Prospective study of community acquired rotavirus infection

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0095-1137/89/092083-08$02.00/0

Prospective Study of Community-Acquired

Rotavirus Infection

BO JIAN ZHENG,l* SIMON K. F. LO,1JOHN S. L.

TAM,lt

MONA LO,2 CHAPYUNGYEUNG,3

ANDMUN HON NG'

Departments ofMicrobiologyl and Paediatrics,2 University ofHong Kong, Pokfulam Road, andKung TongCommunity

Health Programme, UnitedChristian Hospital, Kung Tong,3 Hong Kong Received 27 February1989/Accepted 7 June 1989

We determined titers ofgroupArotaviruscommonantibodiesandneutralizing antibodiesagainstserotypes 1 to4ofprototype human rotavirus (HRV) in cord blood andserumspecimens obtained from 38 infants at

4-month intervals from birth until 2 years ofage. Nineteen of the infants developed one episode of HRV

diarrheaeach,andtheywerematchedbyageand birthweight withthe other 19infants,who didnotdevelop

HRV diarrhea during the follow-up period. We estimated the incidence rate of HRV infection for the two groupsof infants combined to bea minimum of 1.34 episodes per infantper year, which is 22 times more common thanthe occurrenceofovertdisease caused by the virus in this community. The infectionoccurred

constantly throughout the first 2 years ofinfancy, whereas all butone of the 19 episodes ofovert disease occurred before 12 months ofage. Seven of these overt episodes were preceded by at least one episode of subclinical infectionearlier, and the otherseven wereprobably duetoprimaryHRVinfection. Theremaining fiveepisodes occurred before4months ofage,sothatwecouldnotascertainwhethertheywereduetoprimary infections because of thepresenceof maternal antibodies. Weshowed that levels ofHRVantibodiesinserum specimens obtainedbeforeclinicalonsetof diarrheavariedwidely, and,formostinfants inthe diarrhealgroup, levelsoftheseantibodies weresimilar to those in the serum specimensobtainedat thesametimes from the

corresponding age- and birth weight-matched control infants. Nevertheless, the age at which overt disease causedbyHRVwasmostprevalentcoincided with thetimewhen thematernal antibodies haddeclinedtolow

levelsbutthe infants had notyetacquired hightitersof these antibodies in theirsera.

In a prospective study of infantile diarrhea in an urban community, we monitored 371 infants from birthto 2years ofage,foratotal of7,718months. Allthese infants resided inasuburbofHongKong,and40 of themdevelopedatleast

oneepisodeeachofhumanrotavirus(HRV)diarrheaduring

thestudy period. At an incidence rate of0.06episodes per infant year, HRVwas foundto be secondto salmonella as

the most commonof the conventional enteric pathogens of infancy in this community (J. Tam, personal

communica-tion).

As a part of this study, we collected cord blood and, subsequently, serum specimensat4-monthintervals until 2 years of age from this group of infants. We selected 19 infantsfromthiscohort who developedHRVdiarrheaduring

the study period and for whom complete collections of

serum specimens over thisperiod were available. To com-pensatefor theage andseasonalvariation ofHRV diarrhea previously reportedfor this andothercommunities(10, 13), weselected another19infantsfrom thesamecohortwho did notdevelop HRV diarrhea, who were matched by date of

birthand birthweightwiththe infants in thediarrhealgroup.

Wedetermined titersofgroupArotaviruscommon antibod-ies and neutralizing antibodies against serotypes 1 to 4 of

prototypeHRVsintheseserumspecimens. Theresultswere compared to (i) determine the occurrence ofasymptomatic

HRV infections in these infants, (ii) evaluate the effects of

asymptomatic HRVinfections onthedevelopment of HRV diarrhea, and (iii) correlate the levels of the serum HRV

*Corresponding author.

tPresent address:Department ofMicrobiology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.

antibodies of maternal origin or acquired from previous

infections with subsequent developmentof HRV diarrhea. (This research was conducted by B. J. Zheng in partial fulfillmentof therequirementsfor the Ph.D. degreefromthe University of HongKong, Kung Tong.)

