Isolation of a human rotavirus containing a bovine rotavirus VP4 gene that suppresses replication of other rotaviruses in coinfected cells

Arch Virot (1996) 141:615-633

_Archives

Vi?ology © Springer-Verlag 1996 Printed in Austria

Isolation of a human rotavirus containing a bovine rotavirus VP4

gene that suppresses replication of other rotaviruses

in coinfected cells

R. L. Ward 1'*, Q. Jin TM, O. Nakagomi 3, D. S. Sander 1'*, and J. R. Gentsch 2

1 Division of Clinical Virology, James N. Gamble Institute of Medical Research, Cincinnati, Ohio; 2 Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.; 3 Department

of Microbiology, Akita University School of Medicine, Akita, Japan

Accepted October 31, 1995

Summary. Bovine-human reassortant strains containing ten human rotavirus gene segments and segment 4, encoding VP4, of a bovine rotavirus were isolated from the stool of an infected Bangladeshi infant during cell culture adaptation. Two plaque purified variants of this reassortant, one making very large (429-L4) and the other tiny (429-$4) plaques, were further analyzed. The electropherotypes of these variants were identical except for slight mobility differences in segment 4. The predicted sequence of amino acids (aa) 16-280 in VP4 proteins revealed four differences between variants even in this limited region, so no single difference could be linked to plaque size. The small plaque variant $4 was phenotypically unstable and mutated to a large plaque-former within a single cell culture passage. The predicted sequence of aa 16-280 of a large plaque variant derived from $4 revealed six changes, only one of which was common to that of the L4 strain, thus suggesting that multiple amino acid changes in VP4 may affect plaque size. Although the large plaque variant L4 grew faster and was released from cells more rapidly than $4, its replication and that of other rotaviruses tested (i.e. RRV, NCDV and Wa) was suppressed by $4 in coinfected cells. Using an RRV x $4 reassortant containing only RRV segment 4, it was established that suppression was linked to the $4 VP4 protein. This suppression could not be associated with inhibition of viral adsorption and, therefore, appeared

Present addresses: *Division of Infectious Diseases, Children's Hospital Medical Center, Cincinnati, Ohio, U.S.A.; **Institute of Virology, Chinese Academy of Preventive Medicine, Beijing, People's Republic of China.

616 R.L. Ward et al.

to occur following internalization. Thus, a new proper ty of the rotavirus VP4 protein has been identified in a bovine-human rotavirus reas-sortant.

Introduction

The fully encapsulated mature rotavirus particle is composed of three protein shells, the outermost of which contains the two neutral ization proteins, VP7 and VP4 [23]. Al though it has been reported that there are only 60 dimers of VP4 per virion [26], a much smaller number than the predicted 780 molecules of VP7 per virion [22, 35], VP4 has been assigned a variety of roles. These include determinat ion of in vitro growth properties [6, 13] and virulence [12, 20], and involvement in viral a t tachment and penetrat ion [1, 8] as well as hemagglutina- t ion [13, 16]. It has also been suggested that VP4 may be involved in receptor recognit ion and budding through the endoplasmic reticulum during virus matura t ion [26].

In addi t ion to these broad roles for VP4, a new proper ty for a specific VP4 protein gene has been identified. This VP4 is contained within a reassortant rotavirus strain having a single bovine-like rotavirus gene on a h u m a n rotavirus genetic background. When this virus (429-$4) was used to coinfect cultured cells with other rotaviruses, it greatly suppressed replication of the coinfecting strains. Isolation and characterization of the rotavirus strain containing this unique VP4 protein gene is described in this report.

Materials and methods

Cells and rotavirus strains

The 429 strain of rotavirus was isolated from a stool specimen of a 9-month-old child with gastroenteritis in Bangladesh in 1986. The virus was culture adapted by passage in roller tubes of African green monkey kidney cells as previously described [30, 32]. Sub-strains of the 429 isolate were obtained by plaque purification after additional passages in MA 104 cells. Individual strains were characterized by electrophoretic analysis of viral gene segments, serotyping, genogrouping and nucleotide sequencing. Simian rotavirus RRV (G3 [P3]), bovine rotavirus NCDV (G6 [P1]) and human rotavirus Wa (G1 [PSI) are all prototype strains used in previous studies. All were plaque purified and grown in MA104 cells.

Electrophoretic analysis of rotavirus RNAs

RNA genome segments of rotavirus obtained from lysates of infected MA104 cells were extracted with phenol and analyzed by polyacrylamide gel electrophoresis as described previously [31].

Serotypin9 of rotaviruses

Rotaviruses were serotyped by an ELISA using MAbs to VP7 proteins of human rotaviruses [i.e. MAb 5E8 (G1), MAb 1C10 (G2), MAb 159 (G3) and MAb ST-3:I (G4)] by a procedure described previously [331. Rotaviruses were also serotyped by a similar procedure with MAbs to VP4 of human rotaviruses Wa [P8] and ST-3 [P6] as well as porcine rotavirus OSU [P7] using MAbs 2A3 and 2C 11, Mab HS 11, and MAb 2G4 to these strains, respectively. In

Suppressed rotavirus replication associated with VP4 617

short, 96-well microdilution plates were coated with each of the MAbs, followed sequentially by addition of the viruses, preimmune (control) or hyperimmune guinea pig sera to single shelled Wa virus (each in duplicate for each MAb and each virus), biotinylated goat anti-guinea pig IgG, peroxidase conjugated biotin-avidin, and substrate (o-phenylene- diamine, H202). The final enzymatic reaction was stopped with 1 M H2SO 4 and A49 o was determined. MAbs were all provided by H. B. Greenberg (Stanford University) except MAb ST-3:1 which was provided by B. Coulson (Royal Children's Hospital, Parkville, Australia).

Genogroup determination

Characterization of RNA segments of different rotavirus strains by genogroup analysis was performed as already described [34]. The stringency conditions used during hybridization allowed up to approximately 18% mismatch for hybrid formation but much smaller amounts of mismatch would result in an altered (reduced) electrophoretic mobility of the hybrid bands during gel electrophoresis.

Synthesis of PCR products

Rotavirus infected cell lysates of strains 429-NP, 429-L4 and 429-$4 were frozen and thawed three times, clarified at 8 000 x g and viral double-stranded RNA was extracted with phenol- chloroform (1 : 1), followed by adsorption to and elution from glass powder (RNAID, Bio t 01, Inc., LaJolla, CA) as described previously [10]. Synthesis of VP4 gene cDNA was carried out by reverse transcription-polymerase chain reaction (RT-PCR) using oligonucleotide primer pair Con3 (nucleotides 11-32 of strain Wa gene 4) and Con2 (nucleotides 868-887 of strain Wa gene 4) as previously described [10]. The PCR products were resolved by electrophoresis on 1.2% SeaKem GTG agarose gels (FMC Bioproducts, Rockland, ME) and then purified with a Q~AEX Gel extraction Kit (QIAGEN Inc., Chatsworth, CA) according to the manufac- turer's procedure.

Dideoxynucleotide sequencing

Cycle-sequencing of PCR products by the chain termination method was carried out with the Prism Kit (Applied Biosystems, Foster City, CA) which utilizes Amplitaq and dideoxynuc- leotides labeled with four different dyes [24]. Twenty five cycles of PCR (30 sec at 96 °C, 15 sec at 50 °C, and 4 min at 60 °C) with primers Con2 and Con3 was used for sequencing each PCR product. Additional primers were selected from the sequences generated from Con3 and Con2 reactions or from the sequence of strain 1076 to complete the analysis [12]. Cycle- sequencing products were electrophoresed in 6% polyacrylamide gels containing 8 M urea on the ABI model 373A automatic sequencing apparatus.

Sequence data analysis

Sequences were analyzed using the University of Wisconsin Genetics Computer Group (GCG) software [4].

Coinfection of cell cultures with different rotavirus strains and characterization of progeny viruses

Monolayers of MA104 cells in 96-well microdilution plates were washed twice with medium [Dulbecco's-modified MEM containing 4 pg/ml of trypsin (Gibco Laboratories, Grand Island, NY)] and infected (multiplicity of infection of 50) with different rotavirus strains


either individually or simultaneously. Volumes in wells were adjusted to 0.1 ml and plates were centrifuged (1 h, room temperature, 1000 x g). Unadsorbed virus was removed and wells were washed once before addition of medium (0.2 ml). Plates were incubated (37 °C) for approximately 24 h or for the times specified and placed at - 20 °C. When infection with a coinfecting virus was delayed, the unadsorbed first virus was removed after the one hour adsorption period, the cells were washed, and 0.1 ml of medium was added. This medium was removed just before the second virus was added at the appropriate time interval, and all steps were repeated. For analyses by a one-step growth cycle, the medium was first removed and stored frozen for analyses, an additional 0.2 ml of medium was added per well, then the cells were suspended by scraping and stored frozen. Virus yield in all specimens was determined by a plaque assay using MA104 cells. Yield of individual rotavirus strains in coinfected cell cultures was first determined by plaque size after 3-4 days. Subsequently, the etectrophoretic mobilities of viral RNA segments obtained from plaque purified isolates were determined by polyacrylamide gel electrophoresis [3t]. Plaque sizes of individual viruses were always determined simultaneously to control for possible day-to-day variation.

