Molecular evidence for naturally occurring single VP7 gene substitution reassortant between human rotaviruses belonging to two different genogroups

Arch Virol (1991) 119:67-81

_Archives

Vi rology © Springer-Verlag 1991 Printed in Austria

Molecular evidence for naturally occurring

single VP7 gene substitution reassortant between human rotaviruses

belonging to two different genogroups

O. Nakagomi 1 and T. Nakagomi 2

Departments of 1 Laboratory Medicine and of 2 Microbiology, Akita University School of Medicine, Akita, Japan

Accepted December 1, 1990

Summary. Twenty four stool rotaviruses that comprised 22 distinct electropherotypes were selected for genome analysis from the collection of diarrheal specimens obtained over an eight-year period. These 22 electropherotypes were found in 46% of the total electropherotypes identified during the previous studies and represented 328 (64%) of rotavirus specimens in the collection. When genomic RNAs from these stool rotaviruses were hybridized to the 32p_ labeled transcription probes prepared from prototypes representing three human rotavirus genogroups, Wa, DS-1, and AU-1, any one of the isolates showed a high degree of homology only with one of the three probes, which data confirmed and extended our previous observation on the existence of three distinct genogroups among human rotaviruses. Two stool rotaviruses which had an unusual combination of serotype (G1), subgroup (I) and RNA pattern (an identical short electropherotype), however, yielded the hybridization pattern indicative of an intergenogroupic single VP7 gene substitution reassortant. When they were cell culture adapted and analyzed by RNA-RNA hybridization, molecular evidence was obtained indicating that their VP7 gene derived from viruses belonging to the Wa genogroup whereas the remaining 10 genes hybridized with viruses belonging to the DS-1 genogroup. Interestingly, these natural reassortants emerged in the midst of the rotavirus season in which G1 strains predominated.

Introduction

Rotaviruses, a member of the family Reoviridae, are the single most important etiological agents of acute diarrhea of infants and young children worldwide [-25]. The genus Rotavirus has been classified into six groups (serogroups), A to F, based on group-specific antigens [5, 10, 43, 44]. Group A rotaviruses,

68 O. Nakagomi and T. Nakagomi

which constitute the majority of rotaviruses, can be assigned to at least 11 serotypes on the basis of the antigenicity of VP7, the major outer-capsid glycoprotein [3, 7, 11, 23, 29, 30, 48, 52, 53]. The serotype defined by this protein has been proposed to term G type (for glycoprotein) [11]. The other outer- capsid protein, VP4, is also involved in viral neutralization and the serotype defined by this protein is called P type (for protease-sensitive protein) [11]. Systemic nomenclature of P types has yet to be established, however [11]. Distinct from G and P types, another antigenic specificity called subgroup can be identified by enzyme-linked immunosorbent assays with monoclonal antibodies specific for subgroup antigens located on VP6, the most abundant in- nercapsid protein [20].

The genome of rotavirus comprises 11 segments of double-stranded RNA (dsRNA) which are encased within a double-shelled capsid [10, 11]. Analysis by polyacrylamide gel electrophoresis of field specimens in epidemiologic studies has revealed extensive heterogeneity in the migration patterns of these dsRNA segments [12, 25, 40], although two distinct RNA patterns are apparent, short and long, with the short patterns being characterized as an inversion in the migration order of gene segments 10 and 11 [9]. However, to what extent such diversity in electropherotypes reflects the nucleotide sequence diversity of the genome has not yet fully understood. Earlier observations by Flores et al. ]-13, 14] showed the genetic dimorphism of human rotaviruses at the molecular level by RNA-RNA hybridization assays. Thus, genomic RNAs from subgroup I rotaviruses do not form hybrids with the probe prepared from the prototype subgroup II rotavirus (strain Wa), whereas the genomic RNAs from subgroup II human rotaviruses do not form hybrids with the probe prepared from the prototype subgroup I rotavirus (strain DS-1). On the other hand, strains belonging to the same subgroup share a high degree of genetic homology.