MATERIALSANDMETHODS

In a prospective study ofcommunity-acquired diarrhea, we monitored 371 infants from birthfor 2 years. The study subjects were recruited from low- to lower-middle-income

families in a suburb ofHong Kong, and theirdevelopment was monitored monthly in three community clinics located inthat area. We obtained cord blood specimens from these infantsat birth and thenserum specimensat4-month

inter-vals until 2years ofage. Theserumspecimenswere stored

inportionsat -20°Candwereinactivatedby beingheatedat

56°C for 0.5 h before use. Forty infants developed HRV

diarrheaduringthe study period. For thepresent investiga-tion, we selected 19 of these infants for whom complete

collections ofserumspecimenswereavailable.We matched

each of these infantsbyageand birthweightwithoneinfant

selected from the same cohort who did not develop HRV diarrheaduringthe study period, ascontrols.

Themethods for detection of HRV from stoolspecimens, electropherotypingof the viralgenome,andtitration of HRV

antibodies have been described previously (16). Briefly,

HRV present in stool specimens was detected by

enzyme-linkedimmunosorbentassay bythe method of Beardset al. (1). The viral genomes were subject to electrophoresis in

polyacrylamide gel bythe method ofHerringetal. (7), and the results were analyzed as described by Tam etal. (13).

TitrationofserumimmunoglobulinGantibody againstgroup Arotaviruscommonantigenswas asdescribedbyBishopet 2083

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32000-*

~~~~~(C)

(a)

64000-₆₄₀₈₀

~~~~~~~~~~~~~~~~~16000-32000-000

I

1 ~~~~~~~~~~~~~~~~~80001

"

1\00

I

4000"

<

I I

.z

6080

~~~~~~~~~~~~~~~~~~2000-4000 F

20800

O 4 8 ~~~12 16 20 24

Age(Months)

1000-

~~~~~~~~~~~FIG.

1. Development of HRV antibodies. Serium specimens

were obtained from infants at birth and thereafter at 4-month intervals until theywere2 yearsof age. Thespecimensweretitrated for group A rotavirus common antibody (*) and for neutralizing

antibodiesagainstserotype1Wavirus(x), serotype2S2 virus(0),

o.j , serotype3 Yovirus(A),andserotype4Hochi virus

(M).

Oneinfant

o 4 8 12 16 20 24 (infant49) developed HRVdiarrhea at11.2 month of age (a) and

Age(Months) another(infant 283)developedHRV diarrhea at 3.5 months ofage

(b), while infant 310 didnotdevelopHRV diarrheathroughoutthe

(b)

2-year study period

(c).

<- --t < - - ~ * al. (4), except that thebovine rotavirus strain NCDV was

10-\ used as the source of antigen.

Neutralizing

antibodies

,' againstrotavirus were titratedby fluorescence foci neutral-,'`/ization assay, as describedby Beardsetal. (2).The

proto-I ,'type viruses used for the assay were Wa

(serotype 1),

S2

(serotype

2),

Yo

(serotype

3),

and Hochi

(serotype 4),

and they were propagated in MA104 cell cultures as described previously (16).

Themethod fordifferentiating serotypesofHRVisolates bydothybridization, usingasprobescDNAofsegment9of prototype HRV strains Wa, S2, SA11, and Hochi, was as

previously described (17). cDNA probes were

synthesized

by

themethod of

Taylor

et al.

(14).

Dot

hybridization

was > 20002 \\ ,// {' 11 \\ carriedout asdescribed byLinetal. (11, 12)at

42°C

in the

> 2M < /presenceof 65%formamide.

RESULTS

iooo \

Development

> \

/

of HRV antibodies. We determinedlevels of

groupArotaviruscommonantibodies andneutralizing anti-bodies against HRV serotypes 1 to 4 in serum specimens obtained from 38 infants at birth and subsequently at

4-O r 8 W month intervals until 2yearsofage.Figure 1exemplifiesthe

development of HRV antibodies in threeinfants.

Age (Months) (i)Infant 49. One infant(infant 49)

acquired

atbirthall the maternal HRVantibodies describedabove(Fig. la). Except for serotype 3-neutralizing antibodies, the levels ofall the

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other HRV antibodies declined

markedly

andtolow levels

by

the age of4 months.

