Identification by Full Genome Analysis of a Bovine Rotavirus Transmitted Directly to, and Causing Diarrhea in a Human Child.

Transcription

1 JCM Accepts, published online ahead of print on 31 October 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved Identification by Full Genome Analysis of a Bovine Rotavirus Transmitted Directly to, and Causing Diarrhea in a Human Child Yen Hai Doan 1, Toyoko Nakagomi 1, Yair Aboudy 2, Ilana Silberstein 2, Esther Behar- Novat 3, Osamu Nakagomi 1, Lester M. Shulman 2,4 * 1 Department of Molecular Epidemiology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, and the Global Center of Excellence, Nagasaki University, Nagasaki , Japan. 2Central Virology Laboratory, Public Health Services, Israel Ministry of Health, Sheba Medical Center, Tel Hashomer, Israel Histocompatibility, Immunogenetics, and Disease Profiling Laboratory, Stanford Medical School Blood Center, 3373 Hillview Avenue, Palo Alto, CA Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, Running title: Bovine rotavirus infecting a child. *Corresponding author Lester M. Shulman Head, National Center for Viral Gastroenteritis Head Laboratory of Environmental Virology Public Health Services, Israel Ministry of Health Central Virology Laboratory At the Chaim Sheba Medical Center Tel-Hashomer, Phone: Fax: lester.shulman@sheba.health.gov.il

2 Abstract The genome of rotaviruses consists of 11 segments of double-stranded RNA, and each genome segment has multiple genotypes. Thus, the genotype constellation of an isolate is often indicative of its host species. Albeit rarely, however, interspecies transmission occurs either by virions with non-reassorted or reassorted genomic segments. A rotavirus with P[1] genotype, Ro8059, was isolated from the stool of a one-year old during routine characterization of diarrheal specimens from a sentinel clinic in Israel in Since genotype P[1] is generally associated with bovine rotaviruses, and the child developed diarrhea within days of his first contact with calves at an urban farm, the aim of this study was to characterize the whole genomic constellation of Ro8059 and four P[1] bovine strains BRV101, BRV105, BRV 106 and C31/39 by RNA RNA hybridization and full genome sequencing to determine whether some or all of the segments were of bovine origin. The genome constellations of all four bovine P[1] strains were -P[1]-----A3--T6-- for VP7-VP4-VP6-VP1-VP2- VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Ro8059 shared the same genotype constellation with these bovine strains with high nucleotide sequence identities ( %) for each of the 11 segments indicating that Ro8059 represented a direct interspecies whole genome transmission of a non-reassorted rotavirus from a calf to a human infant. We conclude that this was the earliest example with a complete epidemiological link in which an entirely bovine rotavirus directly infected a human child and caused a symptomatic diarrheal illness. Thus, not all bovine rotaviruses are always naturally attenuated to the human host.

3 Key Words Zoonosis, Full genome sequence analysis, Bovine Rotaviruses, human rotaviruses, electropherograms, RNA-RNA hybridization, Phylogenetic analysis. Downloaded from on November 3, 2018 by guest

4 Introduction Group A rotaviruses, the most important etiologic agents of severe diarrhea in infants and young children worldwide, possess a genome consisting of 11 segments of double-stranded RNA encoding six structural viral proteins (VPs) and six nonstructural proteins (NSPs). Each genome segment encodes a single viral protein, except segment 11 that encodes two, NSP5 and NSP6. The outer protein layer of the virion consists of two independent neutralization antigens, VP7 (which defines G-types) and VP4 (which defines P-types). There have been 27 G-types and 35 P-types reported to date (22). However, only five- G and P-type combinations have been commonly associated with human rotaviruses (9, 44). They are G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. Less common combinations, thought to have originated from animal rotaviruses, have also been identified(9, 45). These include human rotaviruses expressing G8, G10, G11 or G12 G-types. The relative frequency of some of these emerging genotypes has been increasing and varies depending on the geographic location. Examination of the whole genomic constellation of these uncommon strains by RNA-RNA hybridization and sequencing of all 11 genome segments revealed two major forms of interspecies transmission of animal rotaviruses to humans (20, 23, 33): genetic reassortants containing segments from a number of animal species and/or humans (interspecies transmission by reasserted animal rotaviruses); and viruses in which all segments were from the same animal species (interspecies transmission by nonreassorted animal rotaviruses). Molecular analysis has documented many more examples of interspecies transmission of genetic reassortants than of intact animal rotaviruses. Among the earliest documented reassortants were two G3P[3] human strains, Ro1845 and HCR3A that were shown to be rotaviruses of canine/feline origin (36, 38, 50). and G10 are the predominant rotavirus serotypes in cattle in most (1, 4, 28, 29, 43, 47), but not all areas of the world (51). Bovine strains were reported in many countries to be associated with P[1], P[5] and P[11] (1, 4, 29, 43, 47). Two G8P[1] strains, NIC522 and B12, were described as evidence of direct transmission of bovine rotaviruses to humans (2, 12). To date, no report has provided evidence

