Virulence-Associated Genome Mutations of Murine RotavirusIdentified by Alternating Serial Passages in Mice and Cell Cultures

Takeshi Tsugawa,a,b Masatoshi Tatsumi,a,b Hiroyuki Tsutsumia

Department of Pediatrics, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japana; Rotavirus Vaccine Development Section, Laboratory of InfectiousDisease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USAb

ABSTRACT

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism oftheir attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pupsor in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cul-tures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds ofamino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably con-served the 3= consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during pas-sages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteinsconfirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated withrotavirus virulence.

IMPORTANCE

Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, whileserial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuatedvirus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vac-cines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirusby alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identifymutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes(VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins con-firmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Rotaviruses, which form one genus within the family Reoviri-dae, are divided into at least seven species/groups (A to G/H)

(1, 2). Although group A to C rotaviruses have been detected inhumans with diarrhea, group A rotavirus is the single most im-portant etiologic agent causing severe diarrhea in infants andyoung children worldwide, resulting in approximately 453,00deaths (37% of deaths attributable to diarrhea and 5% of alldeaths) among children �5 years of age in 2008 (3). In the UnitedStates alone, rotavirus (RV) infections are estimated to cause ap-proximately 20 to 30 deaths, 50,000 to 67,000 hospitalizations,390,000 to 410,000 physician visits, and a more than $890 millionto $1 billion societal cost annually (4, 5). Thus, the introduction ofa RV vaccine capable of alleviating this enormous health burdenhas been an important global public health goal.

The RV genome consisting of 11 segments of double-strandedRNA (dsRNA) encodes six structural proteins (VP1 to VP4, VP6,and VP7) and six nonstructural proteins (NSP1-NSP6). The vi-rion has three concentric protein layers, with the outer layer(outer capsid) formed by VP4 and VP7, the middle layer (innercapsid) formed by VP6, and the inner layer (core shell) formed byVP2. VP1 (the viral RNA-dependent RNA polymerase) and VP3(RNA capping enzyme), as well as the 11-dsRNA genomes arelocated inside the core shell with VP2. Six nonstructural proteins(NSP1 to NSP6) participate in replication (1).

Previously, in a study involving a semihomologous system ofgnotobiotic newborn pigs infected with a virulent porcine RV, anavirulent human RV, or their reassortants, Hoshino et al. demon-

strated that (i) the third (VP3), fourth (VP4), ninth (VP7), andtenth (NSP4) porcine RV genes each played an important role inthe virulence of RV infection in piglets and that (ii) all four of theporcine RV virulence-associated genes were required for the in-duction of diarrhea and the shedding of RV in piglets (6). Thesenovel observations suggested a potential new strategy for attenu-ating wild-type human RV, with the prospect of new safe andeffective vaccines.

Serial passage of a virulent wild-type virus in vitro often resultsin loss of virulence of the virus in an original animal host, whileserial passage of a cell culture-adapted avirulent virus in vivo oftengains virus virulence in an original animal host (7). Actually, live-attenuated virus vaccines (against measles, rubella, mumps,chickenpox, yellow fever, and RV diarrhea) were originally pro-duced by serial passages of viruses in cell cultures. Althoughsignificant clinical efficacy and safety of two live-attenuated RVvaccines, i.e., Rotarix (monovalent human RV vaccine [Glaxo-

Received 9 January 2014 Accepted 25 February 2014

Published ahead of print 5 March 2014

Editor: S. Perlman

Address correspondence to Takeshi Tsugawa, tsugawat@sapmed.ac.jp.

T.T. and M.T. contributed equally to this article.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00041-14

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SmithKline]) and RotaTeq (pentavalent human-bovine RV reas-sortant vaccine [Merck]), was experienced in many countries (8–12), the mechanism of their attenuation is not well understood. Toanalyze mechanisms underlying this phenomenon, we passagedserially a cell culture-adapted murine RV EB strain by an alternat-ing the host (mice in vivo and cell cultures in vitro) repeatedly. Wethen harvested the mouse- and cell culture-passaged viruses andfully sequenced their 11 genes. We sought to determine a correla-tion of nucleotide changes with virulence or avirulence, and wetried to provide insight into the mechanisms of attenuation of RVvaccine.

MATERIALS AND METHODSViruses. Murine RV EB strain (G16-P[16]-I7-R7-C7-M8-A7-N7-T10-E7-H9) was isolated from a suckling mouse with diarrhea in the UnitedStates in 1982 (13). At first, this strain was adapted to grow in primaryAfrican green monkey kidney (10 AGMK) cell roller tube cultures and wassubsequently plaque purified on a secondary AGMK cell monolayer. Afterplaque purification, murine RV EB strain was passaged serially in MA104cell roller tube cultures 10 to 20 times. We used this strain as an original virus(Po) in order to start this passage experiment with a completely cell culture-adapted murine RV strain.

Serial passages in mice and cell cultures. Figure 1 is a schematic dia-gram of serial passage history of the present study. Murine RV EB strainwas passaged serially in 4- to 5-day-pld mouse pups (CD-1 or BALB/c; invivo) or in cell cultures (10 AGMK cell or MA104 cell line; in vitro) alter-nately and repeatedly. A cell culture-adapted avirulent murine RV EBstrain was passaged serially 7 to 27 times in 4- to 5-day-old mouse pups(CD-1 or BALB/c; in vivo) that were born to a dam free of RV-specificantibodies. Eight pups were inoculated orally and inspected daily for di-

arrhea by gentle palpation of their abdomen. When diarrhea was ob-served, the infected pups were euthanized. In cases of no diarrhea, theanimals were euthanized at 96 h postinfection (14). Thereafter, the intes-tines of one selected mouse were homogenized, and 50 �l of 10% homog-enates diluted with phosphate-buffered saline (PBS) was used as a nextpassage inoculum. Mouse experiments were approved by the Animal Careand Use Committee at the National Institute of Allergy and InfectiousDiseases and performed according to Animal Biosafety Level 3 practice atthe National Institutes of Health.

The mouse-adapted virulent murine RV EB strain was passaged seri-ally 18 to 40 times in 10 AGMK cell or MA104 cell roller tube cultures (invitro). When ca. 75% of the infected cells displayed cytopathic effects(CPE) within 3 to 5 days postinfection, the cultures were frozen andthawed once. Afterward, the lysates were passaged (i) undiluted or diluted10�1 before the 20th passage and (ii) diluted arbitrarily from 10�1 to 10�5

(100-�l aliquots) after the 20th passage to avoid gene rearrangements.In all, we selected 20 virus samples, i.e., five low mouse-passaged (A8,

B8, C8, G8, and R8), four high mouse-passaged (A17, B17, B27, and E7),six AGMK cell culture-passaged (aa18, aa40, bb18, bb40, gg40, and ii40),and five MA104 cell culture-passaged (cc40, jj40, ll40, kk40, and kk18-pl-4-3-1-1) viruses (Fig. 1). All viruses, except for aa18 and bb18, were fullysequenced, including their 3= and 5= ends, and compared to the originalvirus (Po).