Evidently,

the infant

might

have

experienced

an

episode

ofinapparent infection withserotype 3 virus in the interim so that the level of neutralizing

antibody against

thatserotypeat4 monthsof age wassimilar

tothatatbirth.

The infant developed HRVdiarrhea atage 11.2 months. Stool

specimens

obtained

during

this

episode

yielded the short

electropherotype

(IA5) whichwas

previously

shown to be characteristic of the genome of subgroup I serotype 2 viruses (6, 9). The infection was accompanied by marked increases inthe levels ofgroup A rotavirus common

anti-body

andserotype

2-neutralizing antibody during

theinterim

period

between8and12monthsofage,whereas the levels of

neutralizing

antibodies

against

serotypes 1, 3, and 4didnot

change significantly during

thesame

period.

Thus, the

viro-logical

and

serological

findings concurred, indicating

that serotype 2viruswasthemost

probable

causeof this

episode

ofinfection.

Infant49

might

have

experienced

twofurther

episodes

of

HRV,

one

occurring

between 12 and 16 months and the other between 16 and 20 months of age. As suggested by the increases in

neutralizing-antibody

titers

during

these

peri-ods,

the first

episode

was

probably

caused

by

serotype 4 virus and the second was probably caused by serotype 3 virus.

(ii)Infant 283.Infant283 had

acquired

abroadspectrumof maternal HRV antibodies at birth

(Fig. lb),

all of

which,

except for serotype

2-neutralizing antibody,

decreased

sig-nificantly

and to low levels

by

age 4 months. The infant

developed

HRV diarrhea at 3.5 months of age. His stool

specimens yielded

theshort

electropherotype (IA5) usually

obtained with serotype 2 viruses. This may

explain why

serotype

2-neutralizing antibody

was sustained at similar levelsat4months ofage andat

birth,

while thelevels ofall theother HRVantibodieshaddecreased

significantly

andto

low levels.

The

antibody profile

suggests that the infant mighthave

additionally experienced

two further

episodes

of

inapparent

HRV infections. The first

episode, probably

caused

by

serotype 3

virus,

might

have occurred intheinterimbetween 4 and 8 months ofage, and the second

episode might

have occurred between 16 and 20 months of age. On the latter

occasion,

the increases in the titers of serotype 1- and

4-neutralizing

antibodies were similar and were more

marked than the increases in the titers of serotype 2- and

3-neutralizing

antibodies.

Thus,

it is

possible

that the infec-tion

might

be caused

by

serotype 1 or 4 virus or both.

Alternatively,

it is also

possible

that the infant may have been

separately

infected

by

both serotypes in the interim

period

between 16and20months ofage.

(iii) Infant 310. Infant310

(Fig.

lc) also

acquired

a broad spectrum ofmaternal HRV antibodies at birth. The infant

might

have

experienced

an

episode

of serotype 2 HRV infection before4months ofage sothat the levelof

neutral-izing

antibody against

that serotype did not

change

signifi-cantly,

whereas all the otherantibodiestested haddeclined

to low levels.

Subsequently,

the infant

might

have

experi-enced at least two further

episodes

of

inapparent

HRV

infection,

one

occurring

between 16 and 20 months of age which may have been caused

by

serotype 1or4 HRV orboth and another

occurring

between 20 and 24 months of age which may have been caused

by

serotype 3 virus.

HRV diarrhea. Table 1 summarizes the

virological

and

serological findings

on 19

episodes

of HRV diarrhea. The infections wereevidenced

by

isolation of the virus from the

TABLE 1. Correlation between serological and virological findingsonHRVdiarrhea

Resultfor specimenbyvirology Serological

Infantno. Hybridization result

Electropherotype withserotype(s)a (serotype)

49 IA5 2 2

131 IAS 2 2

334 IA5 2 2

234 IA6 2 2

243 IA12 ND 2

283 IA5 ND 2

76 IlAi 1 1

140 IIA21 1 1,4

163 IIA7 1 1, 4

97 IIB5 1, 4 1, 3, 4

225 IIA7 1, 4 1,4

323 IIA7 ND 1, 4

95 IIA7 ND 1, 3, 4

248 lIB9 ND 1, 4

205 1IC2 3 3

245 IID2 ND 3

298 IIA8 4 4

314 IIE2 4 4

341 IIA4 ND 4

aND, Notdone becauseofinadequatespecimens.

diarrheal stoolspecimens. Electropherotypingfurther differ-entiated the short electropherotypes usually obtained with subgroup I (designated 1) serotype 2 viruses and the long electropherotypes whichareusuallyobtainedwithsubgroup Il viruses (designated II) ofserotype 1, 3, or 4 (6, 9, 13). Fourteenepisodes occurred afterthe ageof4months,when maternal antibodies had declined to low levels. For these infants,diarrhea wasaccompanied by significant increasesin levels ofgroup Arotaviruscommonantibodyand neutraliz-ing antibodies against one or more of the HRV serotypes. Four of theseinfants(infants49, 131, 234,and 243)yielding short electropherotypes ofHRV showed significant risesin thetiters ofserotype2-neutralizing antibodybut not inthose of the other neutralizing antibodies. Thus, for these epi-sodes,virologicalandserologicalresultsconcurred,

indicat-ing

that serotype 2 HRV is the mostprobable cause ofthe disease.

All of the other 10 infants yielding long-electropherotype HRV also showed significant rises in titers of group A rotavirus common antibody in sera obtained immediately before and after the development of diarrhea. In addition, serotype 1-neutralizing antibody in one infant (infant 76), serotype 3-neutralizing antibody intwo infants (infants 205 and 245), and serotype 4-neutralizing

antibody

in another infant

(infant

298) showed the most marked rises of all neutralizing antibodies,reaching levelsinthecorresponding

postdiarrheal

serawhichwere

significantly higher

than those ofthe other

neutralizing

antibodies. These results thus allow

us todifferentiate these serotypesastheprobablecausesof these episodes. Four of the remaining infants (infants 140, 163, 225, and 248) showed significant rises in titers of neutralizing antibodies against serotypes 1 and 4, reaching levels in the postdiarrheal sera which were significantly higher than the titers of the other neutralizing antibodies. Since the titers of these antibodieswereincreasedtosimilar levels, however, it was not possible to determine whether the infection was most probably caused by serotype 1 or VOL.27, 1989

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serotype 4 virus orboth. For the other twoepisodes(infants 95 and 97), the titers of neutralizing antibodies against all three serotypes of subgroupIl viruses (i.e., serotypes1, 3, and 4) wereincreased significantly andto similar levels, all of which exceededthat ofserotype 2-neutralizing antibody bymorethanfourfold.It was notpossiblein theseinstances to determine, on the basis of serological findings, which serotype ofthe subgroup Il viruses was the mostprobable cause ofthe infection or whether the infants had had more than one episode of infection.

Five infants developed diarrhea before they were 4 months of age; two yielded short-electropherotype virus (infants 283 and 334), and the other three

yielded

long-electropherotype virus (infants 314, 323, and 341). AUl the infants had initially acquired high levels of maternal HRV antibodies.Forthe twoinfantsexcreting short-electrophero-type viruses, the titers of only serotype 2-neutralizing anti-body remained, when tested again at 4 months of age, at

levels similartothose inthe cord bloodspecimens, whereas neutralizing

antibcJies

against the other serotypes had de-creasedsignificantly andtolowlevelsbythatage. Fortwoof three infants excreting long-electropherotype virus (infants 314 and 341), the titers of only serotype

4-neutralizing

antibody remained at similar levels at4 months ofage. The titers ofneutralizing antibodiesagainst serotypes 1and 4of the other infant(infant 323)excreting long-electropherotype virus at 4 months ofage remained similarto those atbirth, while neutralizii,"antibodiesagainstthe other serotypeshad decreased to low levels by that age. These results suggest that serotype 2viruses may be the mostprobablecauses of the twoformer episodes of diarrhea,while serotype 4 virus and serotype 1 or 4 virus or

both, respectively,

were the probable cause ofthe three latter

episodes

ofdiarrhea.