5 for the direct transmission of the most common P[1], P[5] or P[11] bovine rotavirus strains to human children. There were however a few sporadic cases of strains isolated from humans that were combined with P[6], P[9] and P[14] P-types (reviewed by Martella et. al. (20)). All of the human P[6] and P[9] strains were bovine human reassortants, whereas some of human P[14] rotavirus strains may have arisen by direct transmission of ungulate rotaviruses to humans (25). There are also reports of direct transmission of a G3P[14] lapine rotavirus, B4106 (7, 26), and a G9P[6] porcine rotavirus, B001 (55), to human children. During the routine characterization of diarrheal specimens sent from a sentinel clinic in Israel in 1995, one of the initially untypable rotavirus strains was found to be. An inquiry revealed that the rotavirus was derived from a child who developed diarrhea within days of a history of first contact with cows, suggesting that this represented a rare direct transmission of a bovine rotavirus to a human child. The aim of the study was to examine the whole genomic constellation of this strain by RNA RNA hybridization and full genome sequencing to determine whether the virus was in fact a genuine bovine rotavirus. Full genome sequencing of non-reassorted bovine P[1] rotavirus isolates from Japan and India were also performed to enlarge the geographical and temporal data base of P[1] bovine sequences for this comparison.

6 Materials and Methods. Ethics Statement The Ethical Review Board of the Sheba Medical Center, Tel Hashomer approved this study (SMC ). The sample and results were stripped of all links to personal details pertaining to, or which could be used to identify the individual patient. All data were analyzed anonymously. The Ethical Review Board exempted this study from a requirement for obtaining informed consent. Clinical History and initial characterization of rotavirus Ro8059 The parents of a one-year-old urban resident of Jerusalem took him to a rural farm near Petah Tikva that contained cowsheds and animal corners for children to show him cows and calves for the first time in his life. A few days later the infant presented to his pediatrician with diarrhea. A stool sample sent to the lab was found to contain rotavirus. The G-type of the virus, Ro8059, was identified as using a monoclonal antibody specific for that was kindly supplied by Giuseppe Gernea from Pavia, Italy. The rotavirus in the stool sample from the patient, Ro8059, was adapted to MA104 cells and plaque-purified three times according to the methods described by Kutsuzawa et al (18) and Wyatt et al (53), respectively. Bovine and human rotaviruses The following culture-adapted bovine and human P[1] rotavirus strains were used in this study: BRV101, BRV105 and BRV 106 isolated in Japan between 1983 and 1986 (47), C31/39 isolated in India between (14), and NCDV detected in the USA in 1967 (27), UK detected in the UK in 1973 (3), and human rotaviruses Ro8059 detected in Israel in 1995, as well as human, non-p[1] strains AU-1 G3P[9] (35), Wa G1P[8], and DS-1 G2P[4]. Electrophoresis of dsrna and RNA-RNA hybridization assays.

