JOURNAL OF VIROLOGY, July 2004, p. 7795–7802 Vol. 78, No. 140022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.14.7795–7802.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rotavirus Serotype G9 Strains Belonging to VP7 Gene PhylogeneticSequence Lineage 1 May Be More Suitable for Serotype G9

Vaccine Candidates than Those Belongingto Lineage 2 or 3

Yasutaka Hoshino,1* Ronald W. Jones,1 Jerri Ross,1 Shinjiro Honma,1 Norma Santos,2

Jon R. Gentsch,3 and Albert Z. Kapikian1

Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, Maryland1; Instituto de Microbiologia, Universidade Federal do

Rio de Janeiro, Rio de Janeiro, Brazil2; and Viral Gastroenteritis Section, Division of Viral andRickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia3

Received 7 November 2003/Accepted 15 March 2004

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptomsin infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 sero-types G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently,serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analy-sis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages.The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization spec-ificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which borea single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and fivelineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pigantisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strainsas well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Anti-sera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineageviruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less ef-ficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1,024-fold less ef-ficiently than the homotypic lineage 3 viruses. These findings may have important implications for the devel-opment of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) wouldcertainly be the ideal strain for inclusion in a vaccine.

Group A rotavirus is the leading cause of severe diarrhea ininfants and young children worldwide and has been estimatedto be responsible for a median of 440,000 deaths each yearamong children under 5 years of age, predominantly in thedeveloping countries (37). In the United States alone, approx-imately 500,000 physician visits, 50,000 hospitalizations, andabout 20 deaths are estimated to result among approximately2.7 million children under 5 years of age who get affected byrotavirus diarrhea yearly (3, 11, 23, 37). Although 10 VP7 Gserotypes and 13 VP4 P serotypes have been detected in hu-mans thus far (23), only 4 of 10 G serotypes (G1, G2, G3, andG4) and only 1 of 13 P serotypes (P1A[8] and P1B[4]) haveconsistently been shown to be epidemiologically importantworldwide (10, 28; N. Santos and Y. Hoshino, unpublisheddata). For these reasons, we have developed rhesus rotavirus(RRV)- and bovine rotavirus (UK)-based reassortant vaccineswhich are designed to cover G and P serotypes of epidemio-logic significance (15, 17, 31, 32).

The licensure in 1998 by the U.S. Food and Drug Adminis-tration of an RRV-based quadrivalent vaccine (RotaShield;Wyeth-Lederle Vaccines and Pediatrics, Philadelphia, Pa.) (1)was a strong impetus to establish rotavirus strain surveillanceprograms throughout the world to monitor rotavirus straindiversity before and after vaccine introduction. Thus, todaythere are various national (e.g., the U.S. National RotavirusStrain Surveillance Program and the Australian Rotavirus Sur-veillance Program) as well as regional (e.g., the African Rota-virus Network and the Asian Rotavirus Surveillance Network)groups that have been established to conduct studies on rota-virus epidemiology and disease burden (reviewed in reference27). Such studies have repeatedly shown the continuing world-wide distribution and clinical importance of serotypes G1, G2,G3, and G4 and the existence of serotypes other than G1 to G4in various parts of the world, including G5, G6, G8, G9, G10,and G12 (7, 10, 28; Santos and Hoshino, unpublished). Al-though the occurrence of such uncommon serotypes as G5, G6,G8, G10, and G12 is still focal, the G9 strain appears to bedistributed worldwide and to be clinically important (reviewedin references 7, 28, and 40). With regard to its overall distri-bution in comparison to the well-established serotypes G1 toG4, an analysis of a total of 42,757 rotavirus strains collected

* Corresponding author. Mailing address: National Institutes ofHealth, Building 50, Room 6308, 50 South Dr., MSC 8026, Bethesda,MD 20892-8026. Phone: (301) 594-1851. Fax: (301) 480-1387. E-mail:THOSHINO@niaid.nih.gov.

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globally from 108 studies from 50 countries on five continentsthat were published between 1989 and 2003 (Santos andHoshino, unpublished) indicated the relative distribution ofhuman rotavirus G types as follows: G1 (66.0%), G2 (12.1%),G4 (8.6%), G3 (3.5%), and G9 (2.7%). Although G9 viruseshave been detected in association with a variety of P typesincluding P[4], P[6], P[8], P[9], P[11], and P[19], the P[8],G9has been reported to be most widely distributed worldwide(Santos and Hoshino, unpublished). Thus, in order to “beready and prepared,” an RRV- or UK-based serotype G9 aswell as G5, G8, and G10 single VP7 gene substitution reas-sortant vaccine candidates for potential addition to a multiva-lent rotavirus vaccine were recently constructed and character-ized (17).