When there were adequate

specimens,

we

sought

to

confirm the findings described above

by

dot

hybridization,

using as probes cDNA derived from segment 9 of the prototype rotaviruses Wa (serotype 1), S2 (serotype

2),

SA11 (serotype 3), and Hochi (serotype

4).

Under the

stringent hybridization

conditions we

employed,

these probes were specific for the serotype ofHRV from which they were derived (17). We tested

concurrently

short-elec-tt

.bpherotype

virus isolated from four ofthe six

episodes,

andall ofthemhybridized

specifically

with the S2(serotype 2)probe (Table 1).There weresix

episodes yielding

the

long

electropherotype, of whichserologicalresults

suggested

that uiie wasdue to serotype 1

virus,

two wereduetoserotype 3 virus,and three weredue to serotype 4 virus. Fourofthese isolates were tested by dot hybridization, and the results completely agreed with the

serological findings.

Serological

results were not sufficient to differentiate the serotypes of

subgroup

Il virus isolated from the

remaining

seven

epi-sodes. We testedfour ofthese

isolates,

two ofwhich were

foundtohybridize specificallywith theserotype1

probe,

but the othertwoof which

hybridized

with both serotype 1 and 4 probes.

HRVinfection in case andcontrol infants.

Asymptomatic

infections werediagnosedonthebasis of

serological findings

alone,sincewedidnot

investigate

virus

shedding by healthy

infants. On the basis of observations on overt

episodes

of infection described above, the

following

criteria were

adopted to evaluate serological findings. (i) An infection occurringafter4months ofagewasindicatedbya

significant

increase in titer of one or more of the HRV antibodies (by fourfoldormore)in theinterim4months when theinfection wasthought to have occurred.

(ii)

Aserotype(s) was consid-ered to be theprobable causeofaninfection when

neutral-izing antibody

against

that

serotype(s)

wasthe most

mark-edly

increased,

reaching

a level atthe end of the 4-month

period

which exceeded the titers ofthe other

neutralizing

antibodies

by

fourfold or more.

(iii)

A

serotype(s)

was considered to be the

probable

cause of an

episode

of infection

occurring

before4monthsofage whenthe

neutral-izing

antibody against

that serotype was sustained at 4 monthsofageat alevelsimilartothatatbirthof the

infant,

differing by

less than

twofold,

while the levels ofthe other

neutralizing

antibodies had

significantly

declined in the meantime.

By

thecriteria described

above,

wedetectedatotal of83 additional

episodes

ofHRV

infection,

33 in the diarrheal groupand50 in thecontrolgroup.

Of

102

episodes,

81were

accompanied by

a

significant

rise of

neutralizing

antibody

to oneofthe 4serotypes and21

episodes

were

accompanied

by

rises in

neutralizing

antibodiesto two or more serotypes of

subgroup

Il viruses.

Figure

2 compares HRV infections as

they

occurred in infants in the diarrheal group with the

corresponding

age- and birth

weight-matched

infants in the control group. The results show that HRV infectionswere

similarly

commonforthetwogroupsof infants. Therate of HRV

infection, including

the19

episodes

ofovert

infections,

for the two groups of infants combined was 1.34 per infant per year. Since serum

specimens

were obtainedat4-month

intervals

andbecause itwas not

possible

todetermine from the

serological

findings

whether more than one

episode

of HRVinfection had occurred intheinterim

period,

thisvalue is

likely

to be a minimum estimation of the rate of HRV infection in these infants.

Of

the 14

episodes

ofdiarrheawhichoccurredafterthe age of4

months,

7 were

probably

caused

by primary

infection

(infants 163, 205, 225, 234, 243, 245,

and

248).

Therewas no evidence to suggest that the infants had been

previously

infected with HRV.

However,

the other seven infants had hadat leastone

episode

of

asymptomatic

infectionwith the virus before

they

developed

HRVdiarrhea. Forfiveof these infants

(infants 49, 76, 95, 97,

and

140),

the viruses which caused the disease and the

preceding

asymptomatic

infec-tions

belonged

to different

subgroups

of HRV.