7 Genomic RNAs were extracted from culture fluids using a QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Germantown, MD, USA) and their migration patterns were analyzed on 10% polyacrylamide gels by the method described previously (37). The RNA-RNA hybridization was carried out as previously described (34). Briefly, the genomic RNA was transcribed into 11 positive-sense RNAs (i.e., transcription probes) in the presence of [ 32 P]-labeled GTP by using endogenous viral RNA polymerase present in purified double-layered particles of Ro8059 and NCDV. Hybridization was allowed to occur at high stringency conditions (at 65 o C, for 16 h) between the genomic RNAs of Wa, DS-1, Ro8059, NCDV, BRV105, and each of the two probes. Hybrids were then separated by electrophoresis on a 10% polyacrylamide gel, and the dried gels were exposed to imaging plates and read with BAS5000 (Fuji film, Tokyo, Japan). Genome amplification and sequencing A near full-length genome was assembled for Ro8059, BRV101, BRV105, BRV106 and C31/39 strains by a primer walking strategy. Each genome segment was amplified by PCR using common primer sets designed to be complementary to the segment ends. Briefly, genomic RNAs were extracted from either 10% stool suspensions or infected culture fluid by using the QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Germantown, MD, USA) according to the manufacturer s instructions. An 8μl portion of genomic RNA was mixed with random primers and dntps in a total volume of 9.5 μl and denatured at 97 C for 5 min followed be quenching on ice. To this was added the reverse-transcription mixture containing Super Script III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) in a final reaction volume of 20 μl. The thermal profile included incubation at 25 C for 5 min, at 42 C for 60 min and at 70 C for 15 min for reverse transcription. The genes were amplified from 3 μl of cdna using specific primer pairs for VP1 described in Doan et. al.(8); for VP2, VP6, and NSP1 through NSP5 described in Matthijnssens et al., (21); for VP7 described by Gulati et al, (14); the antisense primer for VP3 of Matthijnssens et al., (21) paired with primer VP3-B25F (5'- GTG TTT TAC CTC TGA TGG TG - 3') designed for this study; and the sense primer for VP4 from Doan et. al., (8) paired with primer VP4-B2361R (5'- GTC ACA TCC TCT GTC AGT TG -3') also designed for this study; in a GoTaq Green Master Mix system

8 (Promega Corporation, Madison, WI, USA) reaction mixture. Amplification conditions were 95 C for 5 min, 35 cycles of 94 C for 45 sec; 45 C for 45 sec and 72 C for 2-6 min depending on the length of cdna, followed by final extension at 72 C for 7 min. The amplified products were then purified using an ExoSAP-IT purification kit (USB Products, Cleveland, OH, USA) according to the manufacturer s instructions. Nucleotide sequencing reactions were performed by fluorescent dideoxy chain termination chemistry using the BigDye Terminator Cycle Sequencing Ready Reaction Kit, version 3.1 (Applied Biosystems, Foster City, CA, USA), and nucleotide sequences were determined using an ABI Prism 3730 Genetic Analyzer (Applied Biosystems). To avoid crosscontamination genes from each strain were amplified and purified on separate days. Sequence analyses The full genome sequences obtained in this study were compared with other bovine rotavirus strains (NCVD, BRV033, WC3), caprine rotavirus strain (GO34), animal-like human rotavirus strains (B1711, KF17, Hun5, , B , PA169, B12) and human rotavirus strains (KUN, Wa and AU-1). Nucleotide sequence identities were calculated and phylogenetic trees were drawn using the MEGA 5 software package (49). Multiple sequence alignments were carried out for each segment using the CLUSTALW program, and the genetic distances between sequences were calculated by the Kimura two-parameter method. Phylogenetic trees were then constructed using the neighbor-joining method. The bootstrap probability at a branching point was calculated with 1,000 pseudo-replicate datasets. Genotype classification was based on the RotaC 2.0 automated genotyping tool for group A rotavirus (19). The sequences (n=55) of the segments were submitted to the DDJB/GenBank/EMBL database and were assigned accession numbers AB AB and AB AB