Serotype G9 rotaviruses were not recognized prior to 1983,when the first G9 strain, WI61, was isolated from an 18-month-old child with diarrhea at Children’s Hospital of Philadelphia,Philadelphia, Pa. (5). In that study, 5 of 59 rotavirus-positivediarrhea stools collected during the 1983 to 1984 rotavirusseason were reported to have a WI61 electropherotype. Thesecond G9 strain, F45, was isolated in Osaka, Japan, in 1985(N. Ikegami, K. Akatani, T. Hosaka, and H. Ushijima, Abstr.7th Int. Congr. Virol., p. 113, 1987). An epidemiologic surveyof rotavirus strains reported that a considerable number ofstrains similar to F45 were prevalent nationwide in Japan dur-ing the 1985 to 1986 rotavirus season (Ikegami et al., Abstr. 7thInt. Congr. Virol. 1987). However, in both the United Statesand Japan, soon after their detection, the G9 viruses becameundetectable for about a decade. Both WI61 and F45 strainscarry P1A[8] specificity. Strain 116E, which was shown to carryG9 and P8[11] specificities, was isolated from an asymptomaticneonate in India in 1986 (8, 9). In 1989, two G9 strains bearingP[19] specificity were isolated from diarrheic children in Thai-land (48). The mid-1990s heralded the detection of serotypeG9 rotavirus strains around the world: in India in 1993; in theUnited States and the United Kingdom in 1995; in Bangladesh,Japan, and Thailand in 1996; and in Malawi, Brazil, and Chinain 1997. Since then, the detection of the G9 viruses has beenreported from a number of countries on each of the five con-tinents (reviewed in references 7 and 29; Santos and Hoshino,unpublished).

This emergence or reemergence of the G9 strains hasaroused considerable scientific interest on various aspects ofthis G serotype, including the epidemiology, evolutionary ori-gin, and genetic composition of the virus as well as vaccinedevelopment against this serotype. For example, the VP7 geneof many G9 isolates has been sequenced and analyzed. Suchstudies have demonstrated the existence of at least three phy-logenetic sequence lineages among the G9 rotavirus VP7 genes(2, 19, 25, 29, 30, 35, 40, 42, 43, 52, 53). In addition, differencesin the reactivity pattern of certain G9-specific monoclonal an-tibodies (MAbs) against selected G9 isolates in lineages 1 to3 were reported (6, 25, 39, 52, 53). However, a comparativesystematic characterization of the VP7 neutralization specific-ities of G9 rotavirus strains belonging to lineage 1, 2, or 3 hasnot been made, which forms the aim of this study. This type ofstudy is essential for choosing the best G9 strain (i.e., the strainwith the broadest reactivity) as a vaccine candidate.