However,

oneofthe

remaining

infants

(infant 298)

was

asymptomati-cally

infectedwithaserotype3 virusbefore4months ofage and later

developed

diarrhea caused

by

aserotype4virusat

4.5 months ofage; both ofthese serotypes

belong

to sub-groupIIHRV.The other infant

(infant

131)

wasinfectedon both occasions with serotype 2 viruses. There were five infants who

developed

HRV diarrhea before

they

were 4

months of age. For these

infants,

it was not

possible

to

determine

serologically

whether

they

had been

previously

infectedwith the

virus,

becauseof thepresenceofmaternal antibodies.

Compared

withinfantsin thediarrhealgroup, HRV infec-tionwas

delayed

until laterages for six of the infants in the control group.

However,

the

remaining

13 control infants had hadatleastone

episode

ofinfection

by

theagesatwhich

corresponding

infants in the

diarrheal

group

developed

diar-rhea.

Development

ofHRV antibodies and HRV infections.

Fig-ure 3 compares the

geometric

mean titers of serum HRV antibodies with theoccurrence of HRV infections and diar-rheacaused

by

the virus.

All

the

study

infants

acquired high

levels of maternal HRV antibodiesat

birth,

but the

geomet-ricmeantiters of HRVantibodies

declined

rapidly,

reaching

the lowest levels

by

4 months ofage

(Fig.

3A).

Increasing

levels of HRV antibodies were evidenced

by

8 months of age, and the antibodies reached

high

levels

by

12 to 16

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HRV INFECTIONS OF INFANCY 2087

HRVdiarrhealgroup

£-,4'4.®3 ®,)

J 1D

_

DD

41 -o

A (3~

s .

4'

r'~~~~~~~-(â

Infant Birth No. MoNYr

74 3/84 3.1 78 3/84 2.8 53 2/84 3.4 85 3/84 3.5

170 6/84 3.6 146 5/84 3.5 147 5/84 3.2

155 6/84 3.0

156 6/84 3.3 299 9/84 3.5 290 8/84 3.1

307 9/84 3.7 335 9/84 3.6 268 8/84 3.2

320 9/84 3.5

327 9/84 3.6

310 9/84 2.7

289 8/84 3.0 198 10/84 3.7

Healthy controlgroup ----14

i3~~

&3

93

S~~

O

2 S

l-Dl ---Os

0-4 8 12 16 20 24

Ageuptomonths

0-4 8 12 16 20 24

Ageuptomonths

FIG. 2. Acase-control studyof HRV infectionduring infancy. Fromacohort of 371infants,19 infants whodevelopedHRV diarrhea(A) during thefirst 2yearsofinfancy (diarrheal group)wereselected. Each of themwasmatchedbybirthdate and birthweightwithoneinfant

selected from thesamecohortwho didnotdevelopthediseaseduringthesameperiod.Theserotypes(i.e., 1, 2, 3,or4)of HRVcausingthe diseaseweredeterminedby electropherotypingof the virus isolates andby serologyasdescribed forFig.1. Subclinical infections(O)were

diagnosedonthe basis ofserological findings only.

months ofagewhichwere similartothoseatbirth. Consist-ent with the development of serum HRV antibodies de-scribed above, the infants were found to be subject to a constant rate of infection throughout the first 2 years of

infancy (Fig. 3B), whereas, with one exception, all overt diseases caused by the virus occurred before 12 months of age (Fig. 3C). The rates ofovert and asymptomatic

infec-tions combined observed among infants of the diarrheal group initially exceeded the rates of infection among the

control infants, but at the end of the 2-year study period, infants in the diarrheal and control groups experienced

similar numbers ofepisodesof infection(52and50,

respec-tively).

Preexistingserumantibodyanddevelopmentof HRV diar-rhea. Figure4 compares titers of HRV antibodies in

predi-arrheal seraobtained from the infants in the diarrhealgroup

immediately before onsetof the disease with those in sera

obtained atthe same times from thecorrespondingcontrol

infants.Titers ofgroupArotaviruscommonantibodyvaried

between 1:200 and 1:196,300for cord blood specimensand between 1:40 and 1:2,800 for the other serum specimens (Fig. 4A).Three infants hadsignificantly highertiters of the

antibody in prediarrheal sera, exceeding those in the sera

obtained from the corresponding control infants by more

thanfourfold. Five infants in the diarrheal grouphad signif-icantly lowertitersoftheantibodythan did their correspond-ing controls, but for theremaining 11 case-controlpairs of

infants, the titers ofgroup A rotavirus common antibodies

for the infants in the diarrhealgroupweresimilartothose for thecorrespondingage- and birth weight-matched infantsin the controlgroup.

