence in pediatric population in Hokkaido, Japan. Archives of Virol- ogy, in press.

Wyatt RG, James WD, Bohl EH, Theil KW, Saif LJ, Kalica AR, Greenberg HB, Kapikian AZ, Chanock RM (1980): Human rotavirus type 2: Cultivation in vitro. Science 207:189-191.

Wyatt RG, James HD, Jr, Pittman AL, Hoshino Y, Greenberg HB, Kalica AR, Flores J, Kapikian A 2 (1983): Direct isolation in cell culture of human rotaviruses and their characterization into four serotypes. Joural of Clinical Microbiology 18:310-3 17.

Yolken RH, Wyatt RG, Zissis G, Brandt CD, Rodriguez WJ, Kim HW, Parrott RH, Greenberg HB, Kapikian AZ, Chanock RM (1978): Comparative epidemiology of human rotavirus types 1 and 2 as studied by enzyme-linked immunosorbent assay. New England Journal of Medicine 299: 1156- 1161.

11:31-38.

Zissis G, Lambert JP (1978): Different serotypes of human rotavirus. Lancet I:38-39. Zissis G, Lambert JP (1980): Enzyme linked immunosorbent assay adapted for serotyping of human

rotavirus strains. Journal of Clinical Microbiology 11: 1-5.

production of subgroup-specific monoclonal antibodies against human rotaviruses and their...

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