MATERIALS AND METHODS

Rotavirus strains, cell cultures, culture medium, virus titration, neutraliza-tion assay, and hyperimmune antiserum. Table 1 summarizes the rotavirusstrains employed in this study. Figure 1 depicts the phylogenetic VP7 sequencelineage of each of the nine G9 strains analyzed. Three lineage 1 viruses, each ofwhich bore P[8] specificity, were strain WI61 from the United States (5), andstrains F45 (Ikegami et al., Abstr. 7th Int. Congr. Virol. 1987) and AU32 (33)from Japan. Strain 116E, which was the only lineage 2 virus available, wasisolated in India and carried P[11] specificity (8, 9). Each of the lineage 1 and 2strains in this study displayed a “long” electropherotype (41). Five lineage 3viruses were strains R44 and R143 from Brazil, which bore P[9] and P[6] spec-ificity, respectively (43); strain US1205 from the United States, carrying P[6]specificity (39); strain INL1 from India bearing P[6] specificity (39); and strainBD524 from Bangladesh which bore P[8] specificity (47). Strains R44, INL1, andBD524 had a “long” electropherotype, whereas strains R143 and US1205 had a“short” electropherotype (an electropherotype displaying slow-moving gene seg-ments 10 and 11) (22). The earliest detected G9 strain (WI61) was isolated in1983, and the most recently detected G9 strain (R143) was isolated in 1999.Single VP7 gene substitution rotavirus reassortant D � UK (G1,P7[5]), used inthe present study in the construction of the G9 � UK virus reassortants asdescribed below, was an experimental vaccine suspension (lot HDBRV-1 [i.e.,D � UK]) prepared in fetal rhesus monkey lung diploid cell strain (FRhL-2)cultures (31). Primary cultures of African green monkey kidney (AGMK) cells(Diagnostic Hybrids, Athens, Ohio) or an established monkey kidney MA104 cellline was used for virus amplification, genetic reassortment, and plaque purifica-tion. The MA104 cell line was used for virus titration and plaque reductionneutralization (PRN) assay. Eagle’s minimum essential medium supplementedwith 0.5 �g of trypsin (�-irradiated trypsin; Sigma Chemical, St. Louis, Mo.) perml and antibiotics was used as maintenance medium, and Leibovitz L-15 medium(Quality Biological, Gaithersburg, Md.) supplemented with antibiotics was em-ployed when making virus or serum dilutions. The PRN assay was performed byusing 50 to 60 PFU per 250 �l of the virus as described previously (16). Agarose(SeaKem ME; BMA, Rockland, Maine) was used as a solidifying reagent in theplaque assay. Hyperimmune antiserum to each of the reassortants was raised inspecific-pathogen-free guinea pigs (Charles River, Wilmington, Mass.) whichwere free of rotavirus-neutralizing antibodies (titer of �1:20 versus AU32) asdetermined by PRN assay. Rotavirus immunogens were prepared as previouslydescribed (50). The intramuscular immunization was performed first withFreund complete adjuvant followed (2 weeks later) by two immunizations (2weeks apart) with Freund incomplete adjuvant and finally, 2 weeks after the thirdimmunization, with immunogen without adjuvant. Seven days after the fourthimmunization, guinea pigs were euthanized, and blood was collected. Sera wereinactivated before use by heating at 56°C for 30 min.

Construction, identification, and characterization of single G9 VP7 gene sub-stitution rotavirus reassortants. Roller tube cultures of primary AGMK orMA104 cells were coinfected at a multiplicity of infection of approximately 1 withhuman rotavirus strain WI61, 116E, R44, R143, US1205, INL1, or BD524 andthe reassortant rotavirus D � UK. When approximately 75% of the infected cellsexhibited cytopathic effects, the cultures were frozen and thawed once, and thelysate was plated onto primary AGMK or MA104 cells in a 6-well plate (Costar;Corning Inc., Corning, N.Y.) in the presence of G1-specific VP7 neutralizingMAb 2C9 (44) for selection of the desired UK � G9 (P7[5],G9) reassortants.Coinfection of the DS-1 (P1B[4],G2) strain and WI61 strain was also performedat a multiplicity of infection of approximately 1. The infected cell culture lysatewas plated onto MA104 cells in a 6-well plate in the presence of G2-specificneutralizing MAb S2-2G10 (46) for selection of the desired DS-1 � WI61(P1B[4],G9) reassortant. Each of the desired single VP7 gene substitution reas-sortants was selected and identified and then plaque purified three times. Theorigin of genes of each reassortant was identified by polyacrylamide gel electro-phoresis of its genomic RNAs. The origins of certain genes which were not ableto be determined with certainty by polyacrylamide gel electrophoresis werestudied further by constant denaturant gel electrophoresis as previously de-scribed (21). Generation and characterization of UK � AU32 reassortant vac-cine candidate was reported previously (17). Hyperimmune guinea pig antiserumto each reassortant was analyzed for VP7-specific antibodies to selected humanand animal rotavirus strains by 60% PRN assay (16, 50).

RESULTS

Generation of eight single VP7 gene substitution rotavirusreassortants and guinea pig hyperimmune antiserum to each

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of the eight reassortants. Since the interaction of VP4-VP7outer capsid proteins of rotavirus has been reported to affectthe expression of selected phenotypes of one or both proteins(4, 26, 38), we constructed eight single VP7 gene substitutionrotavirus reassortants, each of which had 10 genes of bovinerotavirus strain UK and only the VP7 gene of human G9rotavirus strain WI61, AU32, 116E, R44, R143, US1205, INL1,or BD524 (Fig. 2). Thus, each UK � G9 rotavirus reassortantcarried an identical genetic and antigenic configuration, exceptfor outer capsid glycoprotein VP7. We also generated guineapig hyperimmune antiserum to each reassortant for use inneutralization characterization of the VP7 protein of each ofnine G9 rotavirus strains (three lineage 1, one lineage 2 andfive lineage 3 strains) (Table 1). At least two guinea pigs wereused for antiserum production against each reassortant in anattempt to obtain a more accurate neutralization profile.

