JOURNAL OF VIROLOGY, May 1989, p. 2191-21970022-538X/89/052191-07$02.00/0Copyright © 1989, American Society for Microbiology

Purification and Characterization of Adult Diarrhea Rotavirus:Identification of Viral Structural Proteins

ZHAO-YIN FANG,l.2* ROGER I. GLASS,' MARIA PENARANDA,1 HONG DONG,2 STEPHAN S. MONROE,'LEYING WEN,2 MARY K. ESTES,3 JOSEPH EIDEN,4 ROBERT H. YOLKEN,4

LINDA SAIF,s VERA GOUVEA,1 AND TAO HUNG2Viral Gastroenteritis Unit, Division of Viral Diseases, Center for Infectious Diseases, Centers for Disease Control,Atlanta, Georgia 303331*; Laboratory of Virus Morphology, Institute of Virology, Chinese Academy of Preventive

Medicine, Beijing, China2; Department of Virology and Epidemiology, Baylor College of Medicine, Houston,Texas 7703O3; Department of Pediatrics, The Johns Hopkins Hospital, Baltimore, Maryland 212054; and

Food Animal Health Research Program, Ohio Agricultural Research and Development Center,The Ohio State University, Wooster, Ohio 446915

Received 21 October 1988/Accepted 1 February 1989

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus thathas been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins presentin the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SAll.EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of threepolypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-o-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particlesremoved the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. Theremaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualizedon sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis whenprobed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant andwas strongly immunoreactive with human sera, animal sera raised against ADRV and against other group Banimal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovineenteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This47-kDa inner capsid polypeptide contains a common group B pntigen and is similar to the VP6 of the group Arotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid thatseems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations onthe basis of similarities to the control preparation of SAll, including a 136-kDa polypeptide designated VP1,a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and severalproteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivityon Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses areantigenically quite distinct.

The rotaviruses have recently been subdivided into groupsthat share the characteristic rotavirus morphology but can bedistinguished by RNA electropherotype and by group-spe-cific antigens (3). The group A rotaviruses are recognized asa major cause of diarrhea in children (12). The non-group Arotaviruses have been identified in a variety of animalspecies with diarrhea but have rarely been found in associ-ation with human disease (4, 6, 8, 19). In 1982 and 1983,epidemics of severe, dehydrating diarrhea affecting tens ofthousands of adults were reported throughout the People'sRepublic of China and were traced to a group B rotaviruscalled adult diarrhea rotavirus (ADRV) (10, 11). Theseoutbreaks have persisted in the People's Republic of Chinaand have raised many questions concerning the origin ofthese strains, their relatedness to other rotaviruses, and theprospects that group B rotaviruses might become epidemicin other parts of the world. Work with human isolates ofnon-group A rotaviruses has been hampered by an inabilityto grow these viruses in cell culture (lOa). Preliminary cDNAcloning experiments with group B rotaviruses were initiated,

* Corresponding author.

but neither the gene coding assignments nor the proteinstructures have been determined (16).We have purified ADRV from human stool specimens

collected during an outbreak in China and have character-ized the virus, with specific emphasis on the viral proteins.Proteins present in the outer capsid, inner capsid, and corehave been identified by disruption of double-shelled virusinto single-shelled particles and cores. Viral proteins thatreact with human, animal, and monoclonal antisera havebeen characterized by using Western (immuno-) blot analy-sis, and the proteins of ADRV have been compared withthose of group A rotaviruses to identify major similaritiesand differences.

MATERIALS AND METHODSRotaviruses and antisera. Fecal specimens containing

ADRV were collected from patients in a single outbreak thatoccurred in Chengde, Hebei Province, the People's Republicof China, in 1987. Specimens that were positive by anenzyme-linked immunosorbent assay for ADRV (25) andconfirmed by electron microscopy (EM) were pooled andfrozen at -20°C until use. For comparison, the standardgroup A rotavirus SA1l (simian rotavirus, serotype 3) was

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2192 FANG Et AL.

grown in MA104 cells in the presence of trypsin (1 ,ug/ml) bystandard methods (9).

Paired serum specimens from patients with acute diarrheacaused by ADRV were collected durirlg an outbreak inQinhuangdao, Hepei Province, the . People's Republic ofChina, in 1987 (Z. Y. Fang, Q. Ye, S. Qing, M. S. Ho,T.Hung, and R. I. Glass, Program Abstr. 28th Intersci. Conf.Antimicrob. Agents Chemother., abstr. no. 504, 1988).Paired sera to group A rotavirus were bbtained from vac-cinees inoculated with rhesus rotavirtis vaccine as part of avaccine trial. Hyperimmune guinea pig antisera to SA1l andADRV were prepared at the Centers for Disease Control.Hyperimmune rabbit antisera to ADRV were prepared at theBaylor College of Medicine, Houston, Tex. (17); pig antiserato porcine group B rotavirus (Ohio strain) and calf antisera tobovine.group B rotavirus (NB strain) were prepared at TheOhio State University, Wooster, Ohio (21, 22); and ratantibody to the group B rotavirus associated with IDIR (7j,guinea pig antisera to bovine enteric syncytial virus (24), andmonoclonal antibodies.(MAbs) to IDIR virus 15S3 (26) wereprepared at the Johns Hopkins Hospital, Baltimore, Md.MAbs to ADRV were provided by Wei-Wei Ye (Institute ofVirology, Beijing, People's Republic of China) and JohnBurns (5).

Virus purification. Group A rotavirus was purified frominfected MA104 cells by previously described methods (14).In brief, rotavirus was released from the cells after threecycles of freezing and thawing followed by sonication for 1min and then mixed with an equal volume of trichlorotriflu-oroethahe (Genetron). The cell lysate was homogenized byshaking, and the phases were separated by centrifugation at1,100 x g for 10 min. The organic phase was reextractedwith an equal volume of Tris-buffered saline (TBS), pH 7.2(20 mM Tris hydrochloride, 140 mM NaCl, 5 mM KCl, 0.4mM Na2HPO4, 6 mM glucose, 0.5 mM MgCI2, and 5 mMCaCl2). The aqueous phases were combined, layered onto 1ml of 30% (wt/vol) sucrose in TBS, and centrifuged in anSW40 rotor (38,000 rpm, 2 h, 4°C). The virus pellet wassuspended in TBS by sonication. CsCl was added to adensity of 1.37 g/ml, and the virus particles were centrifugedto isopycnic equilibrium in an SW50.1 rotor (35,000 rpm, 18h, 4°C). Visible virus bands were collected by side puncture,desalted by using a Centricon 30 (Amicon Corp.), andscanned from 220 to 360 nm with a Beckman DU-6 spectro-photometer.ADRV was purified directly from infected stool specimens

diluted 1:5 with TBS. After the addition of Zwittergent 3-14(Calbiochem-Behring) to 0.5% and extraction with Gene-tron, virus was pelleted in a type 45Ti rotor (40,000 rpm, 60min, 4°C). The virus was suspended and centrifuged througha 25% sucrose cushion at 35,000 rpm for 100 min at 4°C in anSW40 rotor. The suspended virus was layered on top of apreformed metrizamide gradient (Accurate Chemical andScientific) (20 to 50% [wt/vol]) in TBS and centrifuged at45,000 rpm for 7 h (4°C) in an SW50.1 rotor. Bands contain-ing virus were collected and desalted with a Centricon 30. Toobtain highly purified virions, the concentrated virus wascentrifuged through a preformed sucrose gradient (20 to 40%[wt/wt]) in TBS at 20,000 rpm for 3.9 h (5°C) in an SW40rotor. The virus band was collected as described above.Virus density was measured in CsCl, with suspended virusspun to equilibrium in a density gradient (1.37 g/ml) at 4°C asdescribed for group A rotavirus.Treatment with EDTA and CaC12. Intact, purified double-

shelled virions were confirmed by EM. To remove the outercapsid, the virus was incubated for 20 min at 37°C with 10

mM EDTA. CsCl was added to a final density of 1.40 g/ml,and the sample was centrifuged to equilibrium at 35,000 rpmfor 18 h (4°C) in an SW50.1 rotor to yield purified single-shelled virus particles. The successful isolation of single-shelled particles was confirmed by EM. Core particles ofADRV were obtained by treating single-shelled particleswith 1 M CaCl2 for 20 min at room temperature to removethe inner capsid (1).

