Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes

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Copyright C 1988, American Society forMicrobiology

Occurrence

of Changes

in Human

Rotavirus Serotypes with

Concurrent

Changes

in

Genomic RNA Electropherotypes

TOYOKO NAKAGOMI,' KAORU AKATANI,2 NOBUKO IKEGAMI,2 NORIKO KATSUSHIMA,3 AND OSAMUNAKAGOMI4*

Department of Microbiology' andDepartmentof Laboratory Medicine,4 Akita University School of Medicine, Akita 010, ClinicalResearch Institute, Osaka National Hospital, Osaka 540,2 andDepartmentof Pediatrics,

Yamagata CityHospital, Saiseikan, Yamagata 990,3 Japan

Received 8 June 1988/Accepted 12 August 1988

To investigatetheserotypic and genetic diversity of human rotavirus strains,wehave tested513 and 519 fecal rotavirusspecimens,respectively, byanenzyme-linked immunosorbentassaywithserotype-specific monoclo-nal antibodies andby polyacrylamide gel electrophoresis of the segmented RNAgenome.Ofthe513 specimens,

375weretypedasserotype1(47.3%),serotype2 (2.9%),serotype3 (2.9%),orserotype4 (17.7%).Inaddition,

apresumptivenewhumanserotype,tentatively referredtoasserotypeXin thispaper,wasfound in 1.6% of the specimens tested. The remaining 138 specimens (26.9%) were untypeable. Considerable variation in

relative frequencyof circulating serotypeswasobservedwithrespecttogeographic locations and observation periods. Rotavirus RNAs were visualized in 481 of519 specimens tested. Ofthese, 415 were

typed

as33

electropherotypes,manyof whichwereinfrequentlydetectedandwererestrictedtosingleepidemics.Analysis ofthe 291specimens whose electropherotypesandserotypeswereavailable indicated clearly thatagivenRNA patternalways correspondedtoaparticularserotype.Heterogeneityofelectropherotypeswithinaserotypewas

similarly observedinstrains belongingtothefourpreviouslyestablishedserotypes.The results obtainedin this study indicated thatantigenic changesonthe major neutralization antigen occurred always withconcurrent changesofgenomicRNAelectropherotypes. On the other hand,serotypic changes couldnotbe predictedfrom the changesinRNAelectropherotypes.

Rotaviruses are the single most important pathogen of acute gastroenteritis among children in the developing and developed regions of the world (10). Understanding the epidemiology of rotavirus diarrhea would be advanced by describing the circulation of rotavirus strains in a variety of

geographic locations over time. Serotyping of clinical iso-lates has been hampered bydifficulties in adapting rotavirus strainstocellculture. Incontrast, electrophoreticseparation ofthe 11 segmentsofthedouble-stranded RNA genome has

been used extensively as an alternative method to

charac-terizefield specimensinepidemiologic studies (forareview,

see reference9). Studies based on electropherotyping indi-catethefollowing. (i) Great diversity occurs in the

electro-pherotypes of rotavirus. (ii)Despite this variety, two distinct

electrophoretic migration patternsexist, short and long, on the basisoftherelativemobility ofgene segments 10and 11, with the short-pattern viruses restricted to human rotavi-ruses. (iii) During outbreaks, one strain or a few strains of

rotavirusesare predominantat anytime, withcocirculating strains possessing less-common electropherotypes. (iv) Se-quential changes over time fromonepredominant patternto another mayindicateconcurrent antigenic change, asin the case ofinfluenza virus, although differences in

electrophe-rotypesdo not necessarily signifyserotypic differences. Recently, several groupsofinvestigators havedeveloped enzyme-linkedimmunosorbent assays (ELISAs)using

sero-type-specific monoclonal antibodies (1, 4, 8, 11, 14, 28).

These ELISAs enabledusto serotypehumanrotavirusesin stoolspecimenswithoutadaptingthemtocellculture; thus,

theprocedurescanbe used inepidemiologic investigations.

