Diversity in rotavirus genomesKoki Taniguchi and Shozo Urasawa

Genome diversity has been analysed by various means for alarge number of rotavirus strains collected worldwide. Thevariation observed is thought to have resulted from theaccumulated effects of three distinct mechanisms: pointmutation, reassortment and rearrangement. Although theirhost range does not include arthropods, rotaviruses infect avariety of avian and mammalian species including humans,and interspecies transmission may have increased the rangeof variation occurring.

Key words: rotavirus / reassortment / rearrangement /variation / dsRNA structure

ROTAVIRUSES ARE MAJOR causes of acute gastroenteritisin the young of a variety of mammals and birds.Strains isolated from different, and even from thesame species show a range of antigenic and genomicproperties.1,2 There are at least seven rotavirus groups(A–G) with distinct RNA profiles, and lack of sero-logical cross-reactivity.3 In this review, only group Arotaviruses are considered, as data on variation arebest documented for this group.

The rotavirus genome has 11 segments of dsRNAwhich can be separated by PAGE. There are sixstructural proteins (the core proteins VP1, VP2 andVP3, the inner capsid protein VP6, and the outercapsid proteins VP4 and VP7) and five non-structuralproteins (NSP1-NSP5, in order of decreasing size).

Group A rotaviruses are classified according tothree antigenic specificities (subgroup, G serotypeand P serotype). Subgroup specificity is generallydefined by VP6, and four subgroup specificities (I, II,non-I-non-II and I + II) have been identified; however,VP2 also carries subgroup specificity.4 G serotype isassociated with VP7, and 14 different G serotypes havebeen reported. P serotype is defined by VP4; however,since it is difficult to differentiate the P serotypeserologically, the term ‘VP4 genotype’, based on theVP4 sequence, is also proposed as a standard for VP4differentiation.

In general, there is a definite correlation among

subgroup, G serotype and P serotype (VP4 genotype).However, numerous strains have recently beendetected which carry previously unknown combina-tions of the three antigenic specificities, and inde-pendent segregations between VP2 and VP6, andbetween VP4 and VP7, have been found.5,6 Table 1shows the relationship between G serotype and Pserotype (VP4 genotype) in representative humanand animal rotavirus strains. These results imply thatreassortment of rotavirus genome segments hasoccurred, probably as a result of intraspecies andinterspecies mixed infections, as described below.

The rotavirus genome

All 11 segments of simian strain SA11 have beencloned, and the complete sequences have beendetermined.8 The SA11 genome is 18,555 bp inlength, smaller than that of two other mammalianreoviruses, reovirus type 3 (23,549 bp) and blue-tongue virus BTV-10 (19,218 bp). The genome is AU-rich (66.1%), and the segments range in size from3,302 bp (S1) to 663 bp (S11). Once the sequence ofa segment from a given strain has been determined, alarge library of cognate sequences can be assembledby sequencing the mRNAs, readily prepared by in-vitro transcription.9,10 Sequence data thus obtainedshow a common overall structure.1,2,11,12 As with othermembers of the Reoviridae, sequences at the 5' and 3'ends are highly conserved, while segment-specific 5'and 3' subterminal sequences are also present. Theterminal sequence 5'GGC… is found in all strains, and…UGUGACC3', in most. However, compared withother reoviruses, there is more heterogeneity amongthe different segments of a given strain. The 5' and 3'terminal noncoding sequences are short, though theyvary in length among segments. No polyadenylation isfound.

It has been proposed, for wound tumor phyto-reovirus,13 that base pairing between the segment-specific inverted subterminal repeats is involved inreplication and packaging, but such sequences are notcommonly detected in rotaviruses, although a pan-handle structure and a stem double-loop structure

From the Department of Hygiene, Sapporo Medical UniversitySchool of Medicine, Sapporo 060, Japan

seminars in VIROLOGY, Vol 6, 1995: pp 123–131

©1995 Academic Press Ltd1044-5773/95/020123 + 09 $08.00/0

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involving the 5' and 3' terminal regions of S10 and S8,respectively, are found.14

Gene-protein assignments have been established byin-vitro translation, and reassortment analysis.11,12

Table 2 lists the assignments and the gene organiza-tion of each segment.

RNA electropherotype

The characteristic genome segment profile of arotavirus is readily observed in PAGE, and analysis ofsuch profiles (electropherotyping) is widely used tocharacterize rotaviruses in stools and cell cultures.15

Variation in the mobilities of individual segments hasbeen found among rotaviruses from the same as wellas from different species. Three markedly distinctprofiles (long, short and super-short) are associatedwith differences in the mobility of S11 for strains withlong patterns, and of S10 for those with short andsuper-short patterns. They represent one of the mostuseful criteria for classifying rotaviruses.

Generally, the three types of profile are wellcorrelated with subgroup and G serotype specificitiesin human strains; for example, correlations are foundsuch as short profile-subgroup I-G2 serotype, longprofile-subgroup II-G1, G3, G4 or G9 serotype, andsuper-short-subgroup I-G8 serotype. In contrast, long

Table 1. G serotype and P serotype (VP4 genotype) in human and animal rotaviruses.

VP4genotype*

Strain G serotype P serotype† Host species

1 NCDV G6 P6 CalfA5 G8 — Calf

SA11 (4FEM, 4F, 4fM) G3 P6 (Monkey)2 SA11 (4SEM, TN-L2, TN-S1) G3 — Monkey3 RRV G3 P5 Monkey

K9, CU-1 G3 — DogCat97 G3 — CatHCR3 G3 — Human

4 DS-1, RV-5, S2 G2 P1B HumanL26 G12 — Human

5 UK, B641 G6 P7 Calf61A G10 — Calf

6 M37 G1 P2A Human1076 G2 P2A Human

McN13 G3 P2A HumanST-3 G4 P2A Human

Gottfried G4 P2B Pig7 OSU G5 P9 Pig

YM G11 P9 Pig8 KU, Wa G1 P1A Human

P, YO G3 P1A HumanVA70 G4 P1A HumanWI61 G9 P1A Human

9 K8 G1 P3A HumanAU-1 G3 — Human

Cat2, FRV-1 G3 — Cat10 69M G8 P4 Human11 B223, A44, KK3 G10 P8 Calf

116E G9 P8 HumanI321 G10 P8 Human

12 H2, FI-14 G3 — HorseFI23 G14 — Horse

13 MDR13 G3 — Pig14 Mc35 G10 P3B Human15 Lp14 G10 — Sheep16 Eb G3 P10 Mouse17 993/83 G7 — Calf18 PA169

HAL1166G6G8

P11P11

HumanHuman

19 L338 G13 — Horse

*VP4 genotype was determined by comparative VP4 amino acid sequence analysis. VP4 genotype number described in the previous paper7

is slightly modified in this table.†P serotype was indicated only when the strain was characterized by reciprocal or one-way cross-neutralization assays.

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profile-subgroup I linkage is found mostly in strains ofnon-human origin. It is impossible to predict the Gserotype from segment mobilities other than that ofS11, though the S7-9 complex appears to migratemore slowly in G3 serotype strains.

Electropherotyping has been used to identify indi-vidual strains and to study rotavirus epidemiology, andsuch analyses have led to the following conclu-sions.15-18 (1) During the rotavirus epidemic season(winter) in a community, several strains with differingelectropherotypes cocirculate. (2) Within a givenrotavirus season, diversity increases as the seasonadvances. (3) One electropherotype predominates fora year or two, then there is a major shift in pattern. (4)Continuous changes in segment mobility occurduring the course of an epidemic. (5) The strainpredominant in one season sometimes occurs in a‘herald’ wave during the previous season.

Electrophoretic diversity is assumed to stem mainlyfrom point mutations. In some instances,reassortment and rearrangement are involved, asdescribed below. Even a single base substitutioncan alter segment mobility, and a likely cause ofthis is the resulting change in secondary structure.19

PAGE analysis on concentrated (12.5-17.5 %) gelsgives improved resolution, and reveals the co-existence of virus populations with minute vari-ations within a single broader electrophero-type.20

Although electropherotyping has thus proved veryilluminating, profiles produced under different con-ditions of buffer or gel strength give differentresolutions and mobilities, so comparisons should bemade with caution. In addition, there is little associa-tion between electropherotype and severity orduration of the symptoms caused.

Table 2. Rotavirus (strain SA11) gene coding assignments.

Segment No. of non-codingbases (5/3)

Total length (bp) Product Molecular weightof protein

(no. of amino acids)

Remarks

1 18/17 3302 VP1 125,128 (1088)

Inner core protein, RNA polymerase

2 6/28 2690 VP2 102,698(881)

Inner core, protein, myristylation,RNA binding, leucine zipper

3 49/35 2591 VP3 98,142(835)

Inner core protein, guanylyltransferase.

4 9/22 2362 VP4 86,775(776)

Surface protein (dimer), hemagglutinin, P serotypespecificity, proteolytic cleavage, virulence,virus entry

5 30/93 1611 NSP1(NS53)

58,484(495)

Nonstructural, zinc finger, assembly,RNA binding, association with cytoskeleton

6 23/139 1356 VP6 44,903(397)

Inner capsid protein (trimer), myristylation,subgroup specificity

7 25/131 1104 NSP3(NS34)

36,072(312)

Nonstructural, RNA replication, RNA binding

8 46/59 1059 NSP2(NS35)

36,629(317)

Nonstructural, RNA binding, 10S multimer,complex with RNA polymerase, RNA packaging

9 48/33 1062 VP7 37,198(326)

Surface protein, glycosylation, G serotype specificity,signal peptide sequence, Ca2+ binding,cell attachment

10 41/182 751 NSP4(NS28)

20,309(175)

Nonstructural, glycosylation, association with theincrease in intracellular calcium, budding intoendoplasmic reticulum

11 21/49 667 NSP5(NS26)

21,772(198)

Nonstructural, phosphorylation

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Mechanisms generating genome diversity

As found with influenza virus, which also has asegmented genome,21 variation in rotaviruses appearsto involve three different mechanisms, point muta-tion, reassortment and rearrangement. These arediscussed below.

Point mutation

Sequential accumulation of point mutations has beenobserved in isolates obtained during a single epidemicseason, as shown by oligonucleotide mapping andsequencing.22,23 However, the rate of base substitutionper position per virus generation in vitro or in naturehas not been reported for rotaviruses, except that thefrequency of appearance of variants resistant toneutralizing monoclonal antibodies in vitro has beenestimated as approximately 10–5, comparable to ratesin other RNA viruses. Each segment may be subject todifferent immune or host-selection pressures andconstraints, resulting in different rates of variation. Incontrast to the situation with plant reoviruses inuniform crops, immunity of individuals and thecommunity are important factors influencing theselection and perpetuation of variants. The surfaceproteins VP4 and VP7 are affected by host neutral-izing antibodies, and consequently the genes encod-ing these proteins are expected to evolve much morerapidly than those of internal structural proteins. VP7has six hypervariable regions which are associatedwith G serotype specificity. Resistant mutants obtainedby culturing the virus in the presence of monoclonalantibodies carry substitutions resulting in amino acidchanges in these hypervariable regions. Strains withthe same amino acid substitutions as those in themutants obtained in vitro have been detected innature; they are called monotypes,24 and are found inmost G serotypes, particularly G3.

A similar situation has been found for VP4. In thehuman strain L26, this has an amino acid change atposition 392, identical to that of an artificially inducedmutant resistant to a particular neutralizing mono-clonal.25 L26 may have derived from strain L27 bypassing through an individual expressing the antibodythat recognized the epitope containing that residue.

Other examples exist where point mutations pro-duce detectable changes in phenotype. Replacementof only a few amino acids in VP4 of several SA11clones will change plaque size, and virulence insuckling mice,26,27 (Taniguchi et al, unpublished

data); however, no single amino acid change responsi-ble for the altered phenotype has been identified.

Internal structural proteins or non-structural pro-teins are generally more stable because of structuraland functional constraints and lower exposure toimmune selection. Indeed, homology among differ-ent isolates in the genes encoding these proteinsappears to be high, although the number of suchgenes so far examined is limited.

In contrast, the NSP1 gene shows great diversityamong strains from different species, and the propor-tion of silent to total nucleotide changes is high. Thisgene has evolved into host-specific lineages,28 (Kojimaet al, unpublished results), so it is probably subject tohost-specific constraints. Host-specific selection is alsofound in the genes coding for the outer proteins VP4and VP7.29

Tissue-specific variation is also conceivable. Inimmunodeficient children and animals, rotavirusesmay replicate in the liver. In the Wa variant multiplypassaged in HepG2 liver cells, there are eight basechanges resulting in five amino acid changes in thecoding region of the VP4 gene, compared to theparental Wa strain.30 Rotavirus infection in neuronsor in the respiratory tract has also been reported,31,32

and may well involve mutations, although no one hasyet analysed the sequences of isolates with abnormaltissue tropisms, occurring in different tissues of asingle individual.

More systematic sequence analysis of strainsobtained from epidemics, cell cultures or experi-mental animals is required for a precise calculation ofmutation rates for individual rotavirus segments.Development of phylogenetic trees for each segmentwould also establish the extent of their divergentevolutionary paths.

Reassortment

When cells are infected with two different butcompatible rotaviruses, a high percentage of progenycontain novel assortments of gene segments.33 Inother viruses with divided genomes, this process is alsoknown as pseudorecombination. New virus strains canreadily arise, and evolution occurs by such means,which is probably most efficient when the coinfectingviruses are closely related. Coinfection with pairs ofsubgroup II human rotaviruses has been shown toreadily yield reassortants; for example, nearly 50 % ofthe progeny of G serotype 1 and 3 viruses carryingsubgroup II specificity were reassortants.33 The prog-eny of coinfection with strains from the more distantly

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related subgroups I and II yielded far fewerreassortants.34-36

The 5' and 3' noncoding regions are suggested tobe responsible for assembly of the genome segments;sequence divergence in these regions would thengreatly affect reassortment efficiency. In fact, noreassortants have been detected between strains ofdifferent groups (A to G) or between rotaviruses andother members of the Reoviridae. In contrast, theavian rotavirus strains Ch2 and Ty-1, which have themost distinct sequences from other group A strains,especially in the terminal noncoding regions, canreassort with simian rotavirus.37 One reassortant(TyRh) formed between avian Ty-1 and simian RRVpossessed 10 segments of Ty-1 and only S4 of RRV.

Reassortment in mice has also been shown, aftercoinfection with high doses of parent viruses.38 Twelvehours after infection, 25 % of the progeny werereassortants, and this percentage increased to 80–100% within 72–96 h postinfection. There was nearlyrandom distribution of most segments, but segments3 and 5 of SA11 were preferentially selected. In in-vitro experiments also, certain constellations of seg-ments were repeatedly isolated. The close associationsamong electropherotype, subgroup, G serotype and Pserotype imply that at least in nature the segments ortheir encoded proteins are interdependent. Thisresults in coselection of certain groups of segmentsduring reassortment formation.

Recently, however, more and more rotavirus strainswith unusual phenotypic combinations (such as sub-group I-G3-long RNA pattern, or subgroup II-G2-longRNA pattern) have been isolated. Furthermore,strains with G or P serotype specificity, normallylimited to a given animal species, have been isolatedfrom different species.39-48 RNA–RNA hybridizationassays and Northern blots showed clearly that some ormost segments could hybridize with those of strainsfrom different species. This suggests the occurrenceof natural reassortment between viruses even fromdifferent species. Thus, reassortment frequencies arehigher than previously expected. Nevertheless, exam-ples of naturally occurring reassortants are still few, incontrast to the ease of producing them experimen-tally. In nature, some limiting mechanism may be atwork.

Natural reassortants appear to turn up more fre-quently in developing countries. Lower levels ofhygiene and poorer immunological defences ininfants, due to malnutrition, appear to encouragemixed infections and hence more reassortment.There is also closer contact among humans, livestock

and other animals in such countries. We saw muchhigher frequencies of mixed infection in infants andeven in adults after the devastating floods of1988–1989 in Bangladesh, compared with levelsbefore the floods.49 So far, however, prospectivesurveillance for rotavirus infection/diarrhea in farmworkers, their family contacts, and their farm animalsin rural Panama, has shown no correlation betweeninfection rates in these groups.

Reassortment has played a major role in identifyingviral genes responsible for several biological pheno-types. It is interesting that the recipient virus’s geneticbackground, onto which the genes of a donor virusare imposed, can alter the expression of donorgenes.50 Consequently, the phenotypes of reassortantsmay be unexpected in such a case.

With influenza virus, reassortment is an importantmechanism behind the appearance of pandemicstrains in human or animal populations. Such adramatic situation has not been observed with rota-viruses, but there are some examples of new virusescausing epidemics. Strain K8, a natural reassortantbetween the Wa and AU-1 groups, caused an outbreakin school children,51 and Philippine strains such asL26 and L27, which possess new G serotype specificity,have been the major strains prevailing in one commu-nity for several years.25 There is no denying thepotential for more virulent rotavirus strains to emergeas a result of this mechanism.

Rearrangement

The term ‘rearrangement’, perhaps unfamiliar toplant virologists, usually means alterations of con-siderable tracts of sequence within single genomesegments, sometimes in the form of deletions, andoften as duplications. The term does not indicaterecombination between fractions of separate genomesegments, which appears to be still a further possibil-ity, not so far documented in practice.

Electropherotyping shows up genome rearrange-ments when some segments go missing from theirnormal position, and additional bands appear withdifferent mobilities. Numerous viable isolates withaltered segments have been described.52-62 Rearrange-ment was first detected in viruses from immunocom-promized humans,52,54 and later in immunocompe-tent humans with diarrhea and from domesticanimals. Serial passage of bovine rotaviruses at highmultiplicity has also led to isolation of variants withgenome rearrangements;53 these have been observedfor S5-8, 10, and 11, all of which except S6 encode

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non-structural proteins. Rearrangement is most oftenseen in S11.

Recently, the sequences of some rearranged seg-ments have been determined. In most cases, there arepartial duplications of the coding region, includingsome nucleotides downstream of the terminationcodon. The rearranged S11 found in strains with shortRNA patterns has a long AU-rich sequence (about 150

nt) inserted in the 3' noncoding region immediatelyafter the termination codon. Segment length is thusincreased but the original ORF is maintained,although abnormally large proteins are produced in afew strains. The 5' and 3' noncoding regions are alsoconserved. Figure 1 shows the structure of two suchsegments before and after rearrangement. Calcula-tions based on the increased size of some rearranged

Figure 1. Structures of rearranged rotavirus genome segments containing partial ORFduplications. (A) Example of a simple rearrangement found in S10 of an isolate from a chronicallyinfected immunodeficient child.59 This type of rearrangement can occur in most segments. Inmany cases, point mutations are detected in the upstream copy of the ORF, resulting in amino acidchanges or the appearance of a nonsense codon. (B) Example of a complex rearrangement foundin S11 of a sheep rotavirus (strain Alabama) with a super-short RNA profile.57 The rearranged genehas acquired a second initiation site, and potentially a second small ORF.

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segments suggest that up to 2000 additional base pairs(representing 12 %) can be packaged without affect-ing viability.

None of the variant rotavirus genomes analysedappear to be mosaic structures (recombinations, sensustrictu) composed of sequences from more than onesegment, or containing sequences of the host cellgenome, although such examples are known ininfluenza virus.63

Several rearrangement variants with altered pheno-type have recently been isolated,60-62 (Taniguchi et al,manuscript in preparation). A bovine rotavirus (strainbrvA) has a rearranged S5 in which there is duplica-tion of part of the ORF.60 A point mutation at nt 808produces a nonsense codon near the middle of theupstream copy of the ORF, which then encodes atruncated NSP1 of 258, instead of the original 491amino acids. A further variant (brvE) has a rear-ranged S5 expressing an enlarged NSP1 with 728amino acids. Both variants have altered phenotypes,giving 9–60-fold lower yields in one-step growth, and2.5–100 fold reductions in mean plaque size.

We have also isolated a non-defective bovine rota-virus variant (A5–16) with very small plaque size. It hasa 500 bp deletion in S5 that eliminates a sequencecoding for a cysteine-rich tract important for RNApackaging and replication (Taniguchi et al, manu-script in preparation). The deletion also causes aframeshift resulting in a nonsense codon at nt183–185. Further, a sheep rotavirus carries an S6 inwhich the normal ORF is followed by a partialduplication beginning 23 nt after the terminationcodon.62

An explanation for these observed partial duplica-tions would be that the RNA-dependent RNA poly-merase may fall back on its template at the regiondownstream from the termination codon and retrans-cribe to the end.64,65 Indeed, sequence repeats ofseveral nucleotides, compatible with such a mecha-nism, are detected in the junction and reinitiationsites. Examination of more rearranged genes couldthrow light on the mechanisms of RNA transcriptionand replication.

Prospect

Accumulated data from sequences and electrophero-grams have revealed considerable variation amongrotaviruses. Such variation, while presenting a majorobstacle to the development of vaccines, is of greatinterest in studying the ecology and evolution of these

viruses; it may also illuminate the mechanisms behindthe generation of diversity. Sequences of internal andnon-structural proteins should also be analyzed formore strains, in order to better define the functions ofthese proteins in replication, symptom developmentand ecological behavior.

Although such studies are useful, an importantdevelopment would be a system for introducinggenetically engineered nucleic acid into viable virusparticles. This would revolutionize rotavirus studies.Recent establishment of template-dependent in-vitroreplication of rotavirus RNA66 seems promising.

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