Neutralization capabilities of guinea pig hyperimmune an-tisera raised against each of the eight UK � G9 single genesubstitution reassortants. Table 1 summarizes the antigeniccharacterization of outer capsid glycoprotein VP7 of each ofthe eight G9 rotavirus strains belonging to VP7 gene phyloge-netic sequence lineage 1, 2, or 3 by analysis of their guinea pighyperimmune antiserum neutralization profile. Guinea pig hy-perimmune antisera raised against the VP7 protein of strainWI61 or AU32 which belonged to lineage 1 neutralized lineage2 and 3 strains to at least within eightfold of the homotypiclineage 1 viruses, indicating that the VP7 proteins of all nineG9 strains analyzed were closely related antigenically at leastin this one-way direction. The extent of the relatedness amongthe three lineages was analyzed further by examining the re-ciprocal neutralization specificities; antisera to the lineage 2strain 116E VP7 neutralized lineage 1 virus AU32 to withinfourfold of the homotypic value, indicating that these lineage 1and 2 viruses were similar, if not identical, in both directions,as determined by the 20-antibody rule for relatedness (14).However, antisera to lineage 2 strain 116E VP7 neutralizedlineage 1 prototype strain WI61 16- to 64-fold less efficientlythan the homotypic strain, indicating that the VP7 of the lin-eage 2 virus was distantly related to or antigenically distinctfrom the WI61 strain, suggesting that WI61 could be consid-ered the prime strain (at least with the results from one of thetwo guinea pig sera). Antisera to each of the lineage 3 virusVP7s neutralized the WI61 lineage 1 strain 128- to 1,024-foldless efficiently than the homotypic strains, indicating that theWI61 strain was the prime strain for each of the five lineage 3strains. In addition, antisera to four of five lineage 3 strainVP7s neutralized the AU32 and F45 lineage 1 strains 16- to128-fold less efficiently, indicating that the AU32 and F45strains were the prime strains for certain lineage 3 strains. Thelineage 3 R44 VP7 antisera neutralized the AU32 strain withineightfold of the homotypic strain, indicating a two-way anti-genic relationship.

Reassortant DS-1 � WI61 (P1B[4],G9) was generated foruse in analyzing whether the VP4 protein (P1A[8]) of the WI61strain affected the observed reactivity of the WI61 virus againsteach of the 16 guinea pig hyperimmune antisera tested inneutralization. The reassortant DS-1 � WI61 was shown tobehave in neutralization similar to the parental WI61 virus(Table 1), indicating that the observed neutralization charac-teristics of the WI61 were unique to the VP7 of WI61.

TA

BL

E1.

Antigenic

characterizationof

outercapsid

glycoproteinV

P7of

selectedG

9rotavirus

strainsbelonging

toV

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phylogeneticsequence

lineage1,2,or

3

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Rotavirus

characteristicsR

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PRN

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ofguinea

pighyperim

mune

antiserumto

indicatedrotavirus

reassortant(V

P7gene

lineage) b

Country

oforigin

Year

collected

Type

Etype

a

VP7

genelineage

UK

�W

I61(1)

UK

�W

I61(1)

UK

�A

U32

(1)

UK

�A

U32

(1)

UK

�116E(2)

UK

�116E(2)

UK

�R

44(3)

UK

�R

44(3)

UK

�R

143(3)

UK

�R

143(3)

UK

�U

S1205(3)

UK

�U

S1205(3)

UK

�IN

L1

(3)

UK

�IN

L1

(3)

UK

�B

D524

(3)

UK

�B

D524

(3)P

G

WI61

U.S.

1983[8]

9L

120,480

20,48020,480

40,9601,280

16080

�80

640640

�80

80�

8080

�80

160D

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9S

110,240

20,48020,480

40,960640

160160

�80

640640

�80

�80

8080

80160

F45

Japan1985

[8]9

L1

10,24040,960

40,96040,960

5,1202,560

6402,560

2,5605,120

640320

2,5602,560

3205,120

AU

32Japan

1986[8]

9L

140,960

40,96081,920

81,9205,120

2,5601,280

2,5602,560

2,560320

3202,560

2,560640

5,120116E

India1986

[11]9

L2

10,24010,240

20,48020,480

20,48010,240

640640

2,5602,560

640640

1,2802,560

6402,560

R44

Brazil

1997[9]

9L

340,960

20,48040,960

40,96010,240

10,24010,240

20,48040,960

81,92010,240

10,24020,480

81,92040,960

40,960R

143B

razil1999

[6]9

S3

40,96020,480

81,92081,920

20,48010,240

20,48020,480

81,92081,920

10,24020,480

20,48040,960

40,96081,920

US1205

U.S.

1996–1997[6]

9S

340,960

20,48040,960

40,96010,240

10,24010,240

10,24040,960

81,92020,480

10,24020,480

40,96081,920

81,920IN

L1

India1994

[6]9

L3

40,96010,240

81,92081,920

10,24020,480

10,24020,480

81,92081,920

20,48020,480

40,96081,920

40,96081,920

BD

524B

angladesh1995–1996

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1975[5]

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640320

640320

640640

640160

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320640

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640

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ntigeniccharacterization

forstrains

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VOL. 78, 2004 NEUTRALIZATION CHARACTERISTICS OF G9 ROTAVIRUS STRAINS 7797

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It was noteworthy that antisera to lineage 2 strain 116E VP7neutralized lineage 3 viruses to at least twofold of the homo-typic virus, suggesting a close antigenic relationship in thisdirection. However, in the reciprocal analysis, antisera to thelineage 3 virus VP7s neutralized the lineage 2 virus 116E 16- to64-fold less efficiently, indicating that the lineage 2 virus wasthe prime strain for certain lineage 3 viruses.

Finally, antisera to each of the lineage 3 virus VP7s neutral-ized lineage 3 viruses to a high titer regardless of the countryof origin (Bangladesh, Brazil, or the United States) or year ofisolation (1994 to 1999), indicating a high degree of antigenic

relatedness in both directions and serotypic identity by neu-tralization.

DISCUSSION

Among 10 rotavirus G serotypes found in humans (23),serotype G9 displays various unique characteristics, examplesof which are as follows. (i) Soon after its detection in theUnited States (in 1983) and in Japan (in 1985), the G9 viruseswere not reported to occur in either country for about a decadeand reemerged in the mid-1990s, and today they represent the

FIG. 1. Phylogenetic comparison of the VP7 nucleotide sequences of the G9 strains employed in this study determined by the unweighted pairgroup method with arithmetic mean program. Numbers above branches are bootstrap nucleotide percentage values. The GenBank accessionnumbers for the VP7 sequence are as follows: 116E, L140720; WI61, see reference 12; F45, see reference 12; AU32, AB045372; INL1, AJ250277;BD524, AJ250543; R44, AF438227; US1205, AF060487; and R143, AF274969.

FIG. 2. Electrophoretic migration patterns of genomic RNAs of human rotavirus strain WI61 (lane 1), reassortant UK � WI61 (lane 2),reassortant D � UK (lane 3), reassortant UK � AU32 (lane 4), human rotavirus strain AU32 (lane 5), human rotavirus strain 116E (lane 6),reassortant UK � 116E (lane 7), reassortant D � UK (lane 8), reassortant UK � R44 (lane 9), human rotavirus strain R44 (lane 10), humanrotavirus strain R143 (lane 11), reassortant UK � R143 (lane 12), reassortant D � UK (lane 13), reassortant UK � US1205 (lane 14), humanrotavirus strain US1205 (lane 15), human rotavirus strain INL1 (lane 16), reassortant UK � INL1 (lane 17), reassortant D � UK (lane 18),reassortant UK � BD524 (lane 19), and human rotavirus strain BD524 (lane 20) in 10% polyacrylamide gel. Genomic RNAs were electrophoresedat 13 mA for 15 h, and the resulting migration patterns were visualized by staining the gel with silver nitrate.

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fifth most common G serotype of clinical importance through-out the world. (ii) The VP7 gene of many G9 rotavirus isolateshas been sequenced and reported to belong to one of at leastthree VP7 phylogenetic sequence lineages. (iii) Unlike otherglobally common G1, G2, G3, or G4 strains, which are de-tected almost exclusively in conjunction with P[8] or P[4], G9strains have been detected in association with a variety of Ptypes, including P[4], P[6], P[8], P[9], P[11], and P[19]. The G9strains employed in this study, for example, bore either P[6](three isolates), P[8] (four isolates), P[9] (one isolate), or P[11](one isolate) specificity. In addition, the G9 strains have beenisolated in association with a variety of electropherotypes(short or long pattern) and subgroup specificities (I or II).Rapid global spread and successful establishment in variouscommunities of the G9 serotype may be related to this uniqueantigenic “promiscuity.”

Previously, using enzyme immunoassay (EIA), Coulson et al.(6) examined three lineage 1 (WI61, F45, and AU32) and onelineage 2 (116E) G9 strains against a panel of nine MAbsraised to lineage 1 G9 virus (WI61 or F45) VP7 and reportedthat only one of nine MAbs reacted to a high titer with the116E virus. Thus, the 116E strain was suggested to be a mono-type or a subtype of lineage 1 G9 strains. Later, two additionalMAbs to the WI61 or F45 VP7 which reacted in EIA with bothlineage 1 and lineage 2 viruses were reported to also react withselected lineage 3 G9 strains isolated in the United States (39).More recently, using the same set of G9- and G4-specificMAbs, Kirkwood et al. (25) reported that Australian G9 lin-eage 3 viruses could be grouped into three monotypes. UsingEIA, Zhou et al. (53) examined two lineage 1 Japanese and sixlineage 3 Thai G9 strains against three G9-specific MAbs (oneraised to lineage 1 F45 and the other two raised to lineage-unknown Thai G9 strain CM17-1) and reported that two lin-eage 1 strains (F45 and J830 [TK830]) reacted with all threeMAbs, whereas six lineage 3 strains reacted with two MAbsraised to lineage-unknown G9 strain but not with MAb raisedto the lineage 1 F45 strain. Thus, these studies have shown thatalthough all the G9 strains tested share common antigens,subtle differences in antigenic composition of the VP7 proteinmay exist among lineage 1, 2, and 3 strains. Indeed, althoughthe VP7 proteins of G9 strains employed in this study, forexample, were highly related to each other (�90.8% overallamino acid identity), a comparison of the amino acid se-quences in the VP7 variable regions (VRs) (some of whichhave been reported to be antigenic sites through epitope map-ping studies [reviewed in reference 23]) demonstrates the ex-istence of certain amino acid substitution(s) in such region(s)among lineage 1, 2, and 3 viruses or even in strains within thesame lineage. For example, the U.S. lineage 1 strain WI61differs from the Japanese lineage 1 strains F45 and AU32 inVR4 (D) at amino acids (aa) 70, 73, and 74 and strain AU32 inVR7 (B) at aa 149 and from lineage 2 strain 116E in VR4 (D)at aa 68, 73, and 74; in VR5 (A) at aa 100; in VR7 (B) at aa 142and 145; in VR8 (C) at aa 220 and 221; and in VR9 at aa 242(Fig. 3). However, the effect of such amino acid substitution(s)on the qualitative and quantitative nature of anti-VP7 antibod-ies as a whole has not been explored.

A titer obtained by a rotavirus neutralization assay is a sumof neutralization titer due to anti-VP4 neutralizing antibodiesand neutralization titer due to anti-VP7 neutralizing antibod-

ies, because VP4 and VP7 are independent neutralization an-tigens (13, 18, 34). For example, the neutralization titer ofanti-WI61 antiserum is a sum of neutralization titers due toanti-WI61 VP4 (P1A[8]) and anti-WI61 VP7 (G9) antibodies,and the neutralization titer of anti-116E antiserum is a sum ofneutralization titers due to anti-116E VP4 (P8[11]) and anti-116E VP7 (G9) antibodies. Therefore, in order to characterizethe qualitative and quantitative nature of anti-VP7 antibodiesof each of the eight G9 strains employed in this study, theneutralization effect of anti-VP4 antibodies needs to be elim-inated. Thus, we first constructed eight single VP7 gene sub-stitution reassortants, each of which bore a single VP7 geneencoding G9 specificity of one of the eight G9 strains (twolineage 1, one lineage 2, and five lineage 3 strains) and theremaining 10 genes of the bovine rotavirus UK strain and thengenerated two guinea pig hyperimmune antisera to each reas-sortant. The UK strain (49) was chosen because neutralizationspecificities on the UK VP4 were quite distinct from those onhuman rotavirus VP4s of epidemiologic importance (15). Eachhyperimmune antiserum thus generated contained antibodiesto the identical UK VP4 (P7[5]) and antibodies to the VP7 ofone of the eight G9 strains. With such antisera, it became pos-sible to characterize the true nature of neutralization specific-ities on the VP7 protein of each of the eight G9 strains.

As stated earlier, since overall amino acid identity of theVP7 of the eight G9 strains analyzed in this study was �90.8%,it was surprising to find that the nature and spectrum of anti-VP7 neutralizing antibodies generated by each of the eight G9strains were characteristically different (i.e., genotype-specificneutralization profiles were observed). Unexpectedly, antibod-ies to lineage 1 VP7 exhibited the broadest cross-reactivity byneutralizing all nine G9 rotavirus strains to high titers, regard-less of their VP7 phylogenetic lineages. Thus far, the G9 strainsbelonging to lineage 1 have been isolated only in the UnitedStates and Japan. Interestingly, however, in both countries,strains bearing the lineage 1 VP7 have not been detectedduring the last 7 to 10 years. Instead, all the G9 strains isolatedduring that time period in both countries have been reportedto bear lineage 3 VP7. More recently, however, the reemer-gence of the lineage 1 G9 viruses has been reported in Japan(52). Regarding the lineage 2 G9 VP7, it has never been de-tected outside of India, which may suggest its distinct evolu-tionary lineage, different from the lineage 1 or 3 VR7. Indeed,its distinctness is also reflected in the unique amino acid sub-stitutions of the VP7 (e.g., at aa 142 and 145 in VR7 [B]). Theorigin of the lineage 3 G9 VP7, which is carried by rotavirusstrains detected throughout the world since the mid-1990s andwhich is the predominant G9 strain causing diarrhea today, isunknown. Recently, by analyzing a total of 40 G9 strains iso-lated in the United States (1996 to 2001) and in India (1993and 1996 to 1998) using various assays, Laird et al. (29) re-ported that the lineage 3 G9 strains were detected as early as1993 in Indian neonates who shed viruses asymptomatically.

Two (F45 and AU32) of three lineage 1 strains tested wereisolated in Japan during the same rotavirus season; the F45 wasisolated in 1985 in Osaka and the AU32 was isolated in 1986 inYamagata. The two cities are approximately 1,200 km (750miles) apart. It is of interest that the F45 and the AU32 strainsshare an identical VP7 amino acid sequence between residues1 and 75, including mutations at aa 22, 37, 70, 74, and 75

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relative to the WI61 VP7 (lineage 1 U.S. isolate), whereas theWI61 shares an identical amino acid sequence with the F45(but not with the AU32) between residues 76 and 326. Yet theF45 exhibited a neutralization profile almost identical to thatof the AU32 against antisera to lineages 2 and 3 viruses, whichwas different from the neutralization profile demonstrated bythe WI61.

“Antigenic drift” of a virus is thought to be driven by specificimmunologic pressures exerted by the susceptible hosts. Theantihemagglutinin antibodies to type A influenza viruses, forexample, are the driving force to generate influenza virusesbearing the mutant hemagglutinin (antigenic drift in hemag-glutinin) (45). It is well known that antibodies to parentalinfluenza A viruses, from which variant influenza A virusesemerge through antigenic drift, are no longer capable of neu-tralizing the mutant viruses efficiently. Thus, each year, whenspecific mutant influenza A viruses are suspected to becomeprevalent in humans, a new vaccine that is targeted to such

mutant influenza viruses needs to be produced. In general, avaccine constructed against a mutant influenza virus can in-duce antibodies capable of efficiently neutralizing not only themutant influenza viruses but also their parental influenzaviruses. Of note is the finding in this study that antibodies tothe VP7 of lineage 1 viruses (the oldest G9 strains to be iso-lated chronologically) neutralized the lineage 3 viruses (whichemerged 10 or more years after the first detection of lineage 1viruses) as efficiently as the homologous viruses. Surprisingly,however, antibodies to each of the five lineage 3 virus VP7s didnot neutralize the lineage 1 viruses efficiently. Thus, it is un-likely that the lineage 3 VP7 evolved from lineage 1 VP7through antigenic drift. Rather, the lineage 1 and 3 VP7s aremore likely to have independent evolutionary origins. Thisassumption is consistent with the hypothesis that the contem-porary G9 strains belonging to lineage 3 may have evolvedfrom an unknown progenitor strain different from that of lin-eage 1 G9 stains (reviewed in reference 29). It is of interest

FIG. 3. Comparison of the deduced amino acid sequence of the VP7s of nine serotype G9 strains employed in the present study. Three VRs(VR1 to VR3) which are not antigenic sites are marked in black boxes. Six VRs (VR4 to VR9) and seven amino acid residues which have beendemonstrated to be involved in the formation of antigenic sites (shown as letters in parentheses) through epitope mapping studies (reviewed inreference 23) are marked in red boxes.