9 Results Electropherotype and RNA-RNA hybridization assays Genomic RNA from Ro8059 was compared with that from BRV101, BRV105, BRV106, C31/39, UK and NCDV by electrophoresis on 10% polyacrylamide gels. Ro8059 RNA had a long electropherotype typical of G1P[6] bovine rotaviruses from different geographic locations and time periods. Segment 4 of the UK strain, coding for a different P-type, P[5], showed substantially less migration (Fig 1) When 32 P-labeled probes were prepared from Ro8059 and NCDV and allowed to hybridized in solution under high stringency conditions to denatured genomic RNAs from Ro8059, BRV105 and NCDV strains, there were 11 hybrid bands (Fig 2B) that had very similar electrophoretic mobility patterns to the 11 unlabeled homologous segments (Fig 2A). The origin or the cause of an extra hybrid band between Ro8059 RNA and the NCDV probe that did not appear in the reciprocal hybridization was not resolved, although it was a reproducible observation. In contrast, there were only 3 and 1 radiolabeled bands that ran with aberrant mobility when the target was unlabeled RNA from human rotaviruses DS-1 and Wa, respectively. Full genome sequencing We determined near-full nucleotide sequences of all 11 genes of a human rotavirus strain Ro8059 as well as four bovine strains BRV101, BRV105, BRV106 and C31/39. Applying the whole genome-based genotyping system (21) the genotype of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of human rotavirus strains Ro8059 was assigned to -P[1]-----A3--T6--, and as expected, all of four bovine P[1] strains were also assigned to -P[1]---- -A3--T6--. The genome constellation of Ro8059 and all four bovine P[1] strains BRV101, BRV105, BRV106 and C31/39 were identical to that of three typical P[1] bovine rotavirus strains, NCDV, RF, and BRV033 (21). Excluding the G and/or P genotypes, the genome constellation of strain Ro8059 was identical to those of human strains G8P[1] (B12) and P[14] strains (strains 111/05-27, B and PA169). The nucleotide sequences of each of the 11 segments of R08059 were highly

10 homologous with the corresponding segments of non-recombinant bovine rotaviruses of the same genotype (Table 1). They showed lower homology when compared to segments with the same,,,,, or genotypes from non-bovine isolates. Phylogenetic analysis Phylogenetic trees were constructed for each of the 11 genomic segments from cognate sequences from rotavirus isolates that had the same genotypes (Table 1) as Ro8059. Representative trees for VP7, VP6 and NSP4 appear in Fig 3 A-C, respectively, while those for VP4, VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 are presented in the supplementary data (Fig S1 A-H, respectively). The branch topology of the equivalent segments from Ro8059, BRV101, BRV105, BRV106, C31/39 and NCDV fell within the same sub-lineage for all segments, reflecting the high sequence identity reported in the previous paragraph. The highest divergence occurred among the cognate bovine sequences for the segment encoding VP1. There, NCDV and C31/39 formed a cluster separated from Ro8059, BRV101, BRV105 and BRV106 within the same sub-lineage. In contrast, while VP6, VP1, VP2, VP3, NSP2, and NSP4 shared the same genotypes as human DS-1 rotavirus, and NSP1 and NSP5 shared the same genotypes as human rotavirus AU-1, in all cases the human sequences segregated into distantly related sublineages.

11 Discussion Rotavirus Ro8059 was isolated in Israel in 1995 from the stool sample from a one-year old urban child who presented at a health clinic with diarrhea within days of his first contact with calves at a children s corner in a rural farm. The virus was initially characterized as bovine-like, since it had the following bovine like characteristics: it was P[1], it had a long electropherotypes, and it hemagglutinated human type O erythrocytes (unpublished observation), an activity that is generally associated with nonhuman rotaviruses (32). Comparison of RNA-RNA hybridization and full genome sequencing of Ro8059 with Japanese, Indian and US bovine strains revealed that all eleven segments of Ro8059 were of bovine rotavirus origin. To the best of our knowledge this is the first report of the direct whole genome transmission of a non-reassorted P[1] bovine rotavirus to a human child and the only known case of the direct interspecies transmission in which a full detail of the epidemiological link of the affected child with animals has been described. The final link that would have unequivocally demonstrated direct transmission, namely isolation of an Ro8059-like rotavirus from cow feces from the farm at the time of the child s visit, is missing however, since no bovine stool samples were available for analysis. Nevertheless, Israeli bovine rotaviruses would be very similar to those bovine rotavirus strains whose full genome sequences are known, given that all G1P[6] bovine rotavirus strains from Japan, India and the US were shown to be indistinguishably similar. P[1] is one of the most common genotypes detected from cattle (1, 4, 28, 29, 43, 47). However, the full genome sequences for this genotype was available for only three bovine strains: NCDV detected in US in 1967, WC3 detected in USA in 1981, BRV033 detected in Venezuela in In this study, we determined the full genome constellation of four additional P[1] bovine rotavirus strains: BRV101, BRV105, BRV106 detected in Japan between 1983 and 1986, and C31/39 detected in India during the period. All of these seven P[1] bovine rotavirus strains had the same -P[1]-----A3--T6-- genome constellation. For each segment, the seven isolates shared high nucleotide sequence identity and the sequences clustered into the same lineages and sub-lineages. For VP1, two bovine sequences segregated into