Intraditional neutralization assays with hyperimmune anti-sera, rotavirus strains sometimes cross-reacted with more

* Correspondingauthor.

than one hyperimmune serum. SharingofVP3 antigenicity

amongstrains ofdifferent serotypes mayexplain this cross-reaction, since it is now known that both VP7 (gene 8 or 9 product)and VP3 (gene 4 product) (17) elicit aneutralizing antibodyresponse(15, 25). UseofVP7-specific monoclonal antibodies in ELISA, therefore, has made it possible to determine serotypesolelyonthebasis oftheantigenicity of

VP7.

We have used a serotyping ELISA in conjunction with RNApolyacrylamide gel electrophoresis for the analysis of

winter outbreaks of rotavirus diarrhea observed in two

locations in Japan. This paper describes the antigenic and

genetic diversity ofhuman rotaviruses among field

speci-mens collected inJapan.

MATERIALSANDMETHODS

Viruses. The following cell culture-adapted human and animal rotavirus strains were used in this

study:

human

rotavirusesWa(serotype1,subgroup11),KUN(serotype2, subgroupI), DS-1 (serotype2,subgroup I), MO(serotype3, subgroupII), YO (serotype3,subgroup Il),ST3(serotype4, subgroup Il), and simian rotavirus SA11 (serotype 3, sub-group

T).

Atotalof 582 fecal rotavirus

specimens

used in this

study

were collected in Akita from December 1981 to

April

1983 and from January to June 1987 from children with acute

gastroenteritis, inYamagata fromJanuary1986toJune 1987 fromchildrenwithdiarrhea,and inMarch 1987from

patients

in various parts ofJapan. The study

period

included four winter outbreaks ofrotavirus diarrhea. Fecal

suspensions

(approximately 10% in

phosphate-buffered

saline, pH 7.2)

weremixed withanequalvolumeoftrichlorotrifluoroethane

and centrifuged 2,000 x g for 10

min,

and the resultant aqueous layer was tested for rotavirus

antigen by

either a 2586

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reversepassivehemagglutinationassay(22)or alatex agglu-tinationassay(Rotalex; OrionDiagnostica, Espoo, Finland).

Of these 582 rotavirus-positive specimens, 513 were

sub-jected to serotyping by ELISA and 519 were analyzed by

RNAelectrophoresis. For RNA analysis, thicker stool sus-pensions (20to50%)wereextracted withphenol-chloroform

and RNAswere precipitated in ethanol. The serotyping and

RNAanalysis wereconducted independently intwo labora-tories. Data obtained by thesetwomethodswerecompared,

and specimens were retested when the initial results were

ambiguous.

Serotypingtest. Serotypingwasperformedby ELISA bya method previously described (1). In addition to the four monoclonal antibodies specific for serotypes 1 to 4, a fifth

monoclonal antibody was included that specifically

recog-nized the F45strain,apresumptivenewserotypetentatively referred to as serotype X (N. Ikegami, K. Akatani, T. Hosaka, and H. Ushijima, Abstr. VII Inter. Congr. Virol.,p.

113, 1987). Briefly, microtiter plates were coated with a

monoclonal antibody (Osaka National Hospital AH6) which captureddouble-shelled rotavirus particles. Following treat-ment with 1% bovine serum albumin, plates were washed

withphosphate-buffered saline containing 0.05% Tween 20. Stoolextracts(20 ,ul)orinfected tissue culture supernatants (50 pt) were then added and incubated for 90 min at room temperature. After incubation, plateswerewashed and each

well received one of thefollowing biotinylated monoclonal

antibodies: Osaka National Hospital AH49 for serotype 1, AG12forserotype2,AC5 forserotype3, AE18forserotype 4, AJ26 for serotype X, and AB4for double-shelled parti-cles. Following incubation for 90 min, plates were washed

andstreptavidin-peroxidaseconjugateswereadded.

Follow-ingincubation for 10 min, plates werewashed and substrate

(o-phenylenediamine) was added, and the

A500

of each well was measured with a microplate reader. Readings greater than0.05were considered positive.

Electrophoresis of RNA. Genomic double-stranded RNA

was extracted with phenol-chloroform either from stool

suspensions or from the purified virions which were pre-pared from infected MA104 cells by sedimentation through 40%(wtlvol) sucrose, followedby CsCI equilibrium density gradient centrifugation. RNAs were fractionated on a 10%

polyacrylamide gel with a 4% stacking gel in the buffer

systemof Laemmli(20) and thenwerestained with ethidium

bromide.