Results

Isolation and characterization of human-bovine reassortant rotavirus strains obtained from an infected child

Rotavirus strain 429 obtained from the stool of an infected Bangladeshi infant had an RNA electropherotype (i.e. electrophoretic migration pattern of its RNA segments) that was typical of group A human rotaviruses and was identical to that of rotaviruses isolated from three other infected infants in the same study 1-32]. However, unlike the other three isolates whose electropherotypes did not change when they were adapted to grow in cell culture, the RNA pattern of strain 429 contained an additional segment 4 after only a single cell culture passage that migrated much more slowly than the original segment 4 (Fig. 1A). When plaque purified after five cell culture passages, rotavirus isolates with very different plaque size phenotypes were obtained. Isolates with a fast-migrating segment 4, as found for the virus in stool, all made extremely small plaques while isolates with slow-migrating segment 4 produced plaque sizes ranging from extremely tiny to very large. Three representative isolates with slow-migrating segment 4 were plaque purified an additional three times and designated as strains 429-L4 or L4, 429-$1 or $1, and 429-$4 or $4 for very large, medium-sized, and tiny plaque-formers, respectively. A plaque purified isolate with fast-migrating segment 4, like that found in the original stool, was designated strain 429-NP or NP.

When the electropherotypes of these four strains were more carefully examined, additional differences were observed. Segment 4 of the L4 strain migrated slightly faster than the corresponding segment of the $1 or $4 strains, segment 7 of strain S1 migrated faster than this segment of the other three strains, and segment 10 of N P migrated slightly slower than that of the other three strains (Fig. 1B). The only other differences in the electropherotypes of strains NP, L4 and $4 appeared to be in segment 4 which encodes the outer capsid protein VP4.

All four strains belonged to serotype G1 as determined by a serotyping ELISA using monoclonal antibodies (MAbs) to the VP7 proteins of human


Fig. 1. Electrophoretic patterns of viral RNAs of strain 429 isolates. A RNAs from the original stool (O) containing the 429 strain or from passage 2 (P2) of this strain. B RNAs from strain 429 isolates $1, $4, L4 and NP which were obtained after 5 cell culture passages and then plaque purified four times

G1-G4 strains (results not shown). In contrast, using a series of MAbs to the VP4 proteins of human rotaviruses Wa and ST-3 or porcine rotavirus OSU, it was found that only strain NP was recognized by the MAbs to the human strains and only the strains with slow-migrating segment 4 bound to the MAb against OSU VP4 (Table 1). Furthermore, when we attempted to determine the P genotype of these strains by RT-PCR with primers for genotypes [P41, [P61, [PS], [P91 and [P 101 [71, only strain NP (genotype [P6]) could be identified while the other 429 strains were untypable. Together these results suggested that the only segment 4 from a human rotavirus was the fast-migrating segment of strain NP.

Analysis of the 429 strains by genogroup determination revealed that all four formed RNA-RNA hybrids with at least ten segments of the Wa strain and, therefore, at least ten segments of each belonged to the Wa genogroup of human rotavirus (Fig. 2). The migration patterns of the hybrids that formed between Wa and strains $4, L4 and NP were very similar. When tested with other serotype 1 (G1) prototype human rotavirus strains D and M37, strain NP again formed RNA-RNA hybrids with at least eight segments. Few, if any, of the RNA-RNA hybrids that formed between the 429 strains and the three prototype human rotaviruses tested appeared to comigrate with bands formed between

620 R. L. Ward et al.

Table 1. Binding of anti-VP4 MAbs to 429 strains of rotavirus

Reactivities of MAbs a

anti-OSU anti-Wa anti-ST-3

Virus strain MAb 2G4 MAb 2A3 MAb 2Cll MAb HSll

RRV + - - - Wa + + + -- ST-3 -- - - +

Np b _ + + + $1 + - - - $4 + - - - L4 + - - -

aBinding of virus to the different MAbs was determined by an ELISA as described in Materials and methods

b Strain NP was identified as genotype [P6] by RT-PCR while strains S1, $4 and L4 were not typable

homologous segments, thus indicating they were related but not completely homologous. Based on the same criterion, all but segment 4 of strains NP, $4, and L4 were essentially homologous as were all eleven segments of strains $4 and L4. Segment 7 of S1 was similar but not homologous to that of the other 429 strains and segment 4 of this strain was similar but not homologous to that of strains $4 and L4. Taken together, these results imply that all but segment 4 of the 429 strains belong to the Wa genogroup and, therefore, were derived from a human rotavirus. Furthermore, segment 4 of strains Sl, S4 and L4, but not strain NP, all appeared to belong to the same genogroup.

Segment 4 of the 429 strains was further characterized by sequence analyses of nucleotides 55-850 for the NP, $4 and L4 strains. The deduced sequences of amino acids 16-280 encoded within this region were then determined (Fig. 3) along with the percentages of homology of both nucleotide and amino acid sequences with those of published cognate sequences from selected P (VP4) genotypes of rotavirus (Table 2). Strains of 429 with slow-migrating segment 4 (L4 and $4) shared 98.5% amino acid homology (4 amino acid differences) while strain NP shared only 59.6% and 60.3% amino acid homologies with strains L4 and $4, respectively. Furthermore, segment 4 of strain L4 appeared to be derived from a bovine rotavirus because the amino acid sequence encoded by this gene was 90.6% to 95.1% homologous to those of genotype [P1] bovine rotaviruses and was most related to the A5 strain isolated from a calf in Thailand [21, 28], a site in southeast Asia very near the point of origin of the 429 strains. In contrast, the region of segment 4 from NP analyzed in this study encoded an amino acid sequence that was between 91.7% and 93.2% homologous to that of

Suppressed rotavirus replication associated with VP4 62t

Fig. 2. Hybridization patterns between genomic RNAs from the 429 strains or prototype human rotaviruses indicated above each line and the 32p-labeled plus-strand RNA probe prepared from the designated 429 strain, a Ethidium bromide-strained gel under UV light illumination; b corresponding autoradiogram. Approximate positions of the 11 RNA

segments of strain NP are indicated on the left

genotype [P6] asymptomatic strains of human rotavirus, confirming our find- ings using RT-PCR. Comparisons with strains from other selected P genotypes revealed a maximum acid homology of 80.4% with either the L4 or NP strain of 429. Thus, strain NP with fast-migrating segment 4 appeared to be a typical G1 [P6] human rotavirus while the 429 strains with slow-migrating segment 4 appeared to be human x bovine rotavirus reassortants that contained ten genes of human rotavirus origin and segment 4 from a bovine rotavirus.

Phenotypic and genotypic instability of the $4 strain

Plaque purified isolates of 429 strains $4 and L4 both typically grew to titers of approximately 108 plaque-forming units/ml in MA104 cells, but the $4 strain produced extremely small plaques while the L4 strain formed very large plaques. This difference in phenotype was probably due to the comparatively rapid


16 65

L4 VELSDEIQEI GSTKTQSVTI NPGLFAQTSY APVNWGPGET NDSTVVEPVL

$4 ................ R ...... P ..........................

NP ....... ST- --E---N ...... P .... N .... T-SR--V .... TI ....

66 * ** * 115

L4 DGPYQPTTFN PPVSYWMLLA PTNAGVVVEG TNNTNRWLAT ILIEPNVQSV

$4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

NP ....... S-K --SD--I--N ---QQ--L .... KIDI-I-L L-V .... TNQ

116 165

L4 ERTYTLFGQQ VQITVSNDSQ TKWKFVDVSK QTQDGNYSQY GPLLSTPKLY

S4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H ..........

NP S-Q ..... ET K---IE-NTN ..... FEMFR R-VGAEFQHK RT-T-DT--A

166 215

L4 GVMKHGGKIY TYNGETPNAN TGYYSATNYD SVNMTAYCDF YIIPLAQEAK

$4 . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . .

NP -FV-LYNSVW -FH .... H-T -DCS-TS-LS E-ETVIHVE ..... RS--S-

216 * * $ * $ $ 265

L4 CTEYINNGLP PIQNTRNVVP VSISSRSIVH TRAQANEDII VSKTSLWKEM

S4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

NP -V .... T .... M ..... I-- -AL .... VT- Q---V ..... I ........ I

266 280

L4 QYNRDI IIRF KFANS

$4 ...............

NP -H .......... N--

Fig. 3. Deduced amino acid sequences of strains L4, $4 and NP VP4 proteins (residues 16-280). Symbols: + potential trypsin cleavage sites (after residues 241 and 247) between the VP8 and VP5 polypeptides, as well as a potential secondary site (after residue 231); • proline and • cysteine residues conserved between many human and animal rotavirus VP4

proteins [7]

cytopathic effect (cpe) produced by the L4 strain and its release into the cell culture medium as shown by a one-step growth curve (Fig. 4). The $4 strain was not phenotypically stable, however, since even after it was plaque purified four times, a population of large plaques was evident on the plaque plate at approximately 0.1% of the titer of the normal tiny plaque-former. The ratio of small to large plaque-forming units remained relatively constant during further passages at high multiplicity of infection.

Because the VP4 protein has been associated with in vitro growth properties of rotavirus [6,13] and the L4 and $4 strains varied greatly in plaque size but had identical electropherotypes except for segment 4 encoding VP4, it was possible that the difference in phenotype was due to differences in their VP4 proteins. Furthermore, a single amino acid change in VP4 of the $4 strain may have caused its dramatic change in phenotype to a large plaque-former as has been suggested for the pathogenicity of a porcine rotavirus [2]. To make an initial assessment of this possibility, nucleotides 55-850 of a large plaque variant of the $4 strain (designated S4L1) were sequenced and the encoded amino acid sequence was compared with that obtained for the $4 and L4 strains (Table 3).


Table 2. Homologies between VP4 genes (nucleotides 55-850) and encoded proteins for the NP and L4 strains of 429 with those of other rotaviruses

from selected P genotypes

Percentage identity with indicated strain

429-NP 429-L4

Strain P nt aa nt aa Accession # genotype or reference

NP 100 100 62.3 59.6 U32166 L4 62.3 59.6 100 100 U32165 $4 62.3 60.3 99.1 98.5 U32167 C486 1 61.9 58.5 80.1 90.6 Y00127 A5 1 61.8 59.6 95.6 95.1 D13395 SAll4F 1 61.9 59.5 80.8 92.1 X57319 NCDV 1 - - 59.2 --~ 91.7 Nishikawa et al. [19] SAll 2 63.6 57.7 74.3 78 .1 D16346 RRV 3 63.3 60.0 74.4 80.4 M18736 RV-5 4 69.4 65.7 62.4 59.6 M32559 UK 5 60.1 55.9 65.5 68.7 M22306 1076 6 94.4 91.7 62.1 59.6 M88480 ST-3 6 96.3 92.8 67.3 59.5 L33895 M37 6 a 93.2 --~ 57.7 Gorziglia et al. [12] MCN 6 --a 92.0 58.0 Gorziglia et al. [12] OSU 7 61.8 55.8 72.4 77.4 X13190 Wa 8 69.6 67.6 63.3 60.4 M96825 K8 9 58.8 52.8 64.6 61 .1 D90260 69M 10 62.9 60.8 70.2 77.4 M6060() B223 11 48.2 37.7 48.1 41.9 M92986

aNucleotide sequence not submitted to GenBank

Even in this relatively small region of the VP4 protein, there were six amino acid changes f rom $4 to this large p laque variant, only one of which (amino acid 32) was c o m m o n to that found in VP4 of the large p laque-forming L4 strain. Thus, muta t ions at mult iple sites in the V P 4 gene of $4 were rapidly isolated dur ing g rowth in M A 1 0 4 cells, all of which m a y have con t r ibu ted to the observed change in phenotype . Because it was clear that no single amino acid change could be associa ted with a change in pheno type , the remaining sequences of the $4 and S4L1 VP4 genes were no t determined.

Suppression of rotavirus replication in cells coinfected with strain $4

W h e n the 429 strain was adap ted to g row in cell culture, rapid cpe was observed within a single passage, a finding that had no t been noted dur ing pr ior culture adap ta t ion of > 500 h u m a n rotavirus strains. This result could be explained by


109

J 10 8 j -

/ /

10 7

/ /

_ lOa

E / /

105

10 4

/ / / /

/ ./

I t l 8 12 24

Time after Infection (hrs)

48

Fig. 4. One step growth curve of L4 and S4 in MA104 cells. After infection at an moi of 50 as described in Materials and methods, recoverable virus released into the medium or total recoverable infectious virus was measured by a plaque assay. Symbols: • $4 released into medium; (2) IA released into medium; • total $4; [] total L4

Table 3. A comparison of amino acid differences in VP4 (aa 16-280) for the 429 strains L4, $4 and S4L1

Strains compared Site of change Change

L4 vs. $4 32 ser a-~ arg 39 leu --, pro

155 tyr ~ his 191 ala ~ thr

$4 vs. $4L1 b 32 arg ~ ser a 35 ile ~ val

132 ash --* leu 137 lys --+ ile 141 val -~ ala 157 pro ~ leu

~Amino acid common to large plaque-formers bGenBank accession number U32168


the finding that the stool specimen of the child from which strain 429 was isolated contained not only a human rotavirus, but also bovine x human rotavirus reassortants with VP4 protein genes of the bovine virus which permitted the reassortants to replicate efficiently in cell culture. The plaques obtained after a single passage of strain 429 were, however, generally very tiny although a background level of approximately 0.1% of the plaques were extremely large. After a series of 12 high ( > 100) multiplicity of infection (moi) passages, this ratio of small to large plaque-formers changed very little. When the moi during passage was reduced to < 1, however, the titer of large plaque-formers increased from approximately 105 to l0 s pfu/ml and totally obscured detection of small plaque-formers. This suggested that coinfection of cells with large and small plaque-formers obtained after passage of the 429 strain appeared to suppress the replication of the large plaque-formers which would otherwise outgrow the small plaque-formers.

Once a very large (L4) and a tiny ($4) plaque-former was isolated, it was found that coinfection of cells with equal but high (i.e. 50) moi's of L4 and $4 strains caused a 90% reduction in the yield of large plaque-formers but no reduction in small plaque-formers (Table 4). Comparable reductions in the production of large plaque-formers were Observed in cells coinfected with $4 and either Wa, RRV or N C D V (Table 4). To insure that the small plaque-tbrmers contained the S4 VP4 gene as expected, the electropherotypes of tile $4 x RRV

Table 4. Suppression of replication of large plaque-forming rotaviruses when coinfected in MA104 cells with small

plaque-forming strain $4

Yield of virus (pfu/ml)

Infecting strains" Large plaque-former Small plaque-former

$4 1.0 x 108 L4 1.1 x 108 L4 + $4 1.1 x 107 (90%) b 1 x 108 (0%)

RRV 2.0 x 109 RRV + $4 1.5 x l0 s (93%) 1 x 108 (0%)

NCDV 2.1 x l0 s NCDV + $4 8 × 106 (96%) 1 x l0 s (0%)

Wa 8 X 1 0 7

Wa + $4 6 x 106 (93%) 7 x 10 v (30%)

aMA104 cells were infected at an moi of 50 for each virus in all infections

bpercentage inhibition relative to yield in cultures infected with single virus strains


progeny of coinfection (moi = 50:50) were examined. All (10/10) small plaque- formers tested contained the $4 VP4 gene, and all (10/10) large plaque-formers tested contained the RRV VP4 gene (results not shown). However, almost all (17/20) progeny were reassortants based on the migration patterns of other gene segments, but only a small fraction ( < 20%) of the segments in the small plaque-formers were derived from RRV. Furthermore, approximately 30% of the gene segments in the large plaque-formers were derived from $4. Thus, there was an overall large reduction in the quantity of RRV segments incorporated into the progeny of this coinfection, but no reduction in $4 segments.

Because the only gene segment in the L4 and $4 strains that migrated differently was segment 4, it was postulated that suppression of L4 replication and that of the other rotavirus strains in cells coinfected with $4 was caused by a unique property of the VP4 protein of the $4 strain or its gene segment. One of the large plaque-forming reassortants obtained after coinfection with RRV and $4 contained only the RRV VP4 gene and other segments from $4 (Fig. 5). This reassortant, designated as $4-R4, was used in coinfections with RRV, $4 and L4 to determine whether the presence of $4 segment 4 correlated with suppression of the coinfecting rotavirus.

Using moi's of 50 for cells infected with either RRV or $4-R4, RRV grew to a ten-fold higher titer than $4-R4 (Table 5). No reduction in virus yield was found

Fig. g. Electrophoretic patterns of RNAs from RRV, $4, and $4-R4. The conditions for electrophoresis were designed to allow separation of all $4 and RRV segments. These included using a 12.5% acrylamide resolving gel with electrophoresis at 14 amps/gel for 39 h. Each of the three strains analyzed were run both next to each other and together in the same lane to establish the gene origin of segments for the $4-R4 strain

Suppressed rotavirus replication associated with VP4

Table 5. Association between the presence of the 429-$4 VP4 gene and suppression of coinfecting virus replication

Yield of virus (pfu/ml)

Infecting strains a Large plaque-former Small plaque-former

RRV 2.0 × 10 9

$4-R4 b 1.1 x 108 RRV + $4-R4 3 x 109 ( 0 % ) c

$4 1.0 x 10 s $4 + $4-R4 2 x 107 (82%) 1 x l0 s (0%)

aMoi's of 50 were used for each virus in all infections bS4-R4 is a reassortant that contains 10 segments of $4 and

segment 4 of RRV c Percentage inhibition relative to cultures infected with single

virus strains

627

when cells were coinfected with these two virus strains and 26/40 (65%) of the progeny tested contained only RRV gene segments while only 3/40 (7.5%) had the same electropherotype as $4-R4. The other progeny were reassortants between the two parents. In contrast, coinfection with $4 and $4-R4, which differ only in segment 4, resulted in a 90% reduction in large plaque-formers (Table 5), and all (10/10) small plaque-forming progeny tested contained the $4 segment 4. Coinfection of L4 and $4-R4 resulted in no reduction in titer (both viruses form large plaques) and little change in the expected ratio of L4 and $4-R4 progeny based on electropherotype (results not shown). Taken together, these results indicate that the presence of segment 4 of the $4 strain correlated with suppressed replication of the coinfecting virus.

Effects of delayed coinfection on suppression of S4-R4 replication by strain $4

Because the gene encoding VP4 of the $4 strain correlated with suppression of replication and VP4 is believed to be the viral at tachment protein of rotavirus (1, 8), it was possible that the mechanism of $4 suppression of replication was to merely block binding of coinfecting rotaviruses to cells. To test this possibility, cells were coinfected (moi = 50:50) with $4 and $4-R4, but the time of infection with $4 relative to that with $4-R4 was delayed by various time periods. The suppression of the large plaque-forming $4-R4 strain by $4 found after simulta- neous infection was gradually diminished when infection with $4 was delayed (Table 6). However, only when the delay was 3 h or greater was the yield of $4-R4 inhibited < 50%. This result indicated that merely preventing adsorption of $4-R4, and presumably other rotavirus strains, is not the mechanism of suppression by the $4 strain.


Table 6. Effect of time delay in $4 infection on its suppression of the large plaque-former $4-R4 in

coinfected cells

Time of $4 coinfection ~

Yield of large plaque-formers (pfu/ml)

none b 8.5 x 107

0hr 1.5 x 107 (82%) ° 1 hr 2.4 x 10 v (72%) 2 hr 3.2 x 107 (62%) 3 hr 6.0 x 107 (29% 4 hr 8.4 x 107 (1%)

aMA104 cells were infected with strains $4-R4 and $4 (moi = 50 for each) either simultaneously or infection with $4 was delayed for the specified time periods

bCells were infected with strain $4-R4 only °Percentage inhibition of large plaque-formers rela-

tive to the yield from cells infected with strain $4-R4 alone

D i s c u s s i o n

The VP4 protein has been reported to have a number of important roles in the life cycle of rotavirus even though only 60 dimers of this outer capsid protein are found per virus [26]. This is analogous to the o-1 protein of reovirus, the prototype for viruses with double stranded RNA genomes, which exists as 12 dimers or tetramers at vertices of the icosahedral reovirus particle [25]. Both VP4 and o-1 have been reported to be neutralization proteins, the viral hemag- glutinins, the viral at tachment proteins, and determinants of growth properties and pathogenesis. In addition to these properties, it has now been found that the VP4 protein of a least one strain of rotavirus is associated with suppressed replication of other rotaviruses in coinfected cells.

The strain of rotavirus that was able to suppress the replication of other strains was isolated after culture adaptation ofrotavirus present in the stool of an infected Bangladeshi infant. Visualization of the electrophoretic pattern of rotavirus genome segments from this stool sample revealed only 11 segments which suggested the child had been infected with only one virus. After passage in cell culture, however, the presence of an additional coinfecting rotavirus was revealed through an extra, slower-migrating VP4 gene segment. Genogroup and nucleotide sequence analyses of the culture-adapted viruses showed that the original virus was a typical G1 [P6] human rotavirus while the virus with slow-migrating VP4 gene was composed of reassortants. These reassortants all appeared to share ten segments with the originally identified human rotavirus but to contain bovine rotavirus VP4 genes.


Evidence of human infection by animal rotaviruses, once thought to be extremely uncommon, is now being reported with greater regularity [17]. Although human infections have been associated with rotavirus strains whose genes appear to be solely ofanimat rotavirus origin, human x animal reassortant strains have been more commonly found. For example, asymptomatic infections of neonates in India appeared to be due to bovine x human rotavirus reassortants [-3, 9], and among rotaviruses obtained from patients with acute diarrhea in Thailand many appeared to be porcine x human rotavirus reassortants [29]. In addition, bovine x human reassortants were obtained from an infected Cincin- nati infant [18]. Similar to these latter reassortants, bovine x human rotavirus reassortants obtained in the present study were detectable by electrophoretic analysis of viral RNAs only after cell culture passage of the virus. Thus, the reassortant rotavirus strains obtained from both infants were present in stool in much lower concentrations than the human strains. This suggests they probably replicated less well than the human strains in the infected infants and were unlikely to be responsible for their illnesses. The origin of these reassortants is unclear but because multiple bovine rotavirus segments, including segment 4, were obtained from the Cincinnati infant and only bovine rotavirus segment 4 was recoverable from the Bangladeshi infant, it is possible that this was the only bovine rotavirus segment present in the fecal specimen of the infected Bangladeshi child. Formation of this reassortant may have occurred during a previous coinfection with a human (429-NP) and bovine rotavirus, and only segment 4 of the bovine rotavirus was retained as a reassortant which, along with the human strain itself, was able to cause subsequent infections. Confirmation that only segment 4 of the bovine rotavirus was present in the infected child may be possible in future studies using RT-PCR.

Three reassortants with slow-migrating VP4 genes were plaque purified and more fully characterized. All three had distinctive plaque sizes (tiny, small or very large), and the electrophoretic mobility of the VP4 gene of the strain that formed large plaques was slightly faster than this gene segment of the two smaller plaque-formers. Nucleotide sequence analysis of a portion of the VP4 genes of the large (L4 strain) and tiny ($4 strain) plaque-formers revealed four differences in amino acids 16-280 of their VP4 proteins. Thus, no single amino, acid change could be associated with the observed differences in plaque size.

Further analysis of the $4 strain, however, identified two unique properties of this rotavirus. The first was the instability of its phenotype. Even after being plaque purified four consecutive times, when the $4 strain was grown and replaqued, a background level of large plaques were evident at approximately 0.1% the quantity of tiny plaques. Nucleotide sequence analyses of a portion of the VP4 gene of one of these large plaque variants ($4L1) revealed six differences from that of the $4 strain in amino acids 16-280, only one of which was common to that of the L4 strain. Since all six amino acid changes were so rapidly selected, it is possible that most, if not all, were associated with the altered phenotype. Thus, again no single amino acid change in VP4 could be associated with the difference in plaque size.


Such rapid selection of a large number of nucleotide changes within a gene appears to be unusual. For example, when neutralization escape mutants of DS-1 were selected by growth in the presence of neutralizing MAbs to its VP7 protein, typically only a single nucleotide change was observed in the VP7 gene of the selected strains [5]. However, when mutations in several neutralization epitopes were required within rotavirus genes to provide a growth advantage, mutations at multiple sites could be found within the neutralization protein genes. Thirty-nine passages of strain SA- 11 4fM in the presence of homologous polyclonal antisera resulted in numerous mutations in selected progeny within neutralization epitopes of both the VP7 and VP4 protein genes [11]. Because VP4 is associated with in vitro growth properties, it may be possible to rapidly select mutations at sites within the VP4 gene that enhance growth in cell culture. Kitamoto et al. [14] reported changes in five amino acids within the VP4 protein of Wa during adaptation to growth by multiple passages in HepG2 liver cells. In addition, Taniguchi et al. [27] reported that two plaque purified isolates of rotavirus strain SAl l which were obtained from the same source, ( i .e .H.H. Malherbe) but passaged in different laboratories varied greatly in plaque size. These two variants also differed by five amino acids in their VP4 proteins which the authors suggested might be responsible for the difference in plaque size. Thus, if the mutations found in the large plaque variant of $4 provided growth advantages, it is not unexpected that variants with these mutations would be selected, but it is surprising that variants with so many mutations were selected so rapidly, possibly within a single passage.

The second unique property of the $4 strain was its ability to suppress replication of other rotaviruses in coinfected cells. This property was apparently responsible for its ability to continuously replicate to much higher titers than the large plaque-forming variants in coinfected cells. When passaged at high moi (i.e. > 10), the large plaque-forming variants of $4 were maintained at approximately 0.1% of the level of the tiny plaque-formers, but during low moi (i.e. < 1) passage the large plaque variants quickly dominated. Not only did the $4 strain suppress replication of its large plaque variant but it also suppressed replication of the L4 strain as well as all prototype rotaviruses tested (i.e. RRV, NCDV, Wa). In every case, a high moi of the $4 strain was required in order to suppress replication of the coinfecting virus. None of these viruses appeared to suppress the replication of the $4 strain during high moi passages.

In order to establish that the VP4 protein of $4 or its gene segment was responsible for suppressed replication of coinfecting rotaviruses, cells were coinfected with RRV, $4 or L4 and a reassortant ($4-R4) that contained ten gene segments from $4 and the VP4 gene of RRV. Coinfection with RRV and S4-RRV resulted in no detectable suppression in the replication of RRV, thus indicating that the VP4 protein of $4 was responsible for suppressed RRV replication in cells coinfected with RRV and $4. When cells were coinfected with $4 and $4- R4 which differed in only their VP4 gene, replication of the large plaque-forming $4-R4 strain was suppressed. However, the L4 strain, which differed from $4 by only four amino acids in region 16-280 of the VP4 protein, did not


suppress the $4-R4 strain. Thus, the $4 strain appeared to contain a VP4 protein gene with the unique ability to suppress replication of other rotaviruses in coinfected cells.

There are numerous examples of interference between viruses in vitro and in vivo and such interference can occur by a variety of mechanisms. One mechanism is competit ion for viral receptors. For example, it has been suggested that human herpesvirus 7 (HHV-7) and human immunodeficiency virus (HIV-1) cause reciprocal interference by competition for CD4 [151. Therefore, the final experiment in this study was to determine whether the $4 VP4 protein suppressed replication because of its greater affinity for viral receptors than other rotavirus strains which could result in inhibition of adsorption of these other strains. This apparently was not the case because even a delay of two hours in $4 infection of cells previously infected with the S4-R4 strain resulted in > 50% suppression of replication of the large plaque-forming S4-R4 strain. It appeared, therefore, that some event in the rotavirus replication cycle within the cell was inhibited by the presence of the VP4 protein gene of $4. Because VP4 may become associated with incomplete viral particles prior to budding through the rough endoplasmic reticulum and play a role in receptor recognition [26], $4 VP4 may inhibit one or more of these steps during the assembly of other rotaviruses. Alternatively, $4 VP4 may inhibit an early event in replication of other rotaviruses such as transcription. Finally, it is also possible that the VP4 gene of $4 is preferentially selected and packaged during viral assembly. This appears unlikely because even the reassortants found after coinfection of cells within $4 and RRV contained few RRV segments. Thus, there appeared to be an overall reduction in the synthesis of RRV RNA segments or in their incorpor- ation into viral particles following coinfection with the $4 strain. The exact mechanism of inhibition associated with the by $4 VP4 gene in coinfected cells remains to be determined.

Acknowledgements

The stool specimen containing the 429 strain was collected during a study conducted in the Matlab field studies area of the International Centre for Diarrhoeal Disease Research, Bangladesh by J. D. Clemens, D. A. Sack, N. Huda, F. Ahmed and M. Rao.

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Authors' address: Dr. R. L. Ward, Division of Infectious Diseases, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, U.S.A.

Received September 1, 1995

Documents

Isolation of a human rotavirus containing a bovine rotavirus VP4 gene that suppresses replication of other rotaviruses in coinfected cells