12 a separate cluster within the sub-lineage. This indicated that the genome of P[1] bovine rotavirus strains isolated in different regions of the world during a 30 year interval ( ) remained highly conserved. In contrast, the nucleotide sequences of each of the six (VP6, VP1, VP2, VP3, NSP2 and NSP4) and two (NSP1 and NSP5) segments of human prototype rotaviruses KUN and AU-1, respectively, that had the same genotype as the cognate bovine segments, segregated into significantly different lineages. The cognate genomic sequences of the human animal-like rotaviruses (Table 1) were dispersed among lineages and sub-lineages distinct from those of NCDV, WC3, BRV033, BRV101, BRV105, BRV106 and C31/39, some of which also had animal and specifically within the context of this manuscript, other bovine representatives. Such clustering is consistent with separate instances of cross-species transmissions. Fig 3A and 3B represent trees in which cognate human animal-like sequences were widely dispersed among the lineages and sub-lineages, whereas Fig 3C represents a tree where cognate sequences fall within the same sub-lineage. The different branch topology among the trees for the different genomic segments reflects the independent reassortment and post reassortment evolution. The number of rotaviral isolates with available full genomic sequences is still too small to conclude whether any of the patterns of clustering reflect species barrier effects, bottleneck effects from limited opportunities for cross-species transmission and persistent transmission in the new species, and or specific genetic virus-host factors. Zoonotic rotavirus infections of humans with bovine-like rotaviruses or bovines with human-like rotaviruses, although relatively rare, have been previously confirmed by electropherotyping combined with RNA-RNA hybridization and by sequence comparisons (5, 9, 15, 16, 20, 54). However, the majority of these bovinelike rotaviruses recovered from zoonotic infections in humans were intergenogroup reassortant rotaviruses, e.g., rotaviruses in which the nucleotide sequence of only some of the segments were highly related to the sequences of cognate segments of bovine rotaviruses (13, 39). Direct transmission of less common G8P[1] and P[14] bovine rotaviruses to humans has been suggested (2, 12, 25), although most human G8 strains may have originated from complex reassortment events involving human and animal rotaviruses(12), while the P[14] combination has only

13 been found in goats, in a sable antelope, and humans (25). Direct transmissions of a G3P[14] lapine rotavirus (7), a G8P[1] artiodactyl (ruminant and/or camelid) rotavirus (12 ), and a G9P[6] porcine rotavirus (55) have been reported. In the latter case, the father of the infant had been working on a pig farm during the week the infant became ill. Unfortunately, however an epidemiological investigation was not performed and the possibility of human-to-human transmission could not be ruled out. Furthermore, the virus recovered from the child was not a typical bovine rotavirus, but the genome constellation suggested that it was through multiple reassortant events including rotaviruses from another animal species (5, 6, 9, 10, 20, 23, 44). One of two globally licensed rotavirus vaccines, RotaTeq, is a pentavalent vaccine formulated from five bovine (P[7] WC3 strain)- reassortants containing human G1, G2, G3, G4, or P[8] (24). Each bovine-human rotavirus reassortant component and the pentavalent combination used in this live vaccine rarely caused symptomatic infections (52). In fact, most human zoonotic infections with bovine-like rotaviruses have been asymptomatic infections (9, 14, 17, 51). In some cases, however, bovine rotaviruses or bovine non-bovine reassortant in Israel ((13) and this report) and elsewhere (17, 40-42) have caused acute rotaviral gastroenteritis in children that, in some cases, was severe enough to require hospitalization (40). Co-infection of cells with two isolates allows for emergence of natural reassortants. The frequencies of natural reassortants with unusual G- and P-type combinations in humans were shown to correlate with the frequencies of these types in the mixed infections (30). Mixed infections occur in both human and bovine hosts (30, 31, 48). Furthermore, recombination can occur before and/or after zoonotic transfer and the transfer can occur in either direction. Moreover, recombination may facilitate the initial zoonotic transfer and/or subsequent passage in the new species (5, 20). Thus isolates from the rare cases of symptomatic vaccine-related adverse effects should be investigated to rule out emergence new bovinehuman reassortant rotaviruses that may have emerged from mixed infections of vaccine and wild rotaviruses. In conclusion, the RNA-RNA hybridization and full genome sequencing of Ro8059 and the other bovine P[1] strains established that the Ro8059 strain was of