RNA extraction, RT-PCR, and nucleotide sequencing. dsRNA wasextracted with TRIzol (Invitrogen) from 10% intestinal homogenates orinfected cell culture lysates. The primers used for the reverse transcrip-tion-PCR (RT-PCR) are listed in Table 1. The RT-PCR and nucleotidesequencing were performed using a procedure described previously (17).Briefly, the extracted RNA was added to 1 �l of dimethyl sulfoxide. Themixture was denatured at 94°C for 3 min and placed on ice. RT wasperformed at 45°C for 45 min using SuperScript II (Invitrogen), followed

FIG 1 Schematic diagram of serial passage history in the present study. Murine RV EB strain was passaged serially in 4- to 5-day-pld mouse pups (CD-1 orBALB/c; in vivo) or in cell cultures (10 AGMK cell or MA104 cell line; in vitro) alternately and repeatedly. With regard to in vivo passage, eight pups wereinoculated orally and inspected daily for diarrhea by gentle palpation of their abdomens. When diarrhea was observed, infected pups were euthanized. In casesof no diarrhea, the animals were euthanized at 96 h postinfection. Thereafter, the intestines of a selected mouse were homogenized, and 50 �l of 10%homogenates diluted with PBS was used as a next-passage inoculum. With regard to in vitro passage, when ca. 75% of the infected cells displayed CPE within 3to 5 days postinfection, the cultures were frozen and thawed once. Afterward, the lysates were passaged undiluted or diluted 10�1 to 10�5 (100-�l aliquots). Poindicates original murine RV EB strain. It was adapted to grow in 10 AGMK cell roller tube cultures and subsequently plaque purified on a secondary AGMK cellmonolayer. After plaque purification, murine RV EB strain was passaged serially in MA104 cell roller tube cultures 10 to 20 times. Capital (A, B, C, E, G, and R)and lowercase (aa, bb, cc, gg, ii, jj, kk, and ll) letters indicate mouse- and cell culture-passaged viruses. Numbers (7, 8, 17, 18, 27, and 40) indicate the passage level.kk18-pl-4-3-1-1 was plaque purified kk18 consecutively four times in MA104 cells.

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TABLE 1 Summary of primers used for amplification and sequencing of MRV-EB strain cDNAsa

Gene Use Primerb Sense Position Sequence (5=–3=)VP1 RT-PCR VP1-1 � 1–22 GGC TAT TAA AGC TGT ACA ATG G

VP1-M1 � 503–523 TTT ATA AGA GAC GGT TAG ACCVP1-M7 � 1807–1827 AAA GCG GCA AAC TCA ATT GCTVP1-M5 � 915–896 CCC TGC TTT ACT CAT AGC AAVP1-M2 � 2055–2038 TTT CGC TAT TTC GAT CCCVP1-1-4-R � 3302–3282 GGT CAC ATC TAA GCG CTC TAA

FLAC VP1-M15 � 2909–2933 CTT TAG GAG TGC CAA AAA TAG ACG CVP1-M23 � 3079–3098 CCT TTT AAA GGG AAG ATA CCVP1-M21 � 288–269 GGC TAG CTT AGT TTC GAC GGVP1-542m � 542–520 GAC GCC ACG GTG ATG AAT AGG TC

Seq VP1-M12 � 269–288 CCG TCG AAA CTA AGC TAG CCVP1-M24 � 551–571 ATA AGT ACG GTG TTC CAA GACVP1-M4 � 896–915 TTG CTA TGA GTA AAG CAG GGVP1-M18 � 1467–1492 CGC AAA GTC AAC TAG AGA ATA TGC CGVP1-M9 � 2349–2365 GTC AGA TGA TGA AGT TTVP1-M10 � 2955–2973 GGT CTA TGC GCA AGA TAA AVP1-M17 � 1045–1022 CCG CAT CCA CCA TTT TCT GTT TTCVP1-M14 � 1375–1354 CCC TCC GAC CTA ACG GAA TTG GVP1-M11 � 1646–1626 TCT AAC CCC ATG ATG ATA CCCVP1-M19 � 1940–1917 GAG TTA AAT TGA AGC ACA GCG TAGVP1-M8 � 2157–2138 CAG CAC TGC AGC TTG GTC CCVP1-2517m � 2517–2497 CTG TGC TAT TCC TCG TGA CACVP1-M26 � 2943–2920 GTA AGT GTC TGC GTC TAT TTT TGGVP1-M25 � 3286–3267 TCT AAT CTT GAA AGA AAC TGG

VP2 RT-PCR VP2-1 � 1–20 GGC TAT TAA AGG CTC AAT GGVP2-5m � 1337–1356 CCA CGA ATG CAT TAC AGA AAVP2-N2 � 1740–1760 AAT TAA CTT CAG TCA CGT CCCVP2-N1 � 1633–1611 CCC TAA TCG ATT CGA AAG CAA AAVP2-6m � 1991–1966 GGT CAT CCG GAA CAC GTG CAA CAT CGVP2-8 � 2681–2662 GGT CAT ATC TCC ACA GTG GG

FLAC VP2-N4 � 2440–2462 TTT GGT AGC GAA TTA TGA CTG GGVP2-N17 � 2477–2499 ACT AAG GTG TAC AAA CAA ATT CCVP2-N16 � 244–221 AGT CTT GAG AAC TTC GAG TAG CTGVP2-N15 � 282–259 AAA ATT TCA TAC TGT ACT TCT CGC

Seq VP2-N8 � 465–484 AAG ACA CAC TGC CAG ATG GTVP2-N14 � 800–823 CCG CTG AAC AAC GAC ATA ATA TTCVP2-N10 � 1364–1382 CCT CAG ACT CCG TTC CAA AVP2-N13 � 1785–1808 CAG TCA TTC CAA GTC CAC AAA CGCVP2-7m � 1966–1991 CGA TGT TGC ACG TGT TCC GGA TGA CCVP2-N11 � 679–656 CCT TCT GAC CAC GCC TTC GGT CTCVP2-N9 � 1165–1144 TGC CGC AAT TAC TGT CTT AAA AVP2-N3 � 2256–2234 TTT GTC ACT TGA CCA TAG TCT CCVP2-N18 � 2520–2501 GCT CTG AAA TCA AAT TGT TG

VP3 RT-PCR VP3-1 � 1–21 GGC TTT TAA AGC AGT ACC AGTVP3-M17 � 281–305 GCC AAT TTC ACC TAC AAT TTT GAG GVP3-M5 � 976–992 TTT ACC CAC GCC ATT TTVP3-M4m � 1373–1397 GTA TTT ATT CAA AAG CCA TTT AAA GVP3-M12 � 2087–2111 GAA ATA GAG AAA TAC ATT AAT ACG GVP3-495m � 495–471 GTC GCA GCG TTT TGG CAA GTG AAA GVP3-M7 � 1110–1085 CTC CAT TTT TGC CAA TCA ATG TTT CCVP3-M2 � 1647–1628 CCA AAC ATG CCC AGG CAG CCVP3-M11 � 2279–2255 GCG CTT TGA AAC TTT TAA TGA CGT CEB-VP3-End � 2591–2572 GGT CAC ATA GTG ACT GAT GT

FLAC VP3-M10 � 2255–2279 GAC GTC ATT AAA AGT TTC AAA GCG CVP3-M24 � 2375–2398 AAA CTA TAC AAC GCA TTT TAC AAGVP3-M23 � 307–282 GTC CTC AAA ATT GTA GGT GAA ATT GGVP3-495m � Above Above

Seq VP3-M1m � 391–416 CAC AGA TTA TAT ATT TCC AGG CTG GGVP3-M8 � 1915–1942 CAA TTA TTC GTT CGA CCT AAA GAG ATG GVP3-M15 � 603–580 CTG GCG ACT GGA AGT GCT GTA GTC

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TABLE 1 (Continued)

Gene Use Primerb Sense Position Sequence (5=–3=)VP3-M13 � 1397–1373 CTT TAA ATG GCT TTT GAA TAA ATA CVP3-M9 � 1942–1915 CCA TCT CTT TAG GTC GAA CGA ATA ATT GVP3-2580 � 2580–2561 GAC TAG TGT GTT AAG TTT TT

VP4 RT-PCR VP4-Fsm � 1–20 GGC TAT AAA ATG GCT TCA CTVP4-14 � 994–1019 GGC GGT TCG CTA CCC ACT GAC TTC GGVP4-8 � 1671–1690 AGG ACT GGC CGC ATC AGT TTVP4-9m � 1155–1139 CCC TCC AGT GCA TTC GAVP4-15 � 1794–1774 CGA TGA AAC GTC TGT CCA AGCVP4-Rsm � 2359–2343 GGT CAC ATC CTC TAG AC

FLAC VP4-7 � 1951–1969 GCA CAA ATT GCA CCG AAC AVP4-22 � 2063–2086 CTG ACG GAC GTT TCT TCG CAT ACCVP4-21 � 217–196 GGT ATG GTC CAT CGA GCA CTG GVP4-6m � 270–251 GGT CGG TGA GAG TAG AAT AT

Seq VP4-16 � 505–528 GCA GTG GCA AAG CAC ACA GAT CGCVP4-3 � 601–620 TTA ACC GCA CAC TGC GAT TTVP4-18 � 1341–1365 GGG GCT ATA CGG TTT GCC GGC TGC GVP4-10 � 1489–1513 GAG AGA CAA CTA GGC GAA CTA CGC GVP4-11 � 695–671 GTA TTT TGG ATT GGC GGT AAT CCG GVP4-12 � 1365–1341 CGC AGC CGG CAA ACC GTA TAG CCC C

VP6 RT-PCR VP6-F � 1–20 GGC TTT TAA ACG AAG TCT TCVP6-R � 1356–1337 GGT CAC ATC CTC TCA CTA TG

FLAC VP6-1 � 1161–1181 GAC AAC CTA CAA CGC GTA TTTVP6-7 � 1195–1216 TTA GAA GCA TGT TGG TAA AGT GVP6-3 � 268–243 CTG GCT GAC TCA ACA TAA TTT GCG TCVP6-6 � 346–323 CCA TTT CTT TGT GAC TCT CGC ACC

Seq VP6-4 � 498–521 CCA TAT TCA GCA TCA TTC ACA CTGVP6-9 � 913–890 GGT CTC ATC AAT TGA AAC GAA AGG

VP7 RT-PCR Beg9s* � 1–18 GGC TTT AAA AGA GAG AATEnd9s* � 1062–1049 GGT CAC ATC ATA CA

FLAC EB-VP7-1 � 838–854 GGC GGC TCA GAT GTA ATEB-VP7-4 � 871–891 CCA ACA ACT GCA CCA CAA ACCEB-VP7-3 � 304–281 GAT AGT AAA GGC ATA GAG TGG AAGEB-VP7-2 � 390–373 CCC TGT TGG CCA TCC TTT

NSP1 RT-PCR EB-NSP1-1 � 1–21 GGC TTT TTT TAT GAA AAG TCT TEB-NSP1-2 � 1605–1581 GGT TCA CAT TTT TTG CCG GCT AGC G

FLAC NSP1-N3 � 1318–1338 CCA TTT ACT CTC AGC TGT AAANSP1-N9 � 1360–1381 GCA TTA GTA GAG AGA TGG TAT GNSP1-N8 � 223–201 ATT GAC AGA CAT GAT GAA GTG AGNSP1-N2 � 251–230 AAA AAG CAT CTA CCA TAC TGT A

Seq NSP1-N1 � 350–371 CAA TCA ATC ATT CAG TTG TAA ANSP1-N5 � 716–740 CCG TGA AGA CAC TGA TTA ACT CTG GNSP1-N7 � 972–997 CCA ACC AGC ATC TAA AGT ACG CTG CCNSP1-N10 � 547–523 GGT ACG GTA AAT TAG ATT GAT TTG CNSP1-N6 � 898–874 GAT TTA AAT TGT GCG TGA GAA ATG GNSP1-N11 � 1215–1193 AAA ACA GTG AAA TAG AAA GTG AGNSP1-N4 � 1439–1420 TCA ATT AAG CGG TTG GTT GG

NSP2 RT-PCR NSP2-1 � 1–28 GGC TTT TAA AGC GTC TCA GTC GCC GTT TNSP2-2 � 1056–1032 GGT CAC ATA AGC GCT TTC TAT TCT T

FLAC NSP2-N3 � 837–859 TTT GGC AAA ATT GGC ATG CAT TTNSP2-N5 � 877–897 GGG TAA TAC ATT AGA TGT GTGNSP2-N6 � 270–247 TCC GTT TCG AAA TTC ATT CCT CGGNSP2-N2 � 300–281 CAC ACA AGC ATC GCC ACC TT

Seq NSP2-N7 � 427–450 CTC TAA AGA ACT ATT GCT AAA ATCNSP2-N4 � 733–710 AAC TAC ACG ATA GTG GCC CTT CCC

NSP3 RT-PCR NSP3-1 � 1–25 GGC TTT TAA TGC TTT TCA GTT GTT GNSP3-2 � 1072–1046 GGT CAC ATA ACG CCC CTA TAG CCA TTT

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by 94°C for 3 min. PCR was performed using GoTaq DNA polymerase(Promega) under the following conditions: initial denaturation at 95°Cfor 15 min; 40 cycles at 94°C for 45 s, 42 or 50°C for 45 s, and 70°C for 2.5min; and a final extension at 70°C for 7 min. The PCR amplicons werepurified with the Wizard SV Gel and PCR Cleanup system (Promega) andsequenced directly with the BigDye Terminator v3.1 cycle sequencingreaction kit (Applied Biosystems) on an ABI 3730 DNA analyzer (AppliedBiosystems). We confirmed mutations of virus samples (i) by amplifyingone to three different PCR products and (ii) by sequencing them usingtwo to eight different primers.

Determination of the 5=and 3= terminal sequences. To obtain thecomplete nucleotide sequence of all 11 genome segments, 5= and 3= ter-minal sequences of all 11 genome segments were determined using thefull-length amplification of cDNA (FLAC) method described previously(17). Specific primers used for FLAC are given in Table 1.