RESULTS

Serotypic diversity ofhuman rotavirus as revealed by an

ELISAwithserotype-specificmonoclonalantibodies.Wewere

able toclassify 372 of 513rotavirus-positive stool specimens (73%) into the fourpreviously established serotypes (3, 30) or serotype X by using the serotype-specific monoclonal antibodies(Table 1). Three specimens reacted tomore than

one serotype-specific monoclonal antibody. Whether this could betheresultof dual infectionornonspecific binding of

monoclonal antibodies has yet to be determined. The re-maining 138 specimens (26.9%) could not be serotyped. Mostofthese specimens reactedtonone ofthemonoclonal

antibodies, possibly because they lostoutercapsid proteins during storage and processing. For example, addition of EDTA to the buffer results in disintegration of rotavirus outercapsid proteins (26) which bear the serotype-specific epitopes. Of 134 rotavirus-positive specimens collected in Yamagatain theseasonof1986to1987, 72specimens (54%)

were untypeable, probably because those specimens were

TABLE 1. Reactivity ofmonoclonalantibodiesused inserotypingELISA'

A50o for monoclonalantibody' Virus Serotype

AB4 AH49 AG12 AC5 AE18 AJ26

Wa 1 1.198 0.553 0.004 0.008 0.005 0.005

DS-1 2 0.032 0.007 0.213 0.007 0.007 0.004

YO 3 0.638 0.002 0.006 0.175 0.006 0.006

ST3 4 0.033 0.003 0.003 0.006 0.124 0.004 F45 X 0.238 0.000 0.001 0.006 0.003 0.051

aDescribed in Materials and Methods.

b Allmonoclonal antibodiesaredirectedattheoutercapsidprotein. AH49, AC5, and AJ26haveneutralizing activity,whereasAB4,AG12,andAE18do not.

processed with the phosphate-buffered saline containing

EDTAwhichwas suppliedwith the Rotalexkit.

Serotype 1(47.3%)and 4(17.7%) strains predominatedin Japan, with serotype 2(2.9%),3(2.9%),andX(1.6%) strains

less frequentlydetected(Table 2). Therelativefrequencyof

human rotavirus serotypes varied considerably interms of both geographic locations and study periods (Table2). For

example, while serotype 4 strains were most frequently detectedin Yamagata intheepidemic of1985 to 1986, they

were displaced by serotype 1 strains in the next winter

season. Although serotype 1 strains predominated in both Yamagataand Akitaduringtheepidemic of1986to 1987, 27

of61

(44.3%)

rotavirus-positive specimens that were

col-lected from over 20 locations in Japan during a 3-week

period (March1987) in the sameepidemicseason contained

serotype4strains. While serotype 3 strainswerethesecond

mostfrequently detected(32%)in Akitaduringtheepidemic of1981to1982, thisserotypewasveryinfrequently foundin the other epidemics. Furthermore, serotype X, which

ap-peared in 1986, was not detected in any other epidemics.

These results indicate that rotavirus strains of different

serotypescirculated in a sequential fashion among children in Japan.

Genetic diversity ofhuman rotaviruses as determined by electrophoresis of genomic RNA segments.Of481 stool spec-imens in which rotavirus RNAs were visualized, 415 were

classifiedas33electropherotypes, each of whichwas

desig-nated either S (for short patterns) or L (for long patterns)

followed bya numberin the orderofappearance(Fig. 1 to

4). Oftheremaining66rotavirus specimens,37containedL

TABLE 2. Relativefrequencyof human rotavirus serotypes

No.ofspecimensfoundin:

1981- 1982- 1985- 1986- 1986- March

Serotype 1982, 1983, 1986, 1987,

19876

1987,

Akita Akita Yamagata Yamagata Akita various (n =31) (n=47) (n= 114) (n= 134) (n=126) l(onca6ns)

1 14 37 25 45 102 20

2 1 1 4 7 1 1

3 10 0 0 3 0 2

4 4 0 52 7 1 27

X 0 0 8 0 0 0

1 and X 0 0 1 0 0 0

4and X 0 0 2 0 0 0

Untypeable 2 9 22 72 22 il

a Rotavirusspecimens obtained from>20geographic locations throughout Japan.

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A B C D E F G H 1 J K L M N O P Q

2 3 4

5

6

78a

9

10 1

FIG. 1. Polyacrylamide gel electrophoresis ofRNA extractedfrom stool specimens containing serotype 1rotaviruses. Lanes: A, Li (82A012); B,L3(82A003); C,L7(83A003);D,L8(83A005);E,Lii (87H081);F, L10 (86Y0460);G,L19(87Y0184);H, L22(87H003); t,L23 (87T527); J,L24(87T619);K, L25 (87T587);L, L26(87T628); M, L27 (87T624);N, L28(87Y643);O,SA11;P, KUN; Q,MO. Lanes O to Q contain reference strains.RNA segments are indicated at the left.

patterns (with which proper assignment to particular

elec-tropherotypes was not possible because not all of the 11 bands were clearly visualized), 2 contained mixtures of

different L patterns, 7 contained mixtures of S and L patterns, and 20contained only several ofthe 11 rotavirus

RNAsegments. We were not able to detect rotavirus RNA in 20 specimens (3.9%) that were positive for rotavirus

antigen by either a reverse passive hemagglutination assay or a latex agglutination assay. The relative frequency of

A B C D E F G H

23

4

5

6

78

9

10

1

FIG. 2. Polyacrylamide gel electrophoresis of RNA extracted fromstoolspecimens containingserotype2rotaviruses. Lanes: A, KUN; B, MO; C, SA11; D, Si (82A042); E, S2 (83AO01); F, S3 (86Y0395); G,S4 (86Y1329); H,S5(87Y0003). LanesAtoCcontain

reference strains. RNA segmentsareindicatedattheleft.

these electropherotypes shown in Table 3 indicates that manypatterns were infrequentor even unique. Anexample

ofthetemporal distribution of electropherotypes is shownin Table 4. During two consecutive years, RNA

electrophe-rotypes were asheterogeneousatthebeginning of theseason as they were at other times during the epidemic. A few

predominant electropherotypes were seen in each season,

with other electropherotypes appearing concurrently with lowerfrequency. EachRNAelectropherotypewasgenerally

A B C D E F G

1' &

23

4

5 6

78 9 10

11l

FIG. 3. Polyacrylamide gel electrophoresis of RNA extracted fromstoolspecimens containingserotype 3 rotaviruses.Lanes: A, KUN;B, MO; C, SA11; D,L2 (82A001); E, L4(82A017);F, L20 (87Y0228); G,L21(87T612).Lanes AtoCcontain reference strains. RNAsegmentsareindicatedattheleft.

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A B C D E F G H 1 J K L

23 4

5 6

7,

10 11

FIG. 4. Polyacrylamide gel electrophoresis ofRNA extracted from stool specimens containing serotype 4 or serotypeX rotavi-ruses. Lanes: A, KUN; B, MO; C, SA11; D, L5 (82A019); E, L6 (82A035); F, L12 (86Y0089); G, L13 (86Y0113); H,L14(86Y0300);

t, L15 (86Y0301); J, L16 (86YO741); K, L17 (86Y0607); L, L18 (87T537);M, L9(86Y0316).Lanes AtoC, Reference strains;Dto

L, serotype 4 strains; M, serotype X strain. RNA segments are

indicatedatthe left.

restricted to one particular epidemic season. In many in-stances,whatappearedtobe the reappearance ofanearlier electropherotype turned out on coelectrophoresis to be a

distinct new strain. For example, although the L8 pattern

(83Ai25)

thatpredominatedinthe epidemic of1982to 1983

was similarto the

Lii

(87H081)pattern which appeared in

1987, the coelectrophoresis ofthese two patterns revealed slight butdefinite migration differences in gene segments 6

and10(Fig. 5).TheonlyRNA patternthatwasseenintwo

epidemics was the 11 pattern, which appeared in 1986

(86Y023 and 14 additional specimens) andpredominated in thesucceeding epidemicin 1987(87H081and 121additional specimens). In contrast, rotaviruses with identical electro-pherotypes were recovered from different geographic

loca-tions during the same epidemic season. For example, the

L11, L19, and L28 patterns weredetectedin the specimens collected in Akitaand in Yamagata duringthe epidemic of

1986to 1987.

Association of rotavirus serotypes and RNA electrophe-rotypes. For 291 rotavirus specimens, both serotypes and

electropherotypes were available. A clear correlation was found between a given RNA pattern and a particular sero-type (Table 5).Fourteendifferentpatternswithinserotype 1

strains, five patterns within serotype 2strains, four patterns

within serotype 3 strains, and nine patterns within serotype 4 strains weredetected (Fig. 1 to 4). In contrast, all seven

specimensthatcontained serotype Xstrains showed identi-cal RNA electropherotypes. In terms of electropherotypic

variability, serotype 2 strains are as heterogeneous as the other three serotype strains, although only short-pattern

virusesareassociated with this serotype (Fig. 2). However, the association of subgroup I and the short RNA

electro-pherotypedoes not hold true as we have previously reported

(24). In addition to the AU-1 strain which was recovered

from a specimen obtained in 1982 (23), three specimens

TABLE 3. Relativefrequency of human rotavirus electropherotypes

No. ofspecimensfoundin:

Electropherotype 1981- 1982- 1985- 1986- 1986- March 1982, 1983, 1986, 1987, 1987, 1987,

Akita Akita Yamagata Yamagata Akita locations

Li 12

L2 1

L3 4

L4 9

L5 2

L6 2

L7 1

L8 36

L9 il

L10 5

Lii 15 45 77

L12 1

L13 3

L14 19

L15 3

L16 3

L17 15

L18 7 25

L19 35 14 1

L20 1

L21 1 1

L22 25

L23 1

L24 2 5

L25 1

L26 3

L27 1

L28 2 6 4

Si 1

S2 1

S3 4

S4 7 1

S5 1 1

a Rotavirusspecimensobtained from>20geographic locationsthroughout Japan.

(86Y1115, 86Y1135, and 87YO228)obtained duringthe sea-son of 1986 to 1987 possessed long RNA patterns but

belongedtosubgroupI(datanotshown).Interestingly,all of these specimens weretyped as serotype 3. Further studies on these strainsarein progress.

DISCUSSION

The recent development of amethod of serotyping that does not require the adaptation of stool rotaviruses tocell cultureprovides theopportunitytoinvestigatethe serotype

diversity of human rotavirus strains on the basis of the

antigenicity ofVP7(1,4, 8,11,13, 14,28).Inaddition,these

proceduresprecludesometheoretical selection biasin which

particular strains may be quicker to adapt to cell culture. Fourpreviousworks,onecarriedoutinItaly bysolid-phase

immune electron microscopy (12) and the other three per-formed in Australia and in Central African Republic by ELISAs with serotype-specific monoclonal antibodies(4, 7,

11), have shown that although the majority ofstool

rotavi-ruses belong to serotype 1, the frequency of detection of other serotypes varies considerably from one study to an-other.

In addition tofour distinct serotypes of group A human

rotaviruses (3, 30), afifthand a sixth serotype, designated

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TABLE 4. Temporaldistribution of rotavirus electropherotypes No.of specimens found in:

Electropherotype 1986 1987

Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June

L9 9 2

L10 2 1 1 1

Lii 5 2 7 1 1 15 15 8 4 2

L12 1

L13 1 1 1

L14 5 8 5 1

L15 1 1 1

L16 1 2

L17 1 3 6 4 1

L18 5 2

L19 16 6 9 3

L20 1

L21 1

L24 2

L28 2

S3 2 2

S4 5 2

S5 1

69M and W161, respectively, were described previously (5, 21). Ourpanel of monoclonal antibodies includes a reagent

specific forthe F45strain, a serotype X prototype. Whether

theserotype X strains are related serologically to either the 69M ortheW161 strain hasyet to be determined.

In our study, the overall frequency of human rotavirus serotypes was thehighest forserotype 1(47.3%)andsecond

highest for serotype 4 (17.9%). However, the relative fre-quency ofserotypes was not uniform with respect to

geo-graphical locations and study periods. In the epidemic of 1985 to 1986, serotype 4, 1, and X strains circulated in

A B C D

2 34

5 6

10 11

FIG. 5. Coelectrophoresisof RNA extracted from thespecimens containing the L8 or Lii patterns. Lanes: A, SA11 (reference strain);B, L8(83A125); C,L8(83A125)and Lii (87H081); D, Lii

(87HO81).ArrowsindicatetheRNA segments that showadifference

inmigrationbetweentwopatterns. RNA segmentsareindicatedat

theleft.

Yamagata. In thefollowingwinter, serotype 4 strains were

supercededby serotype 1strains. Similarly, while serotypes

1, 3, and 4 circulated in Akita duringthe season of1981 to 1982, serotype 1 strains ofa single RNAelectropherotype

(L8) predominated in the followingepidemic, with most of the other serotypeseliminated. These abrupt changes in the

relative frequency of

circulating

serotypes from one epi-demic to another occurred with the concurrent changes in

thepredominantRNAelectropherotypes. Althougha

defin-itive interpretation of this observation remains to bemade,

theimmuneselectivepressure couldplayarolein

modulat-ing antigenic changes on the neutralization antigen.

Se-quencingof thehyperdivergentareasofthe genecoding for

VP7 would be required to further addressthis question. In

addition, frequent asymptomatic infections among adults (16, 18, 29) may be importantin thetransmission of

rotavi-rusesamongchildren.

Itremains unclear whether thepredominant

electrophero-typeofone seasonis the last strain ofthe past season ora

forerunner ofthesubsequent season(9, 27).Our resultsare notconclusivelikethose reportedby Steele andAlexander (27). Although the

predominant

electropherotype of the

epidemic of1986 to 1987(L11)was seeninthe seasonof 1985

to1986,pattern

Lii

wastheonly

exception

inour

study

that

appeared in twoconsecutive

epidemics.

All the other

elec-tropherotypes were restrictedto single seasons,

suggesting

that a new strain canemergeasthepredominant strain ina

given

epidemic.

As to thetemporal distributionof RNAelectropherotypes,

ourresults showed that RNApatterns were

heterogeneous

throughouttheepidemic. This appearsto oppose the

previ-ous report

by

Konno et al. (19) that a

few,

if not

single,

strains ofdominant electropherotypes

appeared

at the

be-ginning of an epidemic and a wide

variety

of

electrophe-rotypes wereseenthereafter. Since thesetwo studies were done in the same

geographical

location

during

different epidemics, the difference in results may suggest the

com-plexity ofrotavirus epidemiology.

Thepresence of variouselectropherotypeswithina

given

serotype indicates that rotavirus strains

belonging

toeachof

fourserotypesare

similarly

heterogeneous. Itremainstobe

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TABLE 5. Correlation between serotypes and RNA electropherotypes

No. ofspecimens with serotype: Electropherotype

1 2 3 4 X

Li il

L3 3

L7 1

L8 28

L10 2

Lll 93

L19 21

L22 21

L23 1

L24 5

L25 1

L26 3

L27 1

L28 9

Si 1

S2 1

S3 3

S4 3

S5 2

L2 1

L4 9

L20 1

L21 1

L5 2

L6 2

L12 1

L13 3

L14 14

L15 3

L16 3

L17 9

L18 25

L9 7

determined whether this heterogeneity in electropherotypes isrelatedtothepresenceofmonotypeswhicharedefinedby

thedifference inneutralization epitopes detected with mono-clonal antibodies among rotaviruses of the same serotype (7). It isnotclear whether the mutationsrequiredtoalterthe neutralization characteristics ofavirus produce changes in the electrophoretic mobility of the corresponding genome segment.

Althoughtwo rotavirus strains possessing identical RNA electropherotypes but different serotypes were reported

previously (2, 11, 12), the present study, in which both electropherotype and serotype were determined for 291 specimens, showed that a given RNA electropherotype

always corresponded to a particular serotype. Our results

areconsistentwith the observation made by Coulson on90

rotavirus specimens (7)and, therefore,supporttheview that

once the electropherotypes present each year in a given

location have been determined, serotyping of limited

num-bersof each electropherotype would be sufficientto

ascer-tainthe serotypes circulating in the population under study (7). A more extensive comparison, with great care in ana-lyzing electropherotyping results, is required, however, since Clarke and McCrae demonstrated that nucleotide sequence changes at least as great as those found in

seg-mentsshowing electrophoretic mobility variationswerealso detected in segments showing no mobility variation (6).

Further study is also needed to correlate the variations in electrophoretic mobility with changes in coding sequenceor antigenicity.

ACKNOWLEDGMENTS

We thankH.Oyamada(Akita University Hospital) for his excel-lenttechnical assistance; N.Yazaki,M. Sakamoto (YamagataCity Hospital Saiseikan, Yamagata), S. Kuroki (Yuri General Hospital, Honjo),Y. Mochida,T. Yamauchi,andS. Hatano(Special Refer-ence Laboratories, Tokyo) for collectingrotavirus-positive speci-mens; andT. Suto and S. Uesugi for encouragement. We particu-larly thank Roger I. Glass (Centers for Disease Control, Atlanta, Ga.) for his criticalreading ofthemanuscript and helpfulcomments onit.

This work was partly supported by agrant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan.

LITERATURECITED

1. Akatani, K., and N. Ikegami. 1987. Typing of fecal rotavirus

specimens by an enzyme-linked immunosorbent assay using monoclonal antibodies. Clin. Virol. 15:61-68.(In Japanese.) 2. Beards, G. M. 1982. Polymorphism ofgenomic RNAs within

rotavirusserotypes andsubgroups. Arch. Virol. 74:65-70. 3. Beards, G.M., J.N.Pilford,M.E.Thouless, and T. H. Flewett.

1980. Rotavirus serotypes by serum neutralization. J. Med. Virol. 5:231-237.

4. Birch, C. J., R. L. Heath, and I. D. Gust. 1988. Use of serotype-specific monoclonal antibodies to study the epidemi-ology of rotavirus infection.J.Med. Virol. 24:45-53.

5. Clark, H. F., Y. Hoshino, L. M. Bell, J. Groff, G. Hess, P. Bachman,and P. Offit. 1987.Rotavirus isolate W161 represent-ingapresumptivenewhumanserotype.J.Clin. Microbiol.25: 1757-1762.

6. Clarke,I.N.,and M. A.G.McCrae. 1982.Structuralanalysisof electrophoreticvariation in thegenomeprofilesof rotavirus field isolates. Infect. Immun. 36:492-497.

7. Coulson, B. S. 1987. Variation in neutralization epitopes of humanrotaviruses in relationtogenomicRNApolymorphism. Virology 159:209-216.

8. Coulson,B.S., L. E.Unicomb,G. A.Pitson,and R. F. Bishop. 1987.Simpleandspecificenzymeimmunoassayusing monoclo-nalantibodies forserotypinghumanrotaviruses. J. Clin. Micro-biol. 25:509-515.

9. Estes, M. K., D. Y. Graham, and D. H. Dimitrov. 1984. The molecularepidemiology of rotavirusgastroenteritis.Prog. Med. Virol.29:1-22.

10. Flores, J.,O. Nakagomi,T.Nakagomi,R.Glass,M.Gorziglia,J. Askaa,Y. Hoshino, I. Perez-Schael, and A.Z. Kapikian. 1986. The role of rotaviruses in pediatric diarrhea. Pediatr. Infect. Dis. J.5:S53-S62.

11. Georges-Courbot, M. C., A. M. Beraud, G. M. Beards, A. D. Cambell, J.P.Gonzalez,A.J.Georges,and T. H.Flewett.1988. Subgroups, serotypes, and electrophoretypes of rotavirus iso-latedfrom children inBangui,Central AfricanRepublic.J.Clin. Microbiol. 26:668-671.

12. Gerna, G.,S.Arista,N.Passarani,A.Sarasini,andM. Battaglia. 1987. Electropherotype heterogeneity within serotypes of

hu-man rotavirus strainscirculating inItaly. Arch. Virol. 95:129-135.

13. Gerna, G., N. Passarani, A. Sarasini, and M. Battaglia. 1985. Characterization of serotypes of human rotavirus strains by solid-phase immune electron microscopy. J. Infect. Dis. 152: 1143-1151.

14. Heath, R., C. Birch, andI. Gust. 1986. Antigenic analysis of rotavirus isolates using monoclonal antibodies specific for

hu-manserotypes1,2,3, and 4, andSA11.J. Gen.Virol. 67:2455-2466.

15. Hoshino, Y., M. M. Sereno, K. Midthun, J. Flores, A. Z. Kapikian,and R. M.Chanock.1985.Independentsegregation of

twoantigenic specificities (VP3 and VP7) involved in neutrali-zation of rotavirusinfectivity. Proc. Natl. Acad. Sci. USA82: 8701-8704.

16. Hrdy,D. B.1987. Epidemiology of rotaviral infectionin adults. Rev.Infect. Dis. 9:461-469.

17. Kalica,A.R., J. Flores, andH.B. Greenberg.1983. Identifica-tion of the rotaviral gene that codes for hemagglutinin and

on April 11, 2020 by guest

http://jcm.asm.org/

protease-enhanced plaque formation. Virology 125:194-205. 18. Kim, H. W., C. D. Brandt, A. Z. Kapikian, R. G. Wyatt, J. O.

Arrobio, W. J. Rodriguez, R. M. Chanock, and R. H. Parrott. 1977. Human reovirus-like agent infection. Occurrenceinadult contacts ofpediatric patients with gastroenteritis. J. Am. Med. Assoc.238:404 407.

19. Konno, T., T. Sato, H. Suzuki, S. Kitaoka, N. Katsushima, M. Sakamoto, N. Yazaki, and N. Ishida. 1984. Changing RNA patterns in rotaviruses of human origin: demonstration of a single dominant pattern at the start of an epidemic and various patternsthereafter. J. Infect. Dis. 149:683-687.

20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

21. Matsuno, S., A. Hasegawa, A. Mukoyama, and S. Inouye. 1985. Acandidate for a new serotype of human rotavirus. J. Virol. 54: 623-624.

22. Nakagomi, O., A. Nakagomi, T. Suto, H. Suzuki, T. Kutsuzawa, F.Tazawa,T.Konno, and N. Ishida.1982. Detection of human rotavirus byreversed passive hemagglutination (RPHA) using antibody against a cultivable human rotavirus as compared with electron microscopy (EM) and enzyme-linked immunosorbent assay(ELISA). Microbiol. Immunol.26:747-751.

23. Nakagomi, O., T. Nakagomi, Y. Hoshino, J. Flores, and A. Z. Kapikian. 1987. Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup Il human rotaviruses. J. Clin. Microbiol.

25:1159-1164.

24. Nakagomi,O., T. Nakagomi, H. Oyamada, and T. Suto. 1985. Relative frequency ofhuman rotavirus subgroups 1 and 2 in Japanese childrenwithacutegastroenteritis. J. Med. Virol. 17: 29-34.

25. Offit, P. A., and G. Blavat. 1986. Identification of the two rotavirus genesdeterminingneutralization specificities. J. Virol. 57:376-378.

26. Shirley,J. A., G. M. Beards, M. E. Thouless, and T. H. Flewett. 1981.Theinfluence of divalent cations on thestability of human rotavirus. Arch. Virol. 67:1-9.

27. Steele, A. D., and J. J. Alexander. 1987.Molecularepidemiology of rotavirus inblack infants in South Africa. J. Clin. Microbiol. 25:2384-2387.

28. Taniguchi, K., T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa. 1987. Directserotyping of human rotavirus in stools by anenzyme-linked immunosorbent assay using serotype 1-, 2-, 3-, and 4-specific monoclonal antibodiesto VP7.J. Infect. Dis. 155:1159-1166.

29. Weman, W. M., D. Hinde, S. Feltham, M. Gurwith. 1979. Rotavirus infection in adults. Results ofa prospective family study. N.Engl.J. Med. 301:303-306.

30. Wyatt, R.G., H. D. James, Jr., A. L. Pittman, Y. Hoshino, H. B. Greenberg, A. R. Kalica, J. Flores, and A. Z. Kapikian. 1983. Direct isolation incell cultureofhumanrotaviruses and their characterization into four serotypes.J.Clin. Microbiol. 18:310-317.

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