Titers of neutralizing antibodies against the serotypes

causing the diarrhea varied between 1:600 and 1:2,800 for

cord blood specimens and between 1:50 and 1:700 for the other serum specimens (Fig. 4B). Three infants in the

diarrhealgrouphadsignificantlylowertiters of the antibod-ies than did theircorrespondingmatchedcontrols,whileone infant in thediarrhealgrouphadasignificantly highertiter of the antibodies thandid the matched control infant. For the

remaining 15pairsofinfants, however,titers ofneutralizing

antibodies in the prediarrheal sera were similartothose in the serafrom the correspondingcontrol infants.

DISCUSSION

Wedescribedabove part ofaprospective study of infan-tile diarrhea inanurbancommunity. Byserologicalstudyof serum specimens obtained at birth and subsequently at 4-month intervals until 2 years ofage, we estimated HRV infection to occur at a minimum incidence rate of 1.34

episodesperinfantperyear. This is 22 timesmorefrequent

than theoccurrence ofovertdiseases caused by the virus. Whereas overt disease occurredprincipally between 3 and 12 months of age, we showed that HRV infection also

occurred constantly throughoutthe first 2 years of life and

probably beyond. We did not extend our study to older

children, however.

The immunestatusof the studyinfantschangedwithage.

We showed thatallof the infants testedacquired highlevels ofabroad spectrum ofmaternalantibodiesagainstthe virus atbirth. The antibodies decreased tothe lowestlevel when testedagainat4 months ofage. Presumablybecause of the constantexposureof the infantstothevirus,thelevelsof the antibodiesbegantoriseat8monthsofage,reachinglevelsat

between 12and 16 months ofagewhichweresimilar to those atbirth. Thechangingimmune status thusseemsto correlate Infant Birth

No. MoYr Kg 49 2/84 3.0 76 3/84 2.6 95 3/84 3.3 97 4/84 3.2 131 5/84 3.5 140 5/84 3.2 163 6/84 3.5 205 7/84 2.9 225 7/84 3.3 234 8/84 3.6 243 7/84 3.0 245 8/84 3.7

248 8/84 3.6 283 8/84 3.2 298 8/84 3.5 314 9/84 3.2 323 9/84 2.6 334 9/84 3.0 341 10/84 3.8 VOL. 27,1989

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(6)

(A)

0 4 8 . 12 16 20

Age (Months)

33 38 38 38 37 38

NumberofCases

(B)

inversely with the occurrence of overt disease caused by the virus at different ages. It appears that infants were initially protected to some extent by maternal antibodies against overt disease. As levels of maternal antibodies decline during early infancy, the protective role may be gradually assumed by immunity infants acquire as a result of a succession of infections with the virus. The infants seemed tohaveacquired adequate immunity againstHRVbyage 12 months such that overt disease rarely occurred thereafter. The greatest risk of the overtdisease during infancy appears to be in the interim period, after maternal antibodies had declined to low levels at 4 months of age but before the infants had acquired adequate immunity against the virus when they wereabout 1 year old. During this period, infants may benefit from protective effects ascribed to rotavirus vaccines currently beingdeveloped(8, 15).

In an earlier study ofhospitalized patientswith infantile diarrhea, we showed that levels of HRV antibodies con-tained in the serum specimens obcon-tained during the acute phaseof HRV diarrhea varied over awiderangeand thatthe levelsof theseantibodies were similar tothose in the acute serum specimens similarly obtained from control patients S who had diarrheafromother causes (16). Inagreement with these earlierfindings, weshow in the present reportthatthe levels of HRV antibodies in prediarrheal serum specimens obtained immediately before clinical onset ofthe disease also varied over awiderange. In acase-controlcomparison, the levels ofHRV antibodies in the prediarrheal sera were similar tothose in the seraobtainedatthe sametimesfrom the corresponding age- and birth weight-matched control infants who did not develop the disease. Hence, we could not associate levels of preexisting serum HRV antibodies 24 with the

development

ofovert disease.

The findings described above are at variance with the results reported by Chiba et al. (5). In a study of three

37 successive outbreaks of HRV diarrhea in an orphanage, these investigators showed that development ofovert dis-ease was correlated withpreexisting levels of serum HRV antibodies. The correlation was stronger for antibodies against the same serotype as the virus which causes the infection than for antibodies against viruses which were of

--- only the same subgroup as the infecting virus. However,

there was no evidence to suggest that antibodies against virus ofone subgroupmay protectinfantsagainst infection withviruses ofanothersubgroup.Nevertheless, the succes-sive outbreaks were causedby the sameserotype, and, on each of these occasions, the attack rate was very high, involving a large proportion of the infants interned in that orphanage. This may explain the discrepancy between the findings arising from thatstudyand those we described for the hospitalized patients (16) and the community-acquired diarrhea.

Bishop et al. (3) showed that HRV infection during the

(C) FIG. 3. Developmentofrotavirus antibodies inserumand

occur-rence ofrotavirus infection at different ages during infancy. (A) Serumspecimenswereobtained from38infantsatbirth and every 4 months thereafter until 2 yearsofage. Thespecimensweretitrated forantibody againstgroup Arotaviruscommonantigen(S)andfor neutralizing antibodies againstserotype 1 Wavirus (+), serotype 2 S2 virus

(O),

serotype3 Yovirus(A),andserotype4Hochivirus 4 8 12 16 20 24

(El),

as described for Fig. 1. The geometric mean titers of the Ageup tomonths rotavirus antibodies were calculated for the different ages. (B) CumulativeepisodesofsymptomaticandasymptomaticHRV infec-tionscombinedobservedin thediarrhealgroup(-)and the control group(----).(C) Cumulative episodesof HRV diarrhea in 19 infants. 64000

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0 100 400 1600 6400 25600 102400 409600 Titers ofgroupACommon antibodies inControls

0 100 200 400 800 1600 3200 6400 12800

Titers of Neutralizing antibody inControls

FIG. 4. Case-control study of the relationship between preexisting HRV antibodies inserumand development ofovertHRV infection. Cord blood (@ )or serum(0)specimenswereobtained from the infants in the diarrhealgroup asdescribed forFig. 3, immediately before theinfants developed thedisease. The titers ofgroupAcommonantibody (A) and neutralizing antibodies against theserotypes of HRV believedtocausetheinfection(B) in these prediarrheal specimenswereplottedontheyaxis andcompared with titers of these antibodies

inthe specimens obtained fromthe correspondingage-and birth-matched control infantsatthesametimes, plottedonthexaxis. A difference betweenthecasevalues and thecorresponding control value of fourfoldor more wasconsideredsignificant (datum points shown outsidethe solidlines).

neonatal period may protect infants against developing se-vere infection subsequently. Of the 19 episodes of diarrhea

which we investigated, 5 occurred before the age of 4 months. Itwasnotpossibletodetermine whethertheywere due to primary HRV infection, because of the presence of maternal antibodies. However, 7 of the remaining 14 epi-sodes occurring after 4 months ofage were preceded by at

leastoneepisode of subclinical HRV infection. One of these episodes was caused by the same serotype virus as that which had caused the previous infection, and another was caused by HRV belonging to the same subgroup as that

which had caused thatprevious infection. The viruses caus-ing another five episodes of diarrhea were different from thosewhich hadcaused the previous infections. The remain-ingsevenepisodes of diarrheawereprobably duetoprimary HRVinfection.Thus,we arenotabletoascertain from these results whether previous HRV infections may influence subsequent development of uncomplicated and self-limiting cases ofcommunity-acquired diarrhea caused by the virus.

ACKNOWLEDGMENTS

This workwassupported inpartbygrantsfrom theWorld Health Organization, the Croucher Foundation, and Hong Kong Jockey Club (Charity)Ltd.

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