Sequence analysis. Sequence files were analyzed by using Sequencher4.7 (Gene Codes Corp.) and MacVector 9.5.2 (Accelrys). Some virus sam-ples were composed of mixed populations possessing original or mutatedsequences. In such cases, the dominant virus was selected by signal inten-sity of chromatogram. If we could not find a difference between them, wedescribed them as “original � mutated.” The nucleotide sequence data ofmurine RV EB strains reported in the present study have been submittedto the GenBank nucleotide sequence database and the accession numbersfor each individual genome (VP1 to VP4, VP6, VP7, and NSP1 to NSP5/6)are listed in Table 2.

Construction and characterization of recombinant baculovirusesexpressing rotavirus NSP4 proteins. The construction and characteriza-tion of recombinant baculoviruses expressing selected RV NSP4 proteinswere performed using pBlueBac4.5/V5-His-TOPO vector (pBlueBac4.5/V5-His TOPO TA Cloning kit; Invitrogen) according to the manufactur-er’s instructions. The full-length NSP4 gene was amplified by RT-PCR,inserted into baculovirus transfer vector, and expressed in Spodopterafrugiperda 9 (Sf-9) insect cells. The His-tagged rNSP4 proteins were puri-fied by using Ni-NTA agarose (Qiagen) according to the manufacturer’sprotocol and without removal of the His tags. A high-titer viral stock of

TABLE 1 (Continued)

Gene Use Primerb Sense Position Sequence (5=–3=)FLAC NSP3-N3 � 811–837 GGA ACA GCA ACT GAA TTC GAT TGA TTT

NSP3-N7 � 860–883 GAC GAC ATT GAA ACA TTA ATT CGGNSP3-N6 � 254–231 TGT TGT TTA GAG CCT GAT CTA TGGNSP3-N2m � 295–270 GTC GCA CAT CCA ATT TCT GTT CCT AA

Seq NSP3-N1m � 273–293 GGA ACA GAA ATT GGA TGT GCGNSP3-N5 � 569–595 GAG GCA TCA AAA CAG AAA ACG ACA GAGNSP3-N4 � 635–612 CAT TAG TTT TCT TAG CTT TGG CGG

NSP4 RT-PCR 1UG10ad† � 1–19 GGC TTT TAA AAG TTC TGT TCEndG10s† � 750–735 GGT CAC ATT AAG ACC A

FLAC NSP4-M5 � 342–365 GAG TGG TAA AAG AAT TAA GAC AGCNSP4-M7 � 500–524 AAA ACT CTA CAT GAT TGG AAA AAC GNSP4-M3 � 365–342 GCT GTC TTA ATT CTT TTA CCA CTCNSP4-M6 � 450–425 CGT ACG ATC ATC ATA TCA TAT ATT CG

Seq NSP4-M2 � 480–496 AAA CTA ATC AAA AAG CGNSP4-M1 � 288–274 CCA AGC CTC AGC AAA

NSP5 RT-PCR NSP5-5m � 1–20 GGC TTT AAA AGC GCT ACA GTNSP5-3 � 664–646 GGT CAC AAA ACG GGA GTG G

FLAC NSP5-M1 � 280–303 GCT GGC GTG TCT ATG GAT TCA TGCNSP5-M6 � 396–420 GGA CAC CAC AAG GTC AAA AAT TGC GNSP5-M5 � 239–217 CTG GTG AGT GGA TCG TTC GAA GCNSP5-M2 � 354–331 GGC GAG ATC CAC TTG ATT GCA TCC

a Primers used to prepare cDNAs by RT-PCR and FLAC were also used as sequencing primers. For any particular gene, all of the primers identified in the list were used insequencing reactions.b *, Gouvea et al. (15) with modification; †, Horie et al. (16) with modification.

TABLE 2 GenBank accession numbers of the murine rotavirus EBstrains sequenced in this study

Virus strainAccession nos. (VP1 to VP4, VP6,VP7, and NSP1 to NSP5/6)

OriginalPo JF309297-JF309307

Mouse, low passageA8 KJ477105-KJ477115B8 KJ477116-KJ477126C8 KJ477127-KJ477137G8 KJ477138-KJ477148R8 KJ477149-KJ477159

Mouse, high passageA17 KJ477160-KJ477170B17 KJ477171-KJ477181B27 KJ477182-KJ477192E7 KJ477193-KJ477203

AGMK cell passageaa18 KJ477204-KJ477214aa40 KJ477215-KJ477225bb18 KJ477226-KJ477236bb40 KJ477237-KJ477247gg40 KJ477248-KJ477258ii40 KJ477259-KJ477269

MA104 cell passagecc40 KJ477270-KJ477280jj40 KJ477281-KJ477291ll40 KJ477292-KJ477302kk40 KJ477303-KJ477313kk18-pl-4-3-1-1 KJ477314-KJ477324

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the recombinant baculoviruses was prepared in Sf-9 cells, and its titer wasdetermined using a plaque assay according to the manufacturer’s instruc-tions (Bac-N-Blue transfection kit; Invitrogen). A working viral stock of107 PFU/ml was prepared using Grace’s medium (Gibco). Western blotanalysis was used to confirm the specific protein expression of each re-combinant NSP4 (rNSP4) protein in Sf-9 cells. Hyperimmune guinea pigantiserum raised against NSP4 of murine RV EB strain diluted at 1:250reacted with all rNSP4 proteins (18).

Diarrhea induction in neonatal mice. Purified 1 nmol of the rNSP4protein was diluted to 50 �l in sterile, endotoxin-free PBS and was in-jected intraperitoneally into 5- to 6-day-old CD-1 mouse pups by using a30G needle. The neonatal mice were isolated and kept warm and humid.To determine the presence of diarrhea, we examined each neonatal mouseevery 1 h for the first 8 h and then 12 and 24 h after inoculation by gentlypressing its abdomen. The pups were monitored by one researcher. Wa-tery diarrhea and loose yellow stool with some liquid were classified intothe category of diarrhea. Loose yellow stool was not considered diarrhea.All pups were screened at 24 h after inoculation. Endotoxin-free PBS wasused as a negative control. Statistical analysis was performed at the 5%level of significance using a chi-square test with Yates’ correction.

Nucleotide sequence accession numbers. The sequences newly deter-mined in this study were deposited in GenBank under accession numbersKJ477105 to KJ477324 (Table 2).

RESULTSVirulence of RV during serial passages in mice or in cell cul-tures. Figure 1 is a schematic diagram of serial passage history inthe present study. During the initial serial passages in mice fromthe original virus (Po) to A8, infected pups started developingsigns of diarrhea after passage 4, and after four additional serialpassages (for a total of eight passages), all infected pups developeddiarrhea. Then, 10% intestinal homogenate from passage 8 inpups (A8) was inoculated onto 10 AGMK cells and passaged seri-ally 18 times in cell cultures. Subsequently, cell lysate from passage18 in 10 AGMK cells (aa18) was passaged serially in CD-1 mousepups. Infected pups did not develop diarrhea until passage 5. Aftera subsequent three passages (a total of eight passages), all infectedpups developed diarrhea (B8). During in vivo passage, infectedpups started diarrhea after passage 4, and all of them developeddiarrhea after passage 8. Additional passages were done using miceor cell cultures in the same manner.

At the onset, the original virus (Po) was considered to consistof a mixed population, because it had already been passaged seri-ally in MA104 cells 10 to 20 times after plaque purification. There-fore, we could not conclude whether the mutations detected werenewly induced mutations whenever hosts alternated or mutationsselected from closely related, but slightly heterogeneous popula-tions (quasispecies) that were possibly maintained in each inocu-lum. To investigate this question, we obtained kk18-pl-4-3-1-1from cell culture-passaged kk18 virus after four consecutiveplaque purifications, and considered this to be a single avirulentclone. After eight serial passages of kk18-pl-4-3-1-1 in mice, vir-ulent virus, i.e., R8 appeared (Fig. 1); this implied the possibility ofentire newly induced virulence-associated mutations. On the otherhand, purification of mouse-passaged virulent virus from intestinalhomogenate was difficult by plaque purification or limiting dilution.Therefore, we could not conduct the infection experiment with pu-rified virulent virus clone. In the present study, we used 50 �l of 10%intestinal homogenates for in vivo inoculation. The virus inoculumwas not always titrated every time before every in vivo passages; how-ever, we measured the focus-forming unit (FFU) titer of selected virus

inocula together afterward and confirmed the titer to be mostly 2 �106 to 1 � 107 FFU/ml (data not shown).

Mutations and substitutions detected during serial passagesin mice and/or in cell cultures. Mutations and substitutions de-tected during serial passages in mice and/or in cell cultures in thepresent study are summarized in Table 3. At least five mutationswere detected in each of all eleven genes. More than half of themutations were nonsynonymous substitutions in the VP4 (24/31), VP7 (7/10), NSP2 (9/12), NSP4 (11/13), and NSP5/6 (4/5)genes. These five genes also had higher substitution rates (2.03 to6.29%) than the other genes (0.36 to 0.83%).

Summary of dominant mutations detected during serial pas-sages in mice or in cell cultures. To determine the mutations thatcould be associated with virulence or lack of virulence, mutationsdetected exclusively in either mouse (virulent)- or cell culture(avirulent)-passaged viruses were collected. Mutations occurringin more than three virus samples are summarized in Fig. 2. Char-acteristic mutations of mouse-passaged viruses were four muta-tions in the VP4 gene and one mutation each in the VP7 and NSP4genes. Especially noteworthy was that four mutations in the VP4gene and one mutation in the NSP4 gene were detected in five toseven of nine mouse-passaged viruses. R8, which was obtainedfrom serial passages in mice of plaque-purified avirulent virus(kk18-pl-4-3-1-1), also had these characteristic mutations (two inthe VP4 gene and one in the NSP4 gene).

Interestingly, these mutations disappeared again, as confirmedduring the following cell cultures; five mutations in the VP4 orNSP4 genes, which were gained in the mouse-passaged B17 strain,were all lost in the subsequent cell culture-passaged viruses (ii40and jj40). Similarly, three mutations in the VP4 gene in mouse-passaged G8 strain disappeared in the following cell culture-pas-saged virus (gg40). All mutations and substitutions detected ineach of eleven genes during serial passages in mice and/or in cellcultures are summarized in Fig. 3A to K, respectively.

VP4 mutations and substitutions detected during serial pas-sages in mice and/or in cell cultures. Figures 3D and 4A summa-rize the mutations and substitutions detected in the VP4 gene

TABLE 3 Summary of mutations and substitutions detected duringserial passages in mice and/or in cell cultures

Genea

Totallength(bp)

Total no.ofmutations

Mutationrate (%)b

Totalproteinlength(aa)

Total no. ofsubstitutions

Substitutionrate (%)c

VP1 3,302 27 0.82 1088 9 0.83VP2 2,681 15 0.56 878 5 0.57VP3 2,591 13 0.50 835 3 0.36VP4 2,359 31 1.31 775 24 3.10VP6 1,356 6 0.44 397 2 0.50VP7 1,062 10 0.94 326 7 2.15NSP1 1,605 10 0.62 493 3 0.61NSP2 1,056 12 1.14 316 9 2.85NSP3 1,072 11 1.03 310 2 0.65NSP4 750 13 1.73 175 11 6.29NSP5/6 664 5 0.75 197 4 2.03a Underlining indicates genome segments for which the mutation rate was far below thesubstitution rate.b The mutation rate was calculated as the total number of mutations/total length (bp).c The substitution rate was calculated as the total number of substitutions/total length(aa).

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during serial passages in mice and/or in cell cultures. A total of 31mutations were detected during these passages. Of the 31 substi-tutions, 24 were nonsynonymous. Among them, four mutations(substitutions) at nucleotide (nt) 1162 (amino acid [aa] 385), nt1618 (aa 537), nt 1622 (aa 538), and nt 1681 (aa 558), were con-sistently detected in all four high mouse-passaged viruses. Thesubstitution at aa 385 was located in the putative fusion domain(Fig. 4A).

VP7 mutations and substitutions detected during serial pas-sages in mice and/or in cell cultures. Figures 3F and 4B summa-rize the mutations and substitutions detected in the VP7 geneduring serial passages in mice and/or in cell cultures. A total of tenmutations were detected during these passages. Seven of the tensubstitutions were nonsynonymous. Among them, one mutation(substitution) at nt 760 (aa 238) was detected in two of four highmouse-passaged viruses. The substitution at aa 238 was located inan N-glycosylation site, an integrin-binding region, and neutral-ization epitope F (Fig. 4B).

NSP4 mutations and substitutions detected during serialpassages in mice and/or in cell cultures. Figures 3J and 4C sum-marize the mutations and substitutions detected in the NSP4 geneduring serial passages in mice and/or in cell cultures. A total ofthirteen mutations were detected during these passages. Eleven ofthese mutations were nonsynonymous. There were no substitu-tions detected in the enterotoxin domain (aa 114 to 135) in eithergroup. On the other hand, the substitution at aa 37 (Val to Ala)was detected in six of nine mouse-passaged viruses (three of

four high mouse-passaged viruses). This site corresponds tothe H2 domain (Fig. 4C). In addition, substitution at aa 21 (Leuto Ile) was observed in eight of eleven cell culture-passagedviruses (Fig. 3J). This mutation was also observed in two ofnine mouse-passaged viruses (A8 and B27); therefore, it wasnot presented in Fig. 2.

Each of three recombinant NSP4 proteins displayed differ-ent diarrhea-causing capabilities in mouse pups. As mentionedabove, the main substitutions in the NSP4 protein were observedat amino acid positions 21 and 37 (Fig. 3J and 4C). As a result,three representative NSP4 phenotypes were confirmed. The firstwas the NSP4 protein with Ile-21 and Val-37, which was observedin a part of the cell culture-passaged viruses (aa40, bb40, andcc40). The second was the NSP4 protein with Leu-21 and Val-37,which was confirmed in the original virus (Po) and one cell cul-ture-passaged virus (aa18). The third was the NSP4 protein withLeu-21 and Ala-37, which was confirmed in the greater part ofmouse-passaged viruses (R8, A17, B17, and E7).

To test an enterotoxin activity of each NSP4 protein (19), we con-structed rNSP4 proteins, which bore the above-mentioned threekinds of combinations of amino acid positions 21 and 37, using abaculovirus expression system (Table 4). Each of three rNSP4 pro-teins was injected into 5- to 6-day-old CD-1 mouse pups intraperito-neally. The first rNSP4 protein (Ile-21, Val-37) did not cause anydiarrhea in twelve mouse pups. Only one of eight mouse pups inoc-ulated with the second rNSP4 protein (Leu-21, Val-37) developeddiarrhea. On the other hand, eleven of twelve mouse pups inoculated

FIG 2 Summary of mutations detected in more than three virus samples during serial passages in mice or in cell cultures. “nt” and “aa” indicate nucleotides andamino acids, respectively. A blue background indicates nonsynonymous substitution. Yellow and gray backgrounds indicate mutations only detected inmouse-passaged virus and in cell culture-passaged virus, respectively. More than 10 mutations detected both in mice and cell cultures are not presented in thisfigure. All of the mutations detected in eleven genes are summarized in Fig. 3A to K, respectively.

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FIG 3 Summary of mutations and substitutions detected in each of 11 genes (VP1 to VP4, VP6, VP7, and NSP1 to NSP5/6 [A to K, respectively]) during serialpassages in mice and/or in cell cultures. “nt” and “aa” indicate nucleotides and amino acids, respectively. Blue and yellow-green backgrounds indicate nonsyn-onymous substitutions and untranslated regions, respectively. Yellow, gray, and pink backgrounds indicate mutations only detected in mouse-passaged virus, incell culture-passaged virus, or both, respectively. ND, no sequencing data.

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FIG 3 continued

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with the third rNSP4 protein (Leu-21, Ala-37) developed diarrhea.None of the twelve mouse pups inoculated endotoxin-free PBS hadany diarrhea. As a result, Ala-37, which was found in most of mouse-passaged (virulent) viruses, was considered to be the critical substitu-tion to exhibit NSP4 enterotoxin activity.

Alteration of 3=CSs of the NSP1 and NSP3 genes detectedduring serial passages in mice and/or in cell cultures. Nine ge-nome segments did not change their 3= end sequences duringserial passages in mice and cell cultures, the exceptions being theNSP1 and NSP3 genes. In the original virus (Po), the 3= consensus

FIG 4 Schematic diagram of substitutions detected on VP4 (A), VP7 (B), and NSP4 (C). Numbers indicate the amino acid position on each of three genes (1,22, 34–36). Red, blue, and green arrowheads indicate substitutions detected in murine RVs passaged in mouse, cell culture, or both, respectively. Big, middle, andsmall arrowheads indicate substitutions occurring, respectively, in �5, in 3 to 4, or in 1 to 2 strains detected during serial passages in mice and/or in cell cultures.(A) Substitution at aa 538 detected in both passages in mice and passages in cell cultures (see Fig. 3J).

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sequence (3=CS; UGU GACC) in the NSP1 gene had alreadychanged to UGU GAACC (Table 5) (20). However, 3=CS in theNSP1 gene of subsequent mouse-passaged viruses was regainedand invariably conserved. On the other hand, it was lost andchanged in all nine cell culture-passaged viruses (no data for aa18and bb18). In short, it changed to UGU GAACC in seven strains(aa40, bb40, gg40, ii40, jj40, kk40, and kk18-pl-4-3-1-1), to UGUGAUCC in one strain (ll40), and to UGU GACACC in one strain(cc40).

Also in the NSP3 gene of original virus (Po), 3=CS had alreadychanged to UGU GGCC. However, 3=CS was regained in the fol-lowing mouse-passaged virus and conserved in subsequentmouse- or cell culture-passaged viruses, except in four cell cul-ture-passaged viruses (bb40, ll40, kk40, and kk18-pl-4-3-1-1) inwhich it returned to UGU GGCC.

DISCUSSION

In general, there are four patterns for genetic mutation—pointmutation, reassortment, rearrangement, and recombination— ofwhich point mutation and reassortment are important in RV vac-cine development (7). Rotarix (monovalent human RV vaccine;Glaxo-SmithKline) was developed by accumulation of pointmutations during serial passage in cell cultures, and RotaTeq(pentavalent human-bovine RV reassortant vaccine; Merck)was developed by reassortment; however, the mechanism of theirattenuation is not well understood (8, 9, 21). To analyze mecha-nisms underlying this phenomenon, we passaged serially a murineRV EB strain in 4- to 5-day-old mouse pups (CD-1 or BALB/c; invivo) or in cell cultures (10 AGMK cells or MA104 cell line; in vitro)alternately and repeatedly. We then fully sequenced all eleven ge-nome segments of 21 passaged RV isolates and analyzed the cor-relation between point mutations and RV virulence. Using thereciprocal in vivo/in vitro system, we demonstrated the acquisitionand subsequent loss of virulence-associated gene expression ofmurine RV.

In the present study, we showed that virulent mutations couldbe newly induced, e.g., plaque-purified avirulent kk18-pl-4-3-1-1could produce virulent R8 RV after eight serial passages in mice(Fig. 1). On the other hand, we failed to isolate a single virulentclone population from mouse-passaged viruses (A8, B8, G8, andB17) either by plaque purification or by limiting dilution. Thus,we were unable to conduct the infection experiment with purifiedvirulent virus. Therefore, we could not conclude whether the mu-tations detected were either newly induced mutations wheneverhosts alternated or mutations selected from the quasispecies pop-ulation that were possibly maintained in each inoculum. In any

case, the viruses that acquired or preserved virulence-associatedmutations could proliferate well and become predominant inmice, and eventually result in the onset of diarrhea. In contrast,the viruses lacking virulence-associated mutations might havesome kind of growth advantage in cell cultures, becoming thedominant population.

Since the VP4 and VP7 gene products are located on the virusparticle and induced neutralization antibody, it was interestingthat we confirmed consistent or dominant substitutions in theirfusion domain (aa 385 of VP4), glycosylation sites (aa 238 of VP7),or integrin-binding region (aa 238 of VP7) (Fig. 4A and B) (1, 22,23). By using a genetic approach, previous studies showed that atleast eight genome segments (i.e., genes encoding VP3, VP4, VP6,VP7, NSP1, NSP2, NSP3, and NSP4) were involved in the viru-lence of rotaviruses (6, 17, 24, 25). Our analysis of sequence datafrom mouse-passaged (virulent, in vivo) and cell culture-passagedviruses (attenuated, in vitro) was generally consistent with those ofprevious genetic approaches, although there were some dispari-ties.

Sequencing analyses between parental (virulent) and serial cellculture-passaged (attenuated) human RV G1P[8] strains wereperformed for the VP4 gene (26–29). Substitution at aa 385 wasfound in two of four studies (27, 28). When the VP4 sequence ofstrain 89-12 (progenitor of Rotarix) was compared to that of theoriginal strain, five substitutions were detected (aa 51, 167, 331,385, and 695) (27). On the other hand, when strain CDC-9 (at-tenuated live vaccine candidate) was passaged many times, fivesubstitutions (aa 51, 331, 364, 385, and 388) were detected in theVP4 gene (28). In addition to substitution at aa 385, substitutionat aa 51 and 331 was confirmed in both studies. In the analysis ofassociation between amino acid substitutions and the neutraliza-tion resistant mutants using monoclonal antibodies, substitutionat aa 385 in the VP4 gene was also detected (30). The crystal struc-ture analysis revealed that the region between aa 382 and 400 inthe VP4 gene was a potential membrane interaction loop and wasconfirmed to interact with lipid bilayer directly (31, 32). Putting itall together, substitution at aa 385 in the VP4 gene appears to beessentially associated with RV virulence, especially from the aspectof viral attachment or penetration.

With regard to the VP7 gene, sequencing analysis of parental(virulent) and serial cell culture-passaged (attenuated) viruses wasreported in a human RV study, although no substitutions weredetected (28). On the other hand, in the study on neutralizationresistant mutants by using monoclonal antibodies, substitution ataa 238 in the VP7 gene was confirmed, as we saw in our study (30).When aa 238 in the VP7 gene is Asn (N) or Asp (D), it createseither the N-X-T motif (238N) as a N-glycosylation site or theL-D-V motif (238D) as an integrin-binding region (Fig. 4B) (1,22). In the present study, we showed that all cell culture-passagedviruses conserved the N-X-T motif (238N), whereas one of fivelow mouse-passaged (C8) and two of four high mouse-passagedviruses (A17 and B27) acquired the L-D-V motif (238D). Absolutepreservation of the N-X-T motif (238N) as an N-glycosylation sitein cell-cultured viruses implied its superiority for propagation incell culture and agreed with the findings of Graham et al. (33) thatsimian RV SA11 strain, which conserved the N-X-T motif (238N)in the VP7 gene in cell culture, achieved a 10-fold-higher titer thanthe nonconserving strain. In contrast, the significance of our find-ing regarding the acquisition of the L-D-V motif (238D) as an

TABLE 4 Comparison of diarrhea-causing ability in mouse pups giveneach of three recombinant NSP4 proteinsa

Recombinant segmentlength (no. of nt, no.of aa)

Treatment group

Control(PBS) Attenuated

Original(Po) Virulent

101, 21 NA A (Ile) C (Leu) C (Leu)150, 37 NA T (Val) T (Val) C (Ala)a nt, nucleotides; aa, amino acids. Animals with diarrhea (number of mice withdiarrhea/total number of mice) were as follows: control (PBS), 0/12; attenuated, 0/12;original (Po), 1/8; and virulent, 11/12. Statistical analysis was performed at a 5% level ofsignificance using a chi-square test with Yates’ correction: control/virulent, P � 0.01;attenuated/virulent, P � 0.01; and original/virulent, P � 0.01. NA, not applicable.

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TABLE 5 Conservation of the 3= consensus sequence (3=CS) in the NSP1 gene of RVA from GenBank data

Straina Host

Genotype

3=CS (UGU GACC)GenBankaccession no.

Reference(PubMed ID)G/P NSP1

Wild typeEB-mouse Murine G16P[16] A7 Conserved This study This studyB4106 Human G3P[14] A9 Conserved AY740735 16571797DRC86 Human G8P[6] A2 Conserved DQ005119 16672410DRC88 Human G8P[8] A2 Conserved DQ005108 16672410B4633 Human G12P[8] A1 Conserved DQ146644 17166908Dhaka12 Human G12P[6] A1 Conserved DQ146666 17166908Dhaka16 Human G1P[8] A1 Conserved DQ492675 17166908Dhaka25 Human G12P[8] A1 Conserved DQ146655 17166908N26 Human G12P6] A2 Conserved DQ146688 17166908RV161 Human G12P[6] A2 Conserved DQ490540 17166908RV176 Human G12P[6] A2 Conserved DQ490557 17166908Matlab13 Human G12P[6] A1 Conserved DQ146677 17166908B3458 Human G9P[8] A1 Conserved EF990709 18216098TB-Chen Human G2P[4] A2 Conserved AY787647 18329063B1711 Human G6P[6] A2 Conserved EF554088 18796733RC-18-08 Antelope G6P[14] A11 Conserved FJ495130 19153225B383 Bovine G15P[11] A13 Conserved FJ347117 19153225Chubut Guanaco G8P[14] A3 Conserved FJ347106 19153225Rio_Negro Guanaco G8P[1] A11 Conserved FJ347128 19153225GR10924/99 Human G9P[6] A2 Conserved FJ183357 19264638PAH136 Human G3P[9] A3 Conserved GU296410 20409385PAI58 Human G3P[9] A3 Conserved GU296411 20409385BA222 Feline G3P[9] A3 Conserved GU827412 21228122mani-265/07 Human G10P[6] A3 Conserved HM348718 21884295MWI/1473 Human G8P[4] A2 Conserved HQ657133 219158793133WC Human G12P[4] A1 Conserved HQ657144 219158793176WC Human G12P[6] A1 Conserved HQ657155 219158793203WC Human G2P[4] A2 Conserved HQ657166 219158792371WC-B Human G9P[8] A1 Conserved JN013975 220195212371WC-A Human G9P[8] A2 Conserved JN013974 220195211603 Bovine G6P[5] A3 Conserved JN831204 225411631604 Bovine G8P[1] A3 Conserved JN831215 225411631605 Bovine G6P[5] A3 Conserved JN831226 22541163E30 Equine G3P[12] A10 Conserved JF712572 22190012E403 Equine G14P[12] A10 Conserved JF712583 22190012E4040 Equine G14P[12] A10 Conserved JN872871 2219001204V2024 Equine G14P[12] A10 Conserved JN903515 22190012EqRV-SA1 Equine G14P[12] A10 Conserved JQ345493 22190012

Tissue cultureEB-tc Murine G16P[16] A7 UGU GAACC This study This studyEB-tc-ll40 Murine G16P[16] A7 UGU GAUCC This study This studyEB-tc-cc40 Murine G16P[16] A7 UGU GACACC This study This studyKU Human G1P[8] A1 UGU GAACC AB022769 10481750K9 Canine G3P[3] A9 UGU GAACC AF111946 10481750RV198-95 Canine G3P[3] A9 UGU GAACC HQ661140 21609783SA11-4F Simian G3P[2] A5 UGU GAACC AF290883 11160712SA11-30-19 Simian G3P[2] A5 UGU GAACC AF290881 11160712SA11-N2 Simian G3P[2] A5 UGU GAACC JN827248 23263646SA11-N5 Simian G3P[2] A5 UGU GAACC JQ688677 23263646L338 Equine G13P[18] A6 UGU GAACC JF712561 22190012RRV-AUU Simian G3P[3] A9 UGA UUCC AY117049 15372078RRV-CCUU Simian G3P[3] A9 UGCC UUCC AY117050 15372078RRV-CUU Simian G3P[3] A9 UGC UUCC AY117051 15372078RRV-U Simian G3P[3] A9 UG UUCC AY117052 15372078RRV-UU Simian G3P[3] A9 UGU UUCC AY117053 1537207810V0112H5 Avian G23P[37] A16 UAU GACC JX204817 2305239603V0002E10 Avian G22P[35] A16 UAU GACC JX204828 23052396RotaTeq-BrB-9 Vaccine G4P[5] A3 Conserved GU565091 20451234

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integrin-binding region in the VP7 gene in mouse-passaged vi-ruses remains to be elucidated.

NSP4 is a multifunctional protein and is essential for RV rep-lication, transcription, and morphogenesis (19, 34). Apart fromenterotoxin, a lot of NSP4 functions have been mapped to do-mains (Fig. 4C) (34–36). Sequencing analyses of the NSP4 genebetween parental (virulent) and serial cell culture-passaged (at-tenuated) viruses were reported in four human group A rotavirus(RVA) studies, one murine RVA study, and one porcine RVAstudy (28, 37–41). All strains in human RV studies belonged to theG1P[8] genotype, and substitutions at aa 16 (Leu to Ser) and aa 34(Pro to Leu) were detected commonly between parental and serialcell culture-passaged human RV Wa strains, although no clinicaldata were presented (40, 41). In a murine RV study, three murineRV strains (EW, EHP, and EC) were passaged serially in 10 AGMKand MA104 cell cultures (38). Substitution at aa 45 (Thr to Met)was detected commonly between parental and serial cell culture-passaged EW and EHP strains; however, its association with viru-lence was not demonstrated in vivo. In a porcine RV study, twoporcine RV strains (OSU and Gottfried) were passaged serially incell cultures (39), and substitutions at aa 135 (Val to Ala) and aa138 (Pro to Ser) were commonly detected. Although aa 135 waswithin the enterotoxin domain (aa 114 to 135), no association ofthese substitutions with in vivo virulence was presented.

On the other hand, in rNSP4 protein study, we found thatsubstitution at aa 37 (Val to Ala), which was observed dominantlyin the mouse-passaged viruses, was evidently associated with vir-ulence in mice (Table 4). Although aa 37 locates to one of threehydrophobic lesions (H2) and to a transmembrane domain, thefunction of this region, including virulence, remains unknown(Fig. 4C) (34–36). Previously, Zhang et al. reported that aa 138,which is also located outside the enterotoxin domain, was associ-ated with virulence of porcine RV OSU strain based on the studyin mice with rNSP4 proteins (39). Although the mechanism ofrNSP4 virulence was unknown, the difference of substitution po-sition to ours might be due to the different animal hosts used (i.e.,a murine RV EB-homologous animal host versus a porcine RVOSU-heterologous animal host). In addition to these rNSP4 stud-ies, comparative sequencing studies between parental (virulent)and serial cell culture-passaged (attenuated) viruses confirmed

several mutations outside the enterotoxin domain (28, 37–41).On the other hand, no explanation has been offered as to howregions outside the enterotoxin domain regulate enterotoxin ac-tivity. For real insight into this question, future animal studiesusing a universally applicable and fully tractable reverse geneticssystem should be necessary.

All eleven genome segments of RV lack a polyadenylation sig-nal and contain conserved consensus sequences at their 5= and 3=ends. Previously, the 3=CSs (UGU GACC) of eleven genome seg-ments of RV were confirmed as an activator to promote dsRNAsynthesis and translational enhancer to increase the expression ofthe viral proteins (20). Interestingly, in the present study, wefound that the 3=CS of the NSP1 gene was completely conserved inmouse-passaged viruses but lost in all cell culture-passaged vi-ruses. Previous studies reported that the loss of 3=CS of the NSP1gene during serial passages in cell cultures was intricate and de-pended on the combination of RV strain, cell type, and passageconditions (20, 42). Therefore, we investigated 3=CS of the NSP1gene in the GenBank database. We collected relevant informationon 37 wild-type RVs and 29 tissue culture-adapted RVs (Table 5).Interestingly, 3=CSs of the NSP1 gene of 37 wild-type RVs werecompletely conserved, although their hosts and genotypes (VP7/VP4 and NSP1) varied greatly. On the other hand, those of almosthalf of the tissue culture-adapted RVs were lost and had changeddiversely. From the aspect of RV virulence, it was recently revealedthat RVs evaded the innate immune response, especially the inter-feron response through the NSP1 gene promoting the degradationof IRF3 and inhibition of NF-B activity (43). Although no directrelationship between the 3=CS of the NSP1 gene and the virulenceof RV has been described, the conservation of the 3=CS of theNSP1 gene must be associated with the expression of RV virulencenot only in the mouse but also in other hosts.

Although not fully determined, it was strongly suggested thatthe consistent or dominant mutations confirmed here were atleast partly associated with virulence, because they were exhibitedalong with the acquisition of virulence during passages in vivo (inan original animal host, i.e., mice) and disappeared during afterpassages in vitro (in cell cultures). In addition, enterotoxic activityof rNSP4 protein bearing particular substitutions was clearly pre-sented. Currently, we are investigating (i) the presence of major

TABLE 5 (Continued)

Straina Host

Genotype

3=CS (UGU GACC)GenBankaccession no.

Reference(PubMed ID)G/P NSP1

RotaTeq-SC2-9 Vaccine G2P[5] A3 Conserved GU565069 20451234RotaTeq-WI78-8 Vaccine G3P[5] A3 Conserved GU565080 20451234RotaTeq-WI79-4 Vaccine G6P[8] A3 Conserved GU565047 20451234RotaTeq-WI79-9 Vaccine G1P[5] A3 Conserved GU565058 20451234B10 Human G3P[2] A5 Conserved HM627559 21035567DS-1 Human G2P[4] A2 Conserved HQ650120 21600242PA260-97 Human G3P[3] A15 Conserved HQ661118 21609783RV52-96 Canine G3P[3] A9 Conserved HQ661129 2160978330-96 Lapine G3P[14] A9 Conserved DQ205225 16571797RRV Simian G3P[3] A9 Conserved AY117048 15372078PO-13 Avian G18P[17] A4 Conserved AB009633 11325467RF Bovine G6P[1] A3 Conserved M22308 2823457UK Bovine G6P[5] A3 Conserved Not available 15372078

a The EB prefix indicates the murine RV EB strain used in this study. EB-mouse is the murine RV EB strain passaged by mouse pups. EB-tc is the murine RV EB strain passaged bycell cultures except for ll40 and cc40.

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and minor variant (quasispecies) within the original and passagedviruses by next-generation cDNA sequencing and (ii) whether themutations seen in the present study (murine RV EB strain) arealso consistently observed in human RV strains passaged seriallyin cell cultures. At the present stage, we do not have any helpervirus-free, fully tractable reverse genetics system of RVs (44–48),therefore, the analysis of how RV virulence and point mutationsare correlated could be very valuable for the next stage of RVvaccine development.

ACKNOWLEDGMENTS

This study was supported by the Intramural Research Program of theNational Institute of Allergy and Infectious Diseases, National Institutesof Health. There is no conflict of interest to declare.

We thank R. W. Jones for expert technical assistance, A. Z. Kapikianfor reviews of the manuscript, and Y. Hoshino for continuing great sup-port. We also thank P. Olley, University of Alberta, for English languageadvice.

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