14 bovine origin. High resolution molecular analysis combined with the clinical history of this infant is consistent with a direct interspecies transmission from a cow to a human infant. Taken together with previous observations that G8P[1] and P[14] bovine rotaviruses directly transmitted to human and caused diarrhea, this study provided another, more convincing support that typical P[1] bovine rotaviruses are not always naturally attenuated to humans. Interestingly, the sequence analysis also indicated that the genomes of P[1] bovine rotaviruses isolated in different regions of the world during a time span of greater than thirty years were highly conserved. Finally, the increasing use of full genome analysis, and an expanding database of segment sequences of rotaviruses from different host species (11, 46) will shed light on zoonotic infections and rotavirus evolution. Downloaded from on November 3, 2018 by guest

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17 Matthijnssens, J., S. De Grazia, J. Piessens, E. Heylen, M. Zeller, G. M. Giammanco, K. Banyai, C. Buonavoglia, M. Ciarlet, V. Martella, and M. Van Ranst Multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group A rotavirus strains. Infect Genet Evol 11: Matthijnssens, J., D. B. Joelsson, D. J. Warakomski, T. Zhou, P. K. Mathis, M. H. van Maanen, T. S. Ranheim, and M. Ciarlet Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq. Virology 403: Matthijnssens, J., C. A. Potgieter, M. Ciarlet, V. Parreno, V. Martella, K. Banyai, L. Garaicoechea, E. A. Palombo, L. Novo, M. Zeller, S. Arista, G. Gerna, M. Rahman, and M. Van Ranst Are human P[14] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order Artiodactyla? J Virol 83: Matthijnssens, J., M. Rahman, V. Martella, Y. Xuelei, S. De Vos, K. De Leener, M. Ciarlet, C. Buonavoglia, and M. Van Ranst Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission. J Virol 80: Mebus, C. A., M. Kono, N. R. Underdahl, and M. J. Twiehaus Cell culture propagation of neonatal calf diarrhea (scours) virus. Can Vet J 12: Midgley, S. E., K. Banyai, J. Buesa, N. Halaihel, C. K. Hjulsager, F. Jakab, J. Kaplon, L. E. Larsen, M. Monini, M. Poljsak-Prijatelj, P. Pothier, F. M. Ruggeri, A. Steyer, M. Koopmans, and B. Bottiger Diversity and zoonotic potential of rotaviruses in swine and cattle across Europe. Vet Microbiol 156: Monini, M., F. Cappuccini, P. Battista, E. Falcone, A. Lavazza, and F. M. Ruggeri Molecular characterization of bovine rotavirus strains circulating in northern Italy, Vet Microbiol 129: Muhsen, K., L. Shulman, U. Rubinstein, E. Kasem, A. Kremer, S. Goren, I. Zilberstein, G. Chodick, M. Ephros, and D. Cohen Incidence, characteristics, and economic burden of rotavirus gastroenteritis associated with hospitalization of israeli children <5 years of age, J Infect Dis 200 Suppl 1:S Nakagomi, O., Y. Isegawa, R. L. Ward, D. R. Knowlton, E. Kaga, T. Nakagomi, and S. Ueda Naturally occurring dual infection with human and bovine rotaviruses as suggested by the recovery of G1P8 and G1P5 rotaviruses from a single patient. Arch Virol 137: Nakagomi, O., M. Mochizