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J. gen. Virol. (1988), 69, 149-162. Printedin Great Britain 149 Key words: rotavirus, human/reassortment/selection Reassortant Formation and Selection Following Coinfection of Cultured Cells with Subgroup 2 Human Rotaviruses By R I C H A R D L. W A R D * , D O U G L A S R. K N O W L T O N AND P E I - F U N G L. H U R S T James N. Gamble Institute o f Medical Research, 2141 Auburn Avenue, CincinnatL Ohio 45219, U.S.A. (Accepted 24 September 1987) SUMMARY Reassortant formation following coinfection has been suggested as a mechanism of evolution of rotaviruses. This study was designed to examine the selection of reassortants following coinfection of cultured cells with pairs of subgroup 2 human rotaviruses. The three pairs studied (Wa × P, CJN × 31, 62 x 69) were chosen to maximize the number of RNA segments that could be electrophoretically distinguished. After coinfection and multiple passages, reproducible selection of reassortants was observed with each pair. Although more segments were selected from the virus of a pair that grew to higher titre, certain segments were selected independently of the relative growth properties or multiplicities of infection of the coinfecting viruses; selection of other segments was dependent on both. In determining the time and cause of selection it was found that no selection of genomic RNA segments was detectable prior to or during viral particle assembly in coinfected cells. However, selection was evident within the infectious progeny population after a single cycle of replication. Therefore, selection of specific reassortants following coinfection was apparently due to differences in the infectivities of progeny viruses and not in their assembly. This implies that these infectivities were a function of the parental origin of specific genomic segments. INTRODUCTION The genome of rotaviruses is composed of 11 segments of double-stranded RNA. Structural variations in individual segments from different rotavirus strains cause differences in the electrophoretic mobilities of these segments. Electropherotypes of rotaviruses are used to characterize individual strains. Coinfection of a cell with two rotaviruses of different electropherotypes results in progeny viruses carrying genome segments from both parents (reassortants) as determined by electrophoretic mobilities, a phenomenon that is typical of viruses with segmented genomes. It has been observed that multiple rotavirus electropherotypes can circulate within a community during a given season (Estes et al., 1984). At times, 10~ or more of the clinical specimens contain more than 11 rotaviral segments (Spencer et al., 1983; Nicolas et al., 1984; Rodriguez et al., 1983). One explanation for this observation is that persons from whom they were isolated were coinfected with two or more rotavirus strains. If stable reassortants are formed during coinfection, new strains of rotavirus could evolve. Certain human and animal rotavirus strains appear to have originated in this manner (Hoshino et al., 1987; Midthun et al., 1987) and formation of viable reassortants has been demonstrated following coinfection of mice with two strains of animal rotavirus (Gombold & Ramig, 1986). Reassortants have also been formed between human rotaviruses after coinfection of cultured cells (Garbarg-Chenon et al., 1984, 1986; Urasawa et al., 1986) but little is known concerning their competitive growth properties with one another or with the parental strains from which they were derived. 0000-7944 © 1988 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 150 R.L. WARD, D. R. KNOWLTON AND P-F. L. HURST T h e first purpose o f this study was to d e t e r m i n e w h e t h e r r e p r o d u c i b l e s e g m e n t selection occurs after c o i n f e c t i o n of cultured cells w i t h different pairs of h u m a n rotaviruses and w h e t h e r changes in the g r o w t h properties or multiplicities of infection of the c o i n f e c t i n g viruses will alter the selective process. T h e m e t h o d used to p r o m o t e selection o f reassortants w i t h the best g r o w t h properties was to c o i n f e c t cells w i t h plaque-purified strains o f h u m a n rotavirus and carry out m u l t i p l e blind passages of the c o i n f e c t e d cultures. T h e selected p r o g e n y w e r e t h e n identified by electrophoretic analyses. O n c e it was established t h a t reproducible selection of reassortants occurred u n d e r these c o n d i t i o n s for e a c h o f three different pairs o f h u m a n rotaviruses, the cause o f selection was d e t e r m i n e d . It is a n t i c i p a t e d t h a t the selective processes o b s e r v e d in these e x p e r i m e n t s will serve as models for those that take place after c o i n f e c t i o n o f humans. METHODS Viruses and cells. Six strains of human rotavirus were used in this study. All six were shown to belong to subgroup 2 by an ELISA using monoclonal antibodies to subgroup-specific