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that serotype G9 rotavirus strains have been detected in otheranimal species, including pigs and sheep (23). A possible in-volvement of such animal G9 strains in the evolution of humanG9 strains of lineages 1, 2, and 3 is being investigated in ourlaboratory.

Comparative sequence analysis of the VP7 gene of selectedfield isolates of epidemiologic importance has demonstratedthe existence of genetic variation and possible genetic drift ofthe VP7 genes relative to G1, G2, G3, and G4 strains employedto construct RRV-based quadrivalent vaccine (RotaShield, thefirst licensed rotavirus vaccine) (reviewed in reference 36). Jinet al. (20) analyzed neutralization capabilities of serum sam-ples collected from infant vaccinees who received RRV-basedquadrivalent or monovalent (G1) vaccine against prototype G1strain Wa (which had a VP7 sequence almost identical to thatof the strain D in the vaccine) and against a G1 strain (whichby sequence analysis was shown to belong to a phylogeneticlineage different from that of strain D) isolated from vaccine“failures,” i.e., vaccinees who experienced diarrheal episodes.They reported that postvaccination serum neutralizing anti-body titers to the Wa strain were significantly higher than thoseto the circulating G1 strain. Although no correlation was foundbetween neutralizing antibody titers to the clinical G1 isolateand protection against diarrhea in infants who got infectedafter vaccination, that study suggested the presence of anti-genic differences between the circulating G1 strain (isolatedduring the 1991 to 1992 season) and the Wa and the vaccinestrain D (both of which were isolated in 1974). Systematiccomparison between the VP7 antigenic characteristics of G1 toG4 strains present in various existing vaccine candidates andthose of the more contemporary circulating G1 to G4 strainsneeds to be explored.

As stated earlier, serotype G9 has recently established itselfas the fifth globally important rotavirus G type. For example,during the 1998 to 1999 season in Tokyo and Sapporo, Japan,the G9 strains were the most prevalent type, with an incidenceof 52.9 and 71.4%, respectively (51); and during the 2001 to2002 season in Australia, serotype G9 was the most prevalenttype nationwide (40.4%), followed by G1 (38.9%) (24). Al-though the parameters of protection against rotavirus diseasehave not been firmly established, it appears that type specificityof antirotavirus antibodies plays an important role in protec-tion against rotavirus disease; and thus, a G9 component needsto be included in the formulation of an effective rotavirus vac-cine. This study has provided a basis regarding the constructionof G9-specific rotavirus vaccine candidates; the VP7 of a G9strain capable of inducing the most broadly reactive neutraliz-ing antibodies should be utilized for the development of G9-specific vaccine candidates. In this study, we found that anti-bodies to lineage 1 VP7 neutralized not only the lineage 1strains but also the single lineage 2 strain and each of the fivelineage 3 strains to high titers, whereas antibodies to the lin-eage 2 and 3 VP7s were not as efficient in consistently neutral-izing the lineage 1 strains. In several instances, lineage 1 strainswere found to represent the clear prime strains (i.e., 20 U ofantibody to the lineage 1 strain would recognize the lineage 2or 3 strains but not vice versa). Thus, it is important to includea lineage 1 strain as the G9 representative in a multivalentrotavirus vaccine. The recently constructed G9 vaccine can-didates bear the lineage 1 VP7 gene (i.e., UK � AU32 and

RRV � AU32) (17). Safety and immunogenicity as well asefficacy of such candidate vaccines against lineage 2 and 3 G9viruses remain to be evaluated. In addition, it would be inter-esting to analyze sera from infants or young children whounderwent natural primary G9 rotavirus infections by neutral-ization against G9 viruses belonging to lineage 1, 2, or 3 to de-termine if such infections yield similar or differing responses tothose described in this study with hyperimmune guinea pig sera.

ACKNOWLEDGMENTS

We thank Monica Bur, Maria Coelho, and Dana Trageser-Cesler forexpert technical assistance and Osamu Nakagomi (Akita University,Akita, Japan) and H. Fred Clark (Children’s Hospital of Philadelphia)for providing us with rotavirus strains AU32 and WI61, respectively.The F45 strain was a gift from Nobuko Ikegami (Osaka NationalHospital, Osaka, Japan, retired).

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