Endo-O3-N-acetylglucosaminidase H digestion. Purified viruswas denatured by adding 1.7% sodium dodecyl sulfate (SDS)and boiling it for 2 min. This was digested with endo-P-N-acetylglucosaminidase H (Endoglycosidase H; finalconcentration, 0.25 U/ml; ICN Biochemicals, Cleveland,Ohio) for 1.5 h at 37°C. The reaction was stopped by addingan equal volume of 2 x sample buffer and boiling it for 2 minbefore analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).PAGE. SDS-PAGE was performed using 12% polyacryl-

amide slab gels (0.75 mm thick) and Laemmli discontinuousbuffer system (13, 14). Electrophoresis was performed for 5h at 15 to 20 mA per gel. The resulting gels were stained withCoomassie brilliant blue (0.25%). Samples were suspendedin sample buffer (5 mM Tris hydrochloride, pH 6.8, 8%glycerol, 1% SDS, 0.5 M urea, 1% P-mercaptoethanol,0.01% phenol red) and boiled for 2 min before being loadedonto gels.

Molecular weights were estimated by comparing the viralpolypeptides with the following standard reference proteins:Escherichia coli 3-galactosidase (116,250), rabbit musclephosphorylase B (94,400), bovine serum albumin (66,200),hen ovalbumin (42,699), bovine carbonic anhydrase (31,000),and soybean trypsin inhibitor (21,500) (Bio-Rad Laborato-ries).Western blotting. Viral polypeptides were separated by

SDS-PAGE as described above, but no urea was used in theelectrophoretic buffer. The polypeptides were electrotrans-ferred onto a nitrocellulose membrane in Tris-glycine buffercontaining 20% methanol for 2.5 h at 65 V (23); The resultingblots were incubated for 2 h at room temperature withantisera in washing buffer (0.01 M phosphate-buffered salinecontaining 0.3% Tween 20 and 0.25% nonfat milk) and thenwashed four times with washing buffer. The blots wereblocked by incubation with 5% nonfat milk in phosphate-buffered saline and washed. Bound antibodies were detectedby using a rabbit antibody prepared against human immuno-globulin G that was conjugated with horseradish peroxidase(Dako). The reactivity of animal antisera prepared against avariety of different animal rotaviruses was examined, andantibodies were detected by using species-specific peroxi-dase conjugates (goat immunoglobulin to guinea pig [Dako],rabbit immunoglobulin to swine [Accurate], goat immuno-globulin to rat [Accurate], goat immunoglobulin to bovine[Accurate], swine immunoglobulin to rabbit [Dako], andgoat immunoglobulin to mouse [Dako]). After being washed,the blot was developed for 10 min with 0.05% diaminoben-zidine in phosphate-buffered saline containing 0.1% of 3%H202 and washed with H.O. Biotinylated molecUtlar weightmarkers were provided by R. Combs of the Centers forDisease Control.EM. During each step of purification, virus was monitored

by EM. Samples were absorbed for 15 s on carbon-coatedgrids, stained with 2% phosphotungstic acid (pH 4.6) for 15s, and examined in a Phillips 300 electron microscope (18).

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ADRV PROTEINS 2193

C

FIG. 1. Negative staining of various steps of degradation ofgroup B rotavirus ADRV particles. (A) Purified complete virionswith double-shelled capsid. (B) Purified single-shelled particlesprepared by EDTA treatment of double-shelled viruses. (C) Viruscores prepared by incubation of single-shelled virus with 1 M CaCI2for 20 min at room temperature. Samples were absorbed for 15 s oncarbon-coated grids, transferred onto a drop of 2% phosphotungsticacid (pH 4.6) for 15 s, and immediately examined. Magnification,x 124,000.

Density (g/ml)

20

RESULTS

Characterization of purified virions. ADRV has the samedistinctive morphology as group A rotavirus, with a double-shelled capsid and spikes arranged in wheel-like fashion(Fig. 1). The complete particle measures -70 nm in diame-ter, single-shelled viral particles purified in CsCl gradientafter EDTA treatment are -60 nm in diameter, and viralcores prepared by CaCl2 treatment of single-shelled particleshave a hexagonal outline with a diameter of -47 nm.By isopycnic centrifugation in CsCl, complete viral parti-

cles banded at a density of 1.373 g/ml and single-shelledparticles banded at 1.435 g/ml (Fig. 2). Freshly prepareddouble-shelled virus had a maximum UV absorption at 259nm and a minimum UV absorption at 247 nm, and single-shelled particles had a maximum UV absorption at 259 nmand a minimum UV absorption at 244 nm (Fig. 3). Thesevalues are identical to the values observed with the group Arotavirus. The ratio of UV absorbance at 260 nm to that at280 nm (A260/A280), an indicator of RNA content, was -1.43for double-shelled particles and --1.49 for single-shelledparticles.ADRV structural polypeptides. The structural polypeptides

of ADRV were examined by SDS-PAGE, and their molecu-lar weights were estimated by direct comparison with refer-ence markers and with group A rotavirus controls electro-phoresed in companion lanes (Fig. 4). Seven distinctpolypeptide bands were visualized; molecular masses wereapproximately 136, 113, 84, 64, 61, 47, and 41 kilodaltons(kDa). Comparing the sizes and staining intensities of theseproteins with those of the group A rotaviruses suggests thatthe polypeptides of 136, 113, 64, 47, and 41 kDa may becomparable with VP1, VP2, VP5, VP6, and VP7, respec-tively. Several polypeptides smaller than VP7 were visual-ized in the double-shelled group A rotavirus, of which onlyone appeared in the single-shelled particle. The 28-kDapolypeptide reacted strongly with convalescent sera andmay represent VP8 (Fig. 5). Others may represent degrada-tion products since they show a marginal response to con-valescent sera.Removal of the outer capsid ofADRV by EDTA treatment

Abs. (259 nm) Density (g/ml) Abs. (259 nm)

Fraction Fraction

FIG. 2. Profiles of purified group B rotavirus ADRV in CsCI density gradients. Intact ADRV with double-shelled capsid was purified fromstool samples as described in the text and centrifuged on CsCl gradients (A). The peak of virus particles was treated with EDTA and thenbanded in CsCl gradient to yield single-shelled virus preparations (B).

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2194 FANG ET AL.

Absorbance Absorbance Abs.

0.10-

220 248 276 304 332 360

Wavelength (nm)FIG. 3. Absorption spectrum of purified group B rotavirus ADRV

compared with group A rotavirus SAlI (-----).

220 248 276 304 332 360 nm

Wavelength (nm)with double-shelled capsid (A) or with single-shelled capsid (B)

was associated with the loss of three polypeptides withmolecular masses of 64, 61, and 41 kDa (Fig. 4). Similarly,EDTA treatment of group A rotavirus resulted in the loss ofthree proteins, VP5, VP5a, and VP7. Removal of the innercapsid of ADRV by CaCl2 treatment was associated with theloss of one polypeptide with a molecular mass of 47 kDa.Two polypeptides of 136 and 113 kDa remained in the viralcores. Some protein bands were located within the VP3 andVP4 range, but their identities were unclear. Endoglycosi-

Group A Group BIMIS O

M SS DS c-SS DS0-

l 16.3- VP:I-97,4- _m VP2- ._ua

6 6.2- -.-VP5-

-136o - -113

-84

-64

Group AGuinea-

Human pig- ----I --

P A C A

Vp1- -

Vp2- -

Vp53V p 5 - unwip

'I

Vp6- _m"Vp7

Vp8 -

GroupB

Human RabbitI I

I Cff PIA C1 C2

1A3B-_

136- --4113- "

84-

* 64- W

..I

47-EP41441hu

.,'l-'lfmm.

42,7- VP6- mo

VP7- _q0

VP8-31 0 -

FIG. 4. Comparison of structural polypeptides of group A and Brotaviruses and localization of proteins in the outer and inner capsid.Samples of virions (8 to 10 ,ug) purified on CsCl gradients wereanalyzed on a 12% polyacrylamide gel as described in the text.Double-shelled (DS) particles were treated with EDTA to removethe outer capsid, leaving single-shelled (SS) particles. Single-shelledparticles were treated with CaCl, to remove the inner capsid,leaving viral cores. Lane M contains molecular mass markers (inkilodaltons). Numbers on the right represent apparent molecularweights of group B polypeptides (in thousands). The viral proteins(VP) of group A rotavirus are indicated.

FIG. 5. Comparison of human and animal immune responses topolypeptides of group B versus group A rotavirus. Viral polypep-tides were separated by SDS-PAGE and electrotransferred onto anitrocellulose membrane. Blots of group B and group A rotaviruswere probed with acute-phase or preimmune sera (lanes A) or withconvalescent-phase human sera or hyperimmune serum (lanes C)and then developed with peroxidase-conjugated complexes anddiaminobenzidine. Positive control (lanes P) was a human convales-cent-phase serum with 10- to 20-fold-higher titer than paired conva-lescent-phase sera (lane C). Human sera to group A rotavirus (groupA lanes) was obtained from the recipient of rhesus rotavirusvaccine. Guinea pig sera were obtained pre- and postimmunizationwith SAlI. Human sera to ADRV (group B lanes) were obtainedfrom two patients recovering from recent infection. Rabbit serawere obtained from The Baylor College of Medicine, Houston, Tex.(see Materials and Methods).

A--- ADRV- SAl 1

Xmax=259nm

Double ShelledXmi =247 Virus

"N

I 'A C

_.rn -47- 41

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ADRV PROTEINS 2195

Group A Gr onu,p B

M ND D E ND D M

CONVALESCENT

ci' 0 C',.§ .., ,1C.;>N*eVo°,

1 16.3--97.4--66.2-

42.7- _mV7qw

VP7- .-m- 4tw.-4 1 _

3 1 .0 --_m_

FIG. 6. Glycosylation of the 41,000-molecular-weight outer shellpolypeptide of ADRV. Purified group A and B virions were digestedfor 1.5 h at 37°C with 0.25 U of endo-3-N-acetyl-glucosaminidase Hper ml before being analyzed on a 12% polyacrylamide gel asdescribed in the text. Lanes: ND, Not digested; D, digested; M,molecular weight markers. A sample of the endo-,-N-acetyl-glu-cosaminidase H used for the digestion was run in lane E as a control.The arrowheads indicate migration positions of deglycosylatedpolypeptides. VP7 of group A rotavirus was not digested com-pletely.

dase digestion of purified ADRV resulted in deglycosylationof the 41-kDa polypeptide, similar to the deglycosylation ofthe VP7 of SAl (Fig. 6). In ADRV, two deglycosylatedproteins with apparent molecular masses of 35 kDa ap-peared.Western blot analysis of antigenic polypeptides of ADRV.

The viral polypeptides were separated by SDS-PAGE, elec-trotransferred onto nitrocellulose membranes, and probedwith human and animal sera or monoclonal antibodies (Fig.5). All of the structural proteins were detected when probedwith a high-titer convalescent-phase human serum (lane P),indicating the antigenicity of the structural polypeptide.With human sera, a strong reaction to the 113-kDa polypep-tide with both the acute- and the convalescent-phase serawas observed while polypeptides of molecular sizes 47 and41 kDa were ADRV polypeptides which differed betweeninfected patients (lanes Cl and C2), as evidenced by thedifferent ratio of the serum responses to the 47- versus41-kDa polypeptides. Rabbit hyperimmune serum preparedfrom ADRV responded to the 113-, 47- and 41-kDa polypep-tides as well as to a few other proteins. Similar results wereobserved in guinea pig antisera prepared against group Arotavirus.The titer of convalescent-phase serum of one patient was

determined to endpoint (Fig. 7). Antibody to the 47-kDaprotein was present in highest titer (1:1,000), antibody to the113-kDa protein was present in an intermediate titer (1:200),and antibodies to the 136-, 64- and 41-kDa proteins werepresent at the starting dilution (1:100).

Antisera raised against a variety of animal group B rota-viruses and tested in 1:200 dilution against human ADRVdemonstrated strong reactivity to the 47-kDa polypeptideand some reactivity to the 113-kDa polypeptide (Fig. 8).Guinea pig antisera raised against ADRV, however, reactedstrongly with all the viral polypeptides. None of the fourMAbs prepared against ADRV were reactive to ADRV inWestern blot analysis. However, the MAb prepared againstIDIR demonstrated a strong reaction to the 47-kDa polypep-tide.

FIG. 7. Western blots of group B rotavirus polypeptides versustitrations of human acute- and convalescent-phase sera. Immuno-blotting was performed as described in the legend to Fig. 5. Thedilutions of antisera are shown in each lane.

No cross-reactivity could be demonstrated with antiseraagainst group A rotavirus tested by Western blot againstgroup B proteins or antisera against ADRV tested againstgroup A proteins (data not shown).

GPCK RatOKAD)RV IC)IR

A C A C

mm

,

_

GPocBESV

A C

m

Pig Calf MAbC oc CK

RVB RVB IDIR

m

FIG. 8. Western blots of group B rotavirus polypeptides probedwith guinea pig antisera to ADRV (GPaADRV), rat antibodies toIDIR (RatcdDIR), guinea pig antibody to bovine enteric syncytialvirus (GPcxBESV), pig antibody to porcine group B rotavirus(PigaRVB), calf antibody to bovine group B rotavirus (CalfaXRVB),and MAb to IDIR (MAbaIDIR). Lanes A, Acute phase; lanes C,convalescent phase.

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DISCUSSION

The major polypeptides of ADRV migrate in a patterngenerally comparable with that of group A rotavirus (15) andwith that of the recently described group C rotavirus (2),although the apparent molecular weights of each polypeptideare distinct. Treatment of double-shelled particles withEDTA followed by CaCl2 led to degradation of the completevirus into single-shelled particles and cores that were iden-tified by EM. Comparison of the proteins of double, single,and core particles by SDS-PAGE analysis indicated that theouter capsid was composed of polypeptides of 64, 61, and 41kDa and the inner capsid was composed of polypeptides of47 kDa. On the basis of a comparison with the polypeptidesof SAl, we have tentatively designated these polypeptidesVP5, VP5a, and VP7, and VP6, respectively. The 41-kDapolypeptide is a glycoprotein which corresponds to VP7 ofgroup A rotavirus. The 47-kDa polypeptide is the mostabundant structural protein of ADRV and presumably cor-responds to VP6 of group A rotavirus. The 47-kDa polypep-tide is recognized by antisera prepared against a variety ofhuman and animal strains of group B rotavirus and appearsto be common to all of them (20). It is also recognizedstrongly by the MAb prepared to IDIR, suggesting that thismay be a particularly good diagnostic reagent. This 47-kDapolypeptide is most strongly antigenic in human convales-cent-phase sera, perhaps because of its relative abundance inthe virus. It is not removed by EDTA treatment of double-shelled virus but is removed by CaCl2 treatment of single-shelled particles.Other polypeptides of ADRV located in viral cores have

been identified, assigned approximate molecular sizes, andgiven a tentative protein designation on the basis of thecontrol preparation of SAl. These include the 136-kDapolypeptide, proposed to be VP1, a 113-kDa structuralpolypeptide, proposed to be VP2, and several proteinswithin the range of 100 to 72 kDa which may be VP3, VP4,or related products.As determined by Western blot analysis, all the viral

proteins are antigenic when tested with high-titer sera (1:100). As the titer of convalescent-phase sera rose, antibodiesto the 47-kDa polypeptide appeared at highest titer, withantibodies to the 113-, 64-, and 41-kDa polypeptides presentat intermediate titers. The ratio of the antibody response tothe 47- and 41-kDa polypeptides differed markedly betweenpatients, suggesting a parallel with the dissociation of anti-body responses to group A rotavirus between inner-capsidantigen tested by enzyme-linked immunosorbent assay andouter capsid antigen tested as a neutralization response.The lack of cross-reactivity between antisera to group A

and group B rotaviruses has been reported previously instudies using immunoelectron microscopy, immunofluores-cence, and enzyme-linked immunosorbent assays (17, 20,21). These studies suggest that these two viruses, though inthe same family, are antigenically quite distinct. Our dataextend these observations by examining individual proteinsby Western blot. Initially, ADRV was hypothesized to haveemerged by simple reassortment of human group A rotaviruswith animal strains of group B rotavirus. The lack ofcross-reactivity to the individual proteins suggests that ma-jor antigenic differences that we observed reflect a greaterdiversity of the polypeptides.

Efforts at understanding the biology and the origin of thenon-group A rotaviruses, considerations of immunogenicityand control with vaccines, and improved diagnosis of thisnoncultivable agent will require identification of the genes

encoding the inner and outer capsid proteins. This study hasidentified the structural proteins of these capsid structuresand provides a basis for determining the gene-coding assign-ments for these proteins.

ACKNOWLEDGMENTS

We thank Larry Anderson, Bill Bellini, Olen Kew, Marie Morgan,Richard Combs, Will Richardson, Wei Wei Ye, John Burns, Mei-Shang Ho, and Elizabeth Hickey for assistance.

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