epitopes, a gift of Y. Hoshino, National Institutes of Health, Bethesda, Md., U.S.A. Strains Wa and P, prototype strains for serotypes 1 and 3, respectively, were provided by R. Wyatt, N.I.H., Bethesda, Md. Each was grown for approximately 10 passages in MA-104 cells obtained from M. Estes, Baylor College of Medicine, Houston, Tex., U.S.A. Before use in these experiments, both viruses were plaque-purified three times and stock preparations were stored at - 70 °C. The titres of these preparations were 2.3 x 107 (Wa) and 1.1 x 107 (P) p.f.u./ml as measured on MA-104 cell monolayers. The other four strains of human rotavirus were culture-adapted in our laboratory from stools of infected children using methods already described in detail (Ward et aL, 1984). Each was plaque-purified three times after six to 15 passages in cell culture and stock preparations were grown in MA-104 cells before storage at - 70 °C. The titres of these frozen stocks were 7-5 x 10s (CJN), 3-4 x l06 (no. 31), 1.0 × l06 (no. 62) and 1.5 x 106 (no. 69) p.f.u./ml. After 17 additional passages of the CJN virus, a new stock preparation made from an isolate plaque-purified three times had a titre of 2.5 x l0 T p.f.u./ml. Serotypic relationships of these viruses to prototype human strains were determined by plaque neutralization using hyperimmune guinea-pig antisera to Wa (serotype 1), DS-1 (serotype 2), P (serotype 3) and ST-3 (serotype 4) rotaviruses. As previously reported (Ward et al., 1986), CJN was found to share a weak serotypic relationship with all four prototype viruses and strain 31 was found to share a weak serotypic relationship with Wa virus. Strains 62 and 69 were found to belong to serotypes I and 3, respectively. Coinfection and multiple passages withpairs of human rotaviruses. Coinfection with Wa and P, CJN and strain 31, and strains 62 and 69 were performed in tube culture. For this, confluent MA-104 monolayers (4 to 5 days old) were washed three times with Earle's balanced salt solution and inoculated with predetermined concentrations of each virus. The tubes were rolled during a 1 h adsorption period (37 °C) and 2 ml of medium [improved MEM, Richter's modification (Irvine Scientific, Inc., Ca., U.S.A.)] with antibiotics and 2 ~tg/ml trypsin (Gibco, 1:250) was added. Incubation while rolling was continued at 37 °C until significant cytopathic effect was observed (1 to 2 days). The lysates were frozen, thawed and 0.2 ml was used to inoculate tube cultures to make additional passages in the same manner. Control tubes infected with the individual parental viruses were treated in an identical fashion and passaged along with the coinfected cultures. Electrophoretic analysis of viral RNA segments. Tube cultures of rotavirus-infected cells were frozen and 1 ml of the entire lysate was centrifuged at 190000g for 30 min. Pelleted viruses were resuspended in 0.2 ml of 0.1 MNaCI, 0.01 M-Tris-HC1 pH 7.5, 0.001 M-EDTA (STE buffer) with 1~ SDS and extracted with equal volumes of redistilled phenol and chloroform :isoamyl alcohol (25 : 1). After centrifugation (10000 g, 10 rain), the upper phase was collected and mixed with 4 vol. of ethanol. Samples were stored overnight at - 20 °C and RNA was pelleted by centrifugation (10000g, 10 rain). The pellet was dried, then dissolved in distilled water and mixed with an equal volume of 2 x electrophoresis sample buffer (0.125 M-Tris-HC1 pH 6-8, 40~o glycerol, 0-2~o SDS). Slab gels (10~ acrylamide, 0.27~ bisacrylamide) with stacking gel (4~ acrylamide, 0.16~ bisacrylamide) were prepared according to Laemmli (1970) and 20 ~tl of sample was added per slot. Electrophoresis was performed at a constant current (16 mA/gel) for 18 h at room temperature. Fixation and staining of gels was performed by a modification of the method of Bohl e t at. (1984). Gel slabs were fixed with 10~o ethanol, 0.5 ~ glacial acetic acid (three changes, 30 min each) and stained for 2 h in 6.3 mM-silver nitrate. After two washes with distilled water and one with developer (0-75 w-NaOH, 0-037~ formaldehyde), gels were allowed to develop until RNA bands were clearly visible. Developer was then washed from the gels with 5 ~ acetic acid prior to being photographed. RESULTS Selection o f reassortants by multiple passage following coinfection T h e h u m a n rotavirus strains chosen for this study were all s u b g r o u p 2 and w e r e classified as long electropherotypes. T h r e e pairs o f viruses were used ( W a and P, C J N and 3 l , 62 and 69), Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 Rotavirus reassortant selection in vitro P + Wa CJN + CJN 31 Wa 1 1 2,3 2,3 4 151 31 62 62 + 69 69 1 1 1 2,3 2 3 3 2 4 4 4 4 7,8 9 5 5 5 6 6 6 5 6 7 8 7 7 7 9 8 9 8,9 10 8 9 10 11 10 10 10 11 11 11 10 11 11 Fig. 1. Electrophoretic pattern of genomic RNA from the three pairs of human rotaviruses used in coinfection. Segments7 and 8 of CJN and strain 31 and segments 2 and 3 of strains 62 and 69 migrate in reverse order as determined by analysis of individual reassortants (see text). They are numbered according to the migration order of CJN and strain 62. each pair chosen to maximize the number of RNA segments that could be electrophoretically distinguished. In all three combinations, at least 10 out of the 11 sets of segments were sufficiently separable to be distinguished by standard electrophoretic techniques (Fig. 1). Those not separable were segment 6 of the Wa and P strains and segment 11 of the CJN and 31 strains. It should be noted that certain segments were not always totally resolved during coelectrophoresis of RNAs from these three virus pairs. However, these segments could be readily distinguished when electrophoresis of the pairs was conducted in adjacent lanes. The segments whose ideritification was confirmed in this manner included 7 and 11 of strains Wa and P, segments 9 and 10 of CJN and strain 31, and segments 1, 2, 3, 6, 7 and 11 of strains 62 and 69. Although segment 3 of strains 62 and 69 could not be resolved, this segment was shown to be functionally different for these two viruses as will be discussed below and has been appropriately labelled in Fig. 1. Because of this and the fact that segment 2 of these viruses could be distinguished permitted identification of the parental origin of both segments 2 and 3 of strains 62 and 69. The m.o.i, for each virus used in the initial coinfections were 1, 0.2 and 0.2 for Wa x P, CJN x 31 and 62 × 69, respectively. Blind passages were made thereafter. Duplicate cultures were treated in the same manner in order to determine whether the selection process was reproducible for each set of viruses. Passages were made for each set until the electrophoretic profiles of their RNAs did not undergo further changes during several additional passages. Coinfection followed by multiple passages resulted in total or almost total loss of at least one segment from each of the parental strains (Fig. 2). In every case, the parental strain that grew to Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 152 0 q~ ~+~: e/'l °~' ...... .........Ilt ................ 1111 i: ~;i:ii~;I'#I ~+:~:~: III t l i:i i HI i,I | . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " .......... ~.+~ Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 ~i~ !~ ~ ~! Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 62 (c) 62 + 69 69 1 2 3 4 5 6 7 13 62 62 + 69 69 Fig. 2. Electrophoretic profile of genomic RNA from viruses selected during serial passage following coinfection with three pairs of human rotaviruses. Each pattern consists of the individual or mixed parental virus profiles at both ends of the gels as labelled and coinfected cultures in the middle portions with the passages indicated by the numbers. (a) Wa x P; (b) CJN × 31; (c) 62 x 69. U. © 154 R.L. W A R D , D. R. K N O W L T O N A N D P - F . L. H U R S T the higher titre in cell culture, i.e. Wa, 31, 69 (see Methods), contributed the majority of the segments in the selected reassortant population. After 18 passages following infection with Wa and P rotavirus, the only P segments that appeared to dominate the final population were 2 and 9, and segments 3, 4 and 10 of the P strain were undetectable (Fig. 2a). Thirteen passages after coinfection with CJN and 31 strains, segment 5 was almost totally derived from CJN (Fig. 2b). The only other CJN segment detectable in the gel patterns was segment 6. The same number of passages following coinfection of 62 and 69 strains resulted in progeny with segment 10 only from 62, along with portions of segments, 1, 2, 4, 8 and 9 (Fig. 2c). Additional faint bands were observed in Fig. 2(b) and in the section of this gel pattern shown in Fig. 1. Because of their regularity in relation to the main bands, they were probably due to unequal migration of segments in a portion of the gel. They were not observed in other gels of the same materials. In order to determine whether the selection process found for each of the three pairs of rotaviruses during the passages following coinfection was reproducible, the experiments were repeated in quadruplicate. The same selective process was observed as noted from the results shown in Fig. 2. Furthermore, when the initial coinfection and second passage were made in primary African green monkey kidney cells followed by additional passages in MA-104 cells, no differences in the selection of segments was observed. Thus, the selection process observed under these conditions was reproducible and characteristic of the viruses used in the coinfection. Individual progeny viruses obtained after multiple passages following coinfection with each pair of rotaviruses were examined next in order to determine (i) whether the selection of segments from each parent occurred as predicted based on the total population shown in Fig. 2, and (ii) whether the selected RNA segments were distributed independently within the individual progeny. Twenty progeny viruses from each of the three coinfections were plaquepurified after multiple passage and their electropherotypes were determined. The proportion of segments derived from each parent (Table 1) was consistent with that predicted from the total segment population (see Fig. 2). Furthermore, the individual R N A segments selected appeared to reassort independently within the progeny viruses (Table 2); i.e. no significant association was found between any two segments (P > 0.05) as determined by the method of Lubeck et al. (1979). However, many more reassortants would have to be analysed to confirm this observation. It should be noted that the parental genotypes were not present within the 60 progeny viruses picked at random for this analysis. Therefore, all 60 were reassortants. Analysis of the individual reassortants revealed that the migration order of segments 7 and 8 in C J N was reversed in strain 31 and the order of segments 2 and 3 in strain 62 was reversed in strain 69. Changes in migration order have been observed with segments of other rotavirus strains (Garbarg-Chenon et al., 1984; Kalica et al., 1981; Dyall-Smith & Holmes, 1981). Segments have been numbered according to the order of separation of CJN and strain 62 in all Tables and Figures. Modification of RNA segment selection by use of a better adapted parental strain Although all human rotavirus strains used in this study were adapted to grow in cultured cells, there were differences in their abilities to produce infectious progeny. Additional passages caused the more poorly growing strains to become better adapted, i.e. grow to higher titres in MA-104 cells. As previously noted, the parental strains that grew to higher titres were found to contribute the majority of segments in reassortants selected during multiple passages after coinfection. It seemed likely, therefore, that if the poorer grower of a coinfecting pair of rotaviruses was adapted by additional passages to be a better grower, the selection of segments in reassortants formed by these two viruses might shift in favour of this parental strain. To test this possibility, the plaque-purified CJN virus used in the previous experiments was passaged an additional 17 times and a new virus stock, plaque-purified three times, was prepared. The new virus had the same electrophoretic pattern as the old and was neutralized equally by hyperimmune antisera developed against the old virus. The new virus, however, grew to a titre 33-fold greater in cell culture than the old CJN virus and sevenfold greater than strain 31. This new CJN virus was used to coinfect cells with strain 31 and the selection of RNA segments in progeny viruses during multiple serial passages was compared with that previously Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 Rotavirus reassortant selection in vitro 155 Table 1. Distribution of RNA segments in 20 individual progeny viruses plaque-picked after coinfection and multiple passage of each pair Percentage of each parent A t Segment 1 2 3 4 5 6 7 8 9 10 11 Wax P CJN x 31 80 20 0 100 10 90 0 100 100 0 0 100 100 0 0 100 90 10 I00 0 NS* NS 40 60 50 50 15 85 90 10 0 100 30 70 0 100 100 0 0 100 80 20 NS NS * NS, Not separable. 62 x 25 15 0 0 0 0 10 55 0 100 0 69 75 85 1043 100 100 100 90 45 100 0 100 Table 2. Genotypes of individual progeny viruses plaque-picked after coinfection and multiple passage of each pair* Cross WaxP Segments from P 1,2,9 2,9,11 2,9 1,2,7 2 7,9 2,7,8,9 2, 7, 9, 11 2, 6, 7 1, 2, 7, 9 2,5,6,9 2,8,9 2,6,9 5,7 2,7 2,7,9 2, 7, 11 Total Isolates 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 I 1 20 Cross CJN x 31 Segmentsfrom CJN 5 5,6 5,7 5,6,7 Total Cross S e g m e n tfrom s 62 62 x 69 10 8,10 1, 10 1,8,10 7,10 2, 7, 10 1,2,8, i0 2,7,8,10 Total Isolates 10 7 2 1 20 Isolates 6 6 2 2 1 l t 1 20 * Plaques were picked after 18, 13 and 13 passages for Wa × P, CJN x 31, and 62 × 69, respectively. found using strain 31 and old CJN. Coinfection was performed in duplicate cultures at an m.o.i. of 4 with both new C J N and strain 31. The electrophoretic pattern of R N A segments was determined after the 7th, 14th and 21st passages and essentially identical results were found at each passage level. The ratios of segments from the coinfecting virus (Table 3) were quite different from those found previously after coinfection with the old C J N and 31 strains (see Table 1). All C J N segments except 8 and 10 were detectable using the new C J N virus while only segments 5 to 7 of CJN were found after multiple passages following coinfection with strain 31 and old CJN. Since segments 2 and 3 of C J N comigrated as did these segments of strain 31 (see Fig. 1), it was not possible to say whether both of these segments from new C J N were represented in the selected progeny using these techniques. From these results it appeared that selection of certain segments, i.e. segments 1, 2, 3 and 9, under the conditions of these experiments was highly dependent on the relative abilities of the coinfecting viruses to produce infectious progeny in cultured cells. Selection of other segments appeared to be either partially (segments 4 to 7) or totally (segments 8 and 10) independent of the relative growth properties of these two parental strains. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 156 R. L. WARD, D, R. KNOWLTON AND P-F, L. HURST Table 3. RNA segment selection during 21 passages following coinfection with strain 31 and a well adapted (new) CJN Proportion of segments from each parent* A E Segment New CJN 1 2 2 2 3 2t 2 21" 1 4 3 2 0 4 2 3 0 1 2 4 0 4 5 6 7 8 9 31~ 10 0 4 11 NS~ NS * Scale: 0, not detectable; 1, detectable but < 50%; 2, approximately 50%; 3, > 50% but other segment present; 4, the only segment detectable. t Exact amounts of segments 2 and 3 from each parent were not determinable by this analysis (see text for explanation). :~us, Not separable. Table 4.- RNA segment selection during 21 passages following coinfection with the new CJN and strain 31 at different ratios Proportion of segments from each parent & f Segment New CJN 31 (100:1) New CJN 31 (1:100) 1 4 0 1 3 2 3 4 5 6 7 2* 2* 3 4 4 4 2 2 1 0 0 0 0 0 0 4 2 0 4 4 4 0 2 4 8 0 4 0 4 9 10 4 0 0 4 4 0 0 4 11 NSI" NS NS NS * Exact amounts of segments 2 and 3 from each parent not determinable by this analysis. t t~s, Not separable. Effect of different m.o.i, of parental viruses on RNA segment selection In the previous experiments, coinfections were performed using nearly identical m.o.i, for each virus within a pair. Animals would typically be coinfected with unequal amounts of any two viruses. Since these studies in vitro were intended to serve as models for events that occur in vivo, it was of interest to determine whether segment selection in cell culture depends upon the relative m.o.i, of coinfecting strains. The virus pair chosen for this experiment was the new C J N and 31 strains. Coinfections were performed at ratios of 100:1 and 1:100 using an m.o.i, of 4 for the virus strain in the higher concentration. The experiment was performed in duplicate and R N A segment selection was determined by electrophoretic analysis after the 7th, 14th and 21st serial passages. Although some change occurred between passages 7 and 14, little change in electrophoretic pattern was found between passages 14 and 21 after coinfection at either ratio. After 21 passages, segments 5 and 9 from strain 3l and segments 8 and 10 from C J N were undetectable, irrespective of the relative amounts of coinfecting viruses (Table 4). The amounts of the other separable segments Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 Rotavirus reassortant selection in vitro 10 8 1 i i | I 157 I I 10 7 ~- 10~ 10 ~ l0 4 I 6 12 18 Time after infection (h) I ! 24 30 Fig. 3. Growth curves of CJN, strain 31 and their selected reassortants. After coinfection of tube cultures at an m.o.i, of 5 with either CJN ( i ) , strain 31 (~), total selected viruses 23 passages after coinfection with CJN and strain 31 ( , ) , or a plaque-picked CJN x 31 reassortant with the 'preferred' segments (U1), the viruses were allowed to adsorb for 1 h at 37 °C. The cells were washed twice and growth medium without serum was added. The cultures were maintained at 37 °C for the times specified, frozen and examined for infectious virus by plaque assay. The data points represent the averages obtained from duplicate cultures. (1,2 to 3, 4, 6 and 7) from each parental strain depended upon their relative concentrations at the time of coinfection. Duplicate cultures yielded identical results. Thus, segment selection was dependent on the relative m.o.i, of coinfecting parents for some but not all of the segments using this pair of h u m a n rotaviruses. Cause of selection of genome segments Possible causes of the segment selection after coinfection observed in these experiments are that (i) R N A segments are produced in different amounts, (ii) segments are assembled into particles at different rates, (iii) particles with certain segments grow to higher titres, or (iv) particles with certain segments are more stable. I f explanation (iii) or (iv) is correct, reassortants with the favoured combination of segments should produce more infectious progeny than the parental strain from which they originated or these progeny should be more stable than the parental viruses. To test these possibilities, MA-104 cells were infected in duplicate cultures with either the new C J N , strain 31, total selected progeny obtained 23 passages after coinfection with new C J N and strain 31, or an individual reassortant of new C J N x strain 31 which had a 'preferred' distribution of segments, i.e. segments 8 and 10 from strain 31 and segments 5 and 9 from C J N . Yield of progeny viruses was nearly maximal by 12 h after infection with all four viral inocula and little difference in titre was detectable (Fig. 3). Time-dependent loss of infectivity was greater with strain 31 and the infectivity of the other three viral cultures remained essentially equal for the duration of the experiment. This result indicates that both the individual and combined selected reassortants tested were more stable than strain 31 but did not Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 158 R . L . WARD, D. R. KNOWLTON AND P-F. L. HURST Table 5. Parental origin of RNA segments in 33 reassortants plaque-picked after a single replication cycle following coinfection with new CJN and strain 31 Number of segments from each parent A Segment 2 3 4 5 6 7 8 9 10 New CJN 18 14-24" 14-24" 22 29 18 18 12 18 12 11 NSt NS Total 185 145 1 31 15 9-19 9 19 11 4 15 15 21 15 21 * The total of segments 2 and 3 from CJN was 38 and from 31 was 28. t r~s, Not separable. grow to higher titre nor were they more stable than CJN. Thus, the selected reassortants had no obvious growth advantage over the C J N strain. Similar observations were made when growth curves of Wa and P or strains 62 and 69 were compared with their selected reassortants (results not shown). If selection of reassortants occurs during the production of R N A segments [explanation (i)] or assembly of particles [explanation (ii)], segment selection may be evident in individual progeny viruses isolated after a single cycle of replication. This possibility was tested after coinfection of MA-104 cells with new C J N and strain 31. To prevent subsequent replication cycles, cells were infected at an m.o.i, of 4 of each virus, no trypsin was present in the culture medium after the 1 h adsorption period and the viruses were harvested 18 h after infection. Electrophoretic analysis of R N A segments obtained from 100 plaque-picked isolates showed that 33 of these isolates were reassortants. The remainder either contained more than 11 segments (14 isolates) or were parental genotypes (41 CJ N, 12 strain 31). The parental origin of segments in the 33 reassortants was weighted overall in favour of the C J N strain (Table 5). Even though the specific assignment of segments 2 and 3 could not always be made because these segments of C J N comigrate as do those of 31, the total of the two from each parent was determinable. No significant differences from this overall distribution were detectable in the parental origin of seven out of 10 separable segments but there were significant differences in segments 5, 8 and 10 (P < 0.03 as determined by Chi square analysis). These segments were also those found to be most consistently selected from either CJ N (segment 5) or strain 31 (segments 8, 10) during multiple passages (see Tables t and 4). Therefore, non-random distribution of segments was evident in infectious progeny even after only a single cycle of replication. A similar selective process was observed for segments 4 and 10 of Wa virus following a single cycle of replication after coinfection with Wa and P strains (results not shown). These observations suggest that double-stranded R N A segments are either synthesized in different amounts in coinfected cells or are incorporated into assembled particles in different ratios. The first explanation was tested by examination of the relative amounts of R N A genome segments formed during a single cycle of replication following coinfection with new C J N and strain 31. This experiment was performed in the same manner as described above to insure that the progeny R N A s were the products of a single replication cycle. The electrophoretic patterns of genomic R N A obtained after phenol extraction of either the total coinfected cell lysate or a pelleted fraction (190 000 g, 30 min) of this lysate were essentially identical to that obtained after mixing equal volumes of R N A s from singly-infected cell lysates (Fig. 4, lanes 3, 2 and 4, respectively). No segment was found in reduced or increased amounts relative to the same Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 159 Rotavirus reassortant selection in vitro 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 Fig. 4. Gel profiles of genomic RNA from cells either infected individually with CJN or strain 31 or coinfected with these two strains. MA-104 cells were infected (m.o.i. of 5) with CJN, strain 31 or both and, after a 1 h adsorption period, grown at 37 °C for 18 additional h in the absence of trypsin. Singly infected cultures were frozen, thawed, and centrifuged (190000 g, 30 min). Genomic RNA in the pellets was extracted with phenol and analysed by gel electrophoresis. A portion of the coinfected cultures was treated in this same fashion. Another portion was directly extracted without centrifugation then viral particles were banded in CsCI gradients. Particles banding at densities (g/ml) of 1.34 to 1.3555, 1-355to 1-37, 1.37 to 1-39were collected and their RNAs were extracted and analysed_ Lane 1, RNA from CJNinfected cells; lane 2, RNA from the pelleted coinfected culture; lane 3, total RNA from coinfected culture; lane 4, RNAs from CJN and strain 31 individual infections mixed in equal volume; lane 5, RNA from 1-37 to 1.39 density particles of coinfected culture; lane 6, RNA from 1.355 to 1.37 density particles of coinfected culture; lane 7, RNA from 1-34 to 1-355 density particles of coinfected culture; lane 8, RNA from strain 31-infected cells; lanes 9 to 16, RNAs from CJN and strain 31 individual infections mixed in the following ratios: 1 : 1, 1 : 1/2, 1 : 1/4, 1 : 1/8, 1/4 : 1/4, 1/4 : 1/8, 1/4 : 1/ 16, 1/4 : 1/ 32. s e g m e n t from the other parent. Thus, no differences in p r o d u c t i o n of R N A segments were detectable. To d e t e r m i n e whether small differences in the p a r e n t a l origin of a n y two segments could have been detected in this experiment, the singly-infected p a r e n t a l cultures were m i x e d in ratios that varied by twofold i n c r e m e n t s a n d c o m p a r e d after electrophoresis (Fig. 4, lanes 9 to 16). Twofold differences in the quantities of R N A s from the two viruses are clearly e v i d e n t by visual o b s e r v a t i o n of the gel patterns. S c a n n i n g of these gels with a d e n s i t o m e t e r confirmed this observation. Therefore, if the a m o u n t of a particular R N A s e g m e n t m a d e by one virus had been reduced > twofold relative to that of the same s e g m e n t of the second virus in coinfected cells, it should have been e v i d e n t in the gel pattern. The possibility that selection of reassortants occurs d u r i n g p a c k a g i n g of R N A segments into viral particles was e x a m i n e d next. M A - 1 0 4 cells were coinfected with new C J N a n d strain 31 (m.o.i. 4) and, after a single cycle of replication, viral particles were purified by CsC1 gradient centrifugation a n d their R N A molecules were analysed by gel electrophoresis. T h e p a t t e r n from Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 160 R.L. WARD, D. R. K N O W L T O N A N D P - F . L. H U R S T total purified particles was identical to that found after coelectrophoresis of mixed RNAs from the parental viruses (results not shown). When these progeny viruses were separated on CsC1 gradients into dense, single-shelled (density 1-37 to 1.39 g/ml) or light, double-shelled (density 1.34 to 1-37 g/ml) particles, the proportion of segments from the two parents was unequal (Fig. 4, lanes 5 to 7). The dense particles (lane 5) contained considerably more segments from strain 31 and the light particles (lanes 6 and 7) were primarily composed of CJN segments, probably because of the relative instability of strain 31 infectious particles (see Fig. 3). However, specific increases in the amounts of CJN segment 5 or strain 31 segments 8 and 10 were not detectable in either type of particle. Therefore, selection of reassortants with these segments apparently did not take place during assembly. Since the selection of RNA segments consistently observed after multiple passages following coinfection with strains CJN and 31 was not detectable during R N A synthesis or particle assembly but was evident within the infectious (plaque-picked) particle population, it was concluded that particles with segment 5 from C J N and segments 8 and 10 from 31 were more infectious (i.e. better able to form plaques) than particles with these segments from the opposite parent. Thus, segment selection appears to have occurred because of differences in the infectivities of reassortant viruses. DISCUSSION Coinfection of man with more than one strain of rotavirus and generation of reassortant progeny has been suggested as a method of evolution of new human rotaviruses. Although infectious reassortants readily form after coinfection of cultured ceils or animals, little is known about the selective processes involved in their formation and isolation. Gombold & Ramig (1986) found certain constellations of genome segments to be over represented in reassortants formed after coinfection of mice with two strains of simian rotavirus. Graham et al. (1987) made a similar observation after cultured cells were infected with a modified human and a bovine rotavirus. These results indicate that the constellations of genome segments in infectious reassortants isolated after coinfection with two strains of rotavirus can be non-random. As an extension of these findings, it is suggested that the same types of selective mechanisms may be involved in production of new viral strains after coinfection of humans. The present study was designed to determine whether a cell culture system could be used to analyse the selective mechanisms involved in the production of new strains of human rotaviruses. Coinfection with different pairs of subgroup 2 human rotaviruses followed by multiple blind passages caused a reproducible selection of genome segments from each set of parents in progeny viruses. The number of passages required before further selection was not observed varied with the different virus pairs but was essentially complete for all three pairs studied by 13 passages (see Fig. 2). Therefore, the relative amount of each segment from each parental strain found in progeny viruses appeared to be stable within a limited number of passages following coinfection. For each pair of viruses used in these coinfections, one of the parents dominated the final segment selection. This advantage appeared to be related to differences in the abilities of coinfecting viruses to produce infectious viruses in cultured cells. Furthermore, when one member of a pair was adapted through multiple passages to be the better rather than the poorer grower, the overall selection process was changed in its favour (see Tables 1 and 3). Certain segments, however, appeared to be selected independently of the growth properties of the two parents. For example, segments 8 and 10 were from strain 31 even when cells were coinfected with a better adapted CJN strain and segment 5 was almost solely from CJN even when the more poorly adapted C J N virus was used for coinfection. The selection of these particular segments was also maintained when the relative concentrations of coinfecting parents was varied 100-fold in favour of one or the other parent (see Table 4). Although segment selection was reproducible for each of the three pairs of viruses studied, no obvious pattern was discernible regarding the particular segments selected. Segments 2 and 9 from P dominated the selected reassortants and segments 3, 4 and 10 from Wa were highly selected; segment 10 of strain 62 and segments 3 to 7, and 11 of strain 69 were highly selected; as Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53 Rotavirus reassortant selection in vitro 161 already discussed, segment 5 of CJN and segments 8 and 10 of strain 31 were also highly selected. Even segment 4, which has been related to the growth restriction properties of rotaviruses in cultured cells (Greenberg et al., 1983), and virulence in vivo (Offit et al., 1986; Flores et al., 1986; Gorziglia et al., 1986), was not always selected from the better growing virus of a pair (see Table 4). Only segment 10 was consistently selected from one or the other parent of all three pairs. This segment codes for a non-structural glycoprotein of M~ 28 000 that is located in the rough endoplasmic reticulum and may be involved in viral maturation and assembly (Both et al., 1983; Okada et al., 1984; Ericson et al., 1983). Graham et al. (1987) observed that certain segments were selected from each parent after coinfection of cells with a modified human and a bovine rotavirus. Furthermore, they found that the selection process was partially modified by the cell line used for plaque isolation. Although the effects of host factors in the selection process were not examined in detail in our study, they may play a role which should be characterized in future studies. The genotypes of individual progeny viruses were found to be a direct reflection of the overall segment selection observed within the total progeny after coinfection and multiple passages. Sixty individual isolates were examined (20 from each coinfection) and all were reassortants. It was noted, however, that no particular genotype of these reassortants was found in greater abundance than expected based on random distribution of total selected genome segments. Even though many more individual progeny would have to be examined in order to completely verify this conclusion, the results suggest that random distribution of selected segments occurs within the individual progeny of these reassortant populations. Determination of the cause of segment selection following coinfection with human rotaviruses revealed that selection was apparent even after only a single cycle of viral replication. However, selection was not observed during synthesis of R N A genome segments or assembly into particles. Since segment selection was evident within the infectious particle population, however, it must have been due to differences in the infectivities of the assembled virions. Particle/p.f.u. ratios are typically about 100 for enteric viruses that grow to high titres in cultured cells. The particle/tissue culture infectious unit ratios for unadapted human rotaviruses, however, were found to be approximately 50000 and were still approximately 7000 after adaptation (Ward et al., 1984). From the results of the present study, it appears that these ratios are dependent on the parental origin of specific segments in progeny viruses formed in coinfected cells. Although the actual cause for differences in the infectivities of reassortant viruses has not yet been determined, it is anticipated that it will be related to functional variations in the interactions of viral proteins with one another or with their RNAs. It is of interest to note that most if not all progeny viruses formed during multiple passages following coinfection with the human rotaviruses in this study were reassortants. 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I., YOUNG, E. C., SHERWOOD, J. R., KNOWLTON, D. R. & SCHIFF, G. M. (1986). H u m a n rotavirus studies in volunteers: determination of infectious doses and serological response to infection. Journal of Infectious Diseases 154, 871-880. (Received 13 July 1987) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 16:20:53