Until recently, without exception, subgroup I human rotaviruses were found to have short RNA patterns, whereas subgroup II specificity was always as- sociated with human rotaviruses with long RNA patterns [24, 27]. The AU-1 strain isolated in 1982 was the first human rotavirus strain in which this linkage appeared to have been broken [26, 36, 37]. A number of investigators have recently reported the isolation of human rotaviruses with properties similar to those of the AU-1 strain (subgroup I, long RNA pattern) [1, 4, 6, 16, 35, 38- 42, 45, 46]. Molecular analysis by RNA-RNA hybridization assays revealed that the AU-1 and some of the similar strains are much more genetically related to each other than to human strains in other groups represented by strains DS- 1 and Wa [34, 39, 42]. Thus, three human rotavirus genogroups have been defined on the basis of genetic homology with prototype strains Wa, DS- 1, and AU-1 [34, 42].

Genetic reassortment upon mixed infections in a population has been suggested as a mechanism of rapid evolution of rotavirus genome [8]. Our recent analysis by RNA-RNA hybridization of two rotavirus strains isolated in Bang- ladesh provided molecular evidence for natural reassortants of human rota-

Single VP7 gene substitution reassortant in nature 69

viruses belonging to two different genogroups [51-1. To illustrate genetic rela- t ionships among h u m a n rotavirus field isolates and to seek further evidence of natural ly occurring intergenogroupic reassortants, we have examined by RNA- R N A hybridization 24 fecal rotaviruses that were composed of 22 distinct electropherotypes. These stool rotaviruses were selected f rom a collection of diarrheal stool specimens obtained through a series of epidemiologic studies over an eight-year period [35, 37, 39-41]. While it conf i rmed and extended our previous observat ion on the existence of three distinct genogroups, this study provided molecular evidence for single VP7 gene substi tut ion reassortants which were considered to be formed under natural condit ions between rotaviruses belonging to the DS-1 and Wa genogroups.

Materials and methods

Viruses

The following tissue-culture adapted human rotavirus strains were used in this study: Wa, G1, subgroup II [53]; KUN, G2, subgroup I [28]; AU-1, G3, subgroup I [26, 36]; P, G3, subgroup II [52]; ST3, (34, subgroup II [52]; N-l, G1, subgroup II (isolated in Bethesda, Maryland, in 1984 from a Japanese infant with diarrhea). In addition, we used genetic reassortants D x UK 47-1-1 and ST3 x UK 52-2-1 (made by K. Midthun and provided by R.I. Glass) [32] in which only VP7 gene derives from human rotavirus strain D (G1) and the remaining 10 genes from bovine rotavirus strain UK.

The electropherotypes, G types, and subgroups of 24 fecal rotavirus specimens used in this study are shown in Fig. 1. They were derived from the collection of diarrheal stool specimens obtained through the four epidemiologic studies performed previously in our laboratory [35, 37, 39-41]. Specimens were selected on the basis of distinctness of their electropherotypes and the amount of feces available for molecular studies (at least 1 g). These fecal rotaviruses comprised 22 electrophoretically distinct rotavirus strains which represented 46% of the electropherotypes identified during the previous studies. If an assumption was made that electrophoretically identical strains were descendants of a single strain, the fecal rotaviruses used in this study represented 328 of the 511 rotavirus isolates (64%) in the collection.

Preparation of dsRNA

Genomic dsRNA was extracted with phenol-chIoroform from the partially purified virions which were prepared from infected MA104 cells by pelleting them at 38,000 rpm for 3 h in a Hitachi RP42 rotor and then by sedimentation through 30% (wt/vol) sucrose at 38,000 rpm for 3 h in a Hitachi RPS40T rotor. Approximately 1 g from each of 24 fecal rotavirus specimens was extracted with fluorocarbon and processed for dsRNA by sedimentation through 30% (wt/vol) sucrose at 38,000 rpm for 3 h in a Hitachi RPS40T rotor.

Preparation of ssRNA transcripts

Single-stranded RNA (ssRNA) probes were prepared by in vitro transcription of rotavirus genomic RNA in the presence of [32p]-GTP as described previously [34].

RNA-RNA hybridization

RNA-RNA hybridization in solution was performed as previously described [34]. Briefly, genomic RNA was denatured by 2min of incubation at 100 °C, followed by quenching on


Fig. 1. a Electropherotypes, b G types (serotypes), and c subgroups of 24 stool rotaviruses selected for this study. Wa, KUN, and AU-1 are cell-culture adapted reference strains. 89Y042 and 89Y382 contained rotaviruses with an identical electropherotype. 89Y064 and

89Y067 also showed an identical electropherotype

ice for 2 min. 32p-labeled probes (20,000 cpm for each denatured dsRNA) were added; and hybridization was allowed to occur at 65°C for 16h in a buffer containing 5mM Tris acetate, 150 mM NaC1, 1 mM EDTA, and 1% sodium dodecyl sulfate (pH 7.5). The resulting hybrids were fractionated on a 10% polyacrylamide gel. The gels were stained with ethidium bromide and then exposed to X-Omat AR films (Eastman Kodak Co., Rochester, NY).

Results

When genomic RNAs from 22 field rotaviruses were hybridized with 32p-labeled single-stranded RNAs prepared from Wa, K U N (representative of the DS-1 genogroup) or AU-1, any one of the isolates showed a high degree of homology (as indicated by the number of hybrid bands) only with one of the three probes (Fig. 2). Thus, 13 field rotavirus isolates that had long RNA patterns, subgroup II specificity and any one of G1, G3, G4, or G9 type exhibited a significantly higher level of homology with the Wa probe than with either the K U N or AU- 1 probe. A band formed between 82A043 (G3) and the AU-1 (G3) probe was most likely to be the gene that codes for VP7, because our previous experiment in which a single VP7(AU-1) gene substitution reassortant AU-1 x UK-33 showed that the hybrid band formed between AU-1 and G3 human rotaviruses was the VP7 gene [36]. Five field rotaviruses that had short R N A patterns, G2, and subgroup I specificity hybridized almost exclusively with the K U N


probe. Although formation of a hybrid band was noticed between genomic RNAs from some of these viruses and the Wa probe, whether this indicated intergenogroupic exchange of genes was not clear. Three field rotaviruses which had an unusual combination of G type (G3), subgroup (I), and RNA patterns (long) hybridized almost exclusively to the AU-1 probe and not to either the Wa or KUN probe.

While homologous hybrids comigrate with the genomic RNA segments visualized by ethidium bromide staining, hybrids formed between the genes that are related but not completely homologous can be identified as aberrantly migrating bands with lesser intensity. The degree of homology among viruses belonging to the Wa genogroup appeared to be variable accordingly, since the number of hybrids formed with the Wa probe varied from six to 10. On the other hand, viruses belonging to either the DS-1 or AU-1 genogroup seemed more homogeneous (Fig. 2).

An interesting crossover pattern of the hybridization emerged when genomic RNAs from two stool rotaviruses, 89Y064 and 89Y067, which displayed an unusual combination of serotype (G1) and electropherotype (long pattern) were hybridized to the three probes described above. To confirm and further analyze the result with fecal specimens, these rotaviruses were adapted to MA104 cells by the method described previously [28] and were designated AU64 and AU67, respectively. These two cell-culture isolates showed an identical long RNA electropherotype upon coelectrophoresis of the genomic RNAs (data not shown), suggesting that they were derived from a single strain. Figure 3 showed that the Wa probe formed only one hybrid band with genomic RNAs from AU64 and AU67, whereas the KUN probe formed 10 hybrids with them. On the other hand, the AU-1 probe did not hybridize with any of the genes from these two strains (Fig. 4). A corresponding reciprocal result was obtained when the 32p-labeled probe from AU67 was hybridized with genomic RNAs from Wa and KUN (Fig. 3). The 11 hybrid bands which were formed between genomic RNAs from AU64 and the AU67 probe comigrated with the homologous (AU67 x AU67) bands (Fig. 3), suggesting again that these two viruses were derived from a single strain. The hybridization data indicated that AU64 and AU67 were derived from a naturally occurring intergenogroupic reassortant between Wa-like and KUN (or DS-1)-like viruses.

When the G-type specificity (G1) of AU64 and AU67 was correlated with the hybridization results, it was tempting to assume that the gene involved in the formation of the hybrid between the Wa probe and genomic RNAs from these two viruses was the VP7 gene (gene segments 7, 8, or 9 depending on the strain) which codes for the G-type specificity of rotavirus. The absence of the hybrid comigrating with gene segment 9 was noted when the KUN probe was hybridized to genomic RNAs from AU64 and AU67. This also suggested re- placement of the VP7 genes between Wa-like and KUN-like viruses (Fig. 3). To prove this assumption, an additional set of hybridization was performed in which the AU67 probe was hybridized to genomic RNAs from N-1 (G1),


Fig.2. Hybridization patterns obtained between the a, d Wa, b, e KUN, and c, f AU-1 probes and genomic RNAs from indicated rotavirus field isolates, a-c Pictures of ethidium bromide stained gels under UV light illumination, d-f Corresponding autoradiograms.

RNA segments are indicated to the left of each panel


D x U K 47-1-1 (G1), Wa (G1), P (G3), and ST3 (G4). As shown in Fig. 5, the AU67 probe formed a single hybrid band only with G1 strains but not with either G3 or G4 strains. The result obtained between the AU67 probe and the genomic RNAs from reassortant D x U K 47-1-1 was particularly persuasive,


Fig.3. Hybridization pattern obtained between the Wa, KUN, and AU67 probes and genomic RNAs from indicated rotavirus strains, a A picture of ethidium bromide stained gel under UV light illumination, b Corresponding autoradiogram. RNA segments are

indicated to the left of each panel

because this reassortant derives only its VP7 gene f rom the G1 h u m a n rotavirus strain D and the remaining 10 genes f rom a bovine strain U K [32]. Since genomic R N A s f rom ST3 x U K 52-2-1 (in which only its VP7 gene derives f rom ST3 and the remaining 10 genes f rom UK) [32] did not form hybrids with the


Fig. 4. Hybridization pattern obtained between the AU-1 probe and genomic RNAs from indicated rotavirus strains, a A picture of ethidium bromide stained gel under UV light illumination, b Corresponding autoradiogram. RNA segments are indicated to the left of

each panel

Fig. 5. Hybridization pattern obtained between the AU67 probe and genomic RNAs from indicated rotavirus strains and reassortant D x UK 47-7-1. G type specificity of each virus is given in the parenthesis, a A picture of ethidium bromide stained gel under UV light illumination, b Corresponding autoradiogram. RNA segments are indicated to the left of

each panel


AU67 probe, the hybrid observed between the reassortant and AU67 was interpreted as indicating homology between the VP7 gene of D and the AU67 probe. These hybridization data indicated that AU67 (and AU64) was a naturally occurring single VP7 gene substitution reassortant between viruses belonging to the Wa and DS-1 genogroups.

Discussion

Considerable knowledge of genetic relationships among human rotaviruses has emerged through the studies employing molecular hybridization assays under stringent conditions. Two earlier works, one performed in New Zealand by Street et al. [47] and the other done in the United States of America by Flores etal. [13, 14], have shown the low sequence relatedness between subgroup I human rotaviruses with short RNA patterns and subgroup II human rotaviruses with long RNA patterns. These earlier observations were confirmed and extended with a new emphasis on the possible role of interspecies transmission of rotaviruses from animals to humans when we reported a third human rotavirus genogroup represented by AU-1 which showed a significantly higher level of homology with feline rotaviruses than with any other rotaviruses belonging to previously defined two human rotavirus genogroups [42]. Thus, there are currently three rotavirus genogroups among human rotaviruses as identified by RNA-RNA hybridization [34, 42]. The number of genogroups among human rotaviruses may increase in the future because human strains apparently not belonging to any of these three genogroups have been reported [16, 38]. The fact that members of a genogroup, by definition, share a high degree of genetic relatedness but significantly less homology with members of other genogroups suggests the following. (/) There are certain sets of optimal gene constellations among rotaviruses circulating in nature. (ii) If natural reassortants are formed between rotaviruses of different genogroups, the ability of such reassortants to survive various selection pressures including their ability to compete effectively with other individuals of the population may be limited.

By analogy with influenza A virus, both antigenic "shift" which is resulted from interchange of gene segments upon coinfection with two different viruses (reassortment) and "drift" which is the slow progressive accumulation of changes in nucleotide sequence (point mutations) have been suggested as mech- anisms to explain the genomic diversity ofrotaviruses [8, 47]. Increasing number of molecular evidence has been obtained to support the hypothesis that genetic reassortment may have occurred under natural conditions [14, 21, 22, 31, 33, 51]. Scanning some examples of such naturally occurring reassortants has revealed two types of reassortment: one that occurs between viruses belonging to the same genogroup and the other that occurs between viruses belonging to two different genogroups. Some examples for the former type of reassortants are M37 [22, 33] and SB-1A [21, 33], although the latter is a porcine rotavirus. It is probable that reassortants within a genogroup may occur much more frequently than we previously thought. Their identification may be difficult,


however, by our RNA-RNA hybridization assay, because our assay does not distinguish genes that are highly homologous (less than 18% of nucleotide mismatch) 1-34]. Ward et al. [50] have recently shown that coinfection of cultured cells with pairs of human rotaviruses belonging to the Wa genogroup consistently resulted in loss of parental genotypes and selection of reassortants during multiple passages. We thereby reason that great diversity of electropherotypes observed among rotavirus field isolates are more frequently gen- erated by genome interchange among viruses belonging to the same genogroup than by progressive evolutionary divergence due to accumulation of point mutations in descendants of multiple strains. A remarkable degree of nucleotide sequence conservation of VP7 gene of strains belonging to the same serotype [15, 18, 19] also suggests that "drift" may not have occurred at least as frequently as in the case of influenza A virus.

The other type of reassortants, i.e., the ones that are formed between viruses belonging to different genogroups have been reported by Flores etal. [14], Mascarenhas et al. 1-31], and Ward et al. [51], despite the fact that such reassortants are shown by in vitro studies not to be able to effectively compete with the parental strains during multiple cell culture passages [49]. Analyses of genomes of natural reassortants indicated multiple exchange of gene segments between viruses belonging to the Wa and DS- 1 genogroups. Two strains isolated by Ward et al. were particularly well studied by RNA-RNA hybridization [51]. Thus, strain 248 appeared to inherit seven genes from the Wa-like viruses and four genes from the KUN-like viruses (representative of the DS-1 genogroup) [51]. Similarly, strain 456 appeared to derive its three genes from the Wa-like viruses and the remaining eight genes from the KUN-like viruses [51]. AU64 and AU67 reported in this study are included in this type of reassortants. Of particular interest is the finding that AU64 and AU67 were single VP7 gene substitution reassortants in which only VP7 gene derived from viruses of the Wa genogroup and the remaining 10 genes from viruses of the DS-1 genogroup. These two isolates, which were considered to be derived from a single strain because of their identical electropherotype, hybridization pattern, and serologic properties, were obtained from two hospitalized patients (20-month old and 13-month old boys) with diarrhea on the same day in the midst of the t988- 1989 rotavirus season in which G1 strains predominated (55.6%) [35]. If known incubation time of rotavirus (1-2 days) is taken into consideration, it is rea- sonable to speculate that these two patients got infected with this reassortant virus from yet unidentified source(s) rather than to speculate that reassortant formation has occurred in one of these two patients and the resulting reassortant was transmitted to the other. This reassortant hence survived competitively at least for a limited period of time with co-circulating G1 strains. This is puzzling because the emergence of AU64 and AU67 had no apparent selective advantage over the other predominant G1 strains co-circulating in the population with respect to G type specificity which is considered to be most susceptible to antibody selection pressure. This may imply that antibody selection may not


domina te evolut ionary change of rotavirus genomes. In R N A viruses other than influenza A, the emerging data suggest no evidence o f progressive antigenic drift in response to an t ibody pressure [2]. Explana t ion for evolut ion and var- iation o f rotavirus genome by analogy with influenza A virus m a y also need re-evaluation.

Acknowledgements

We thank Dr. Noriko Katsushima for her continuous efforts in collecting diarrheal stool specimens for more than a decade in Yamagata City Hospital Saiseikan, and we wish to dedicate this paper to her on the occasion of her retirement from the hospital.

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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Authors' address: Dr. O. Nakagomi, Department of Laboratory Medicine, versity School of Medicine, Hondo, Akita 010, Japan.

Received November 10, 1990

Akita Uni-

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Molecular evidence for naturally occurring single VP7 gene substitution reassortant between human rotaviruses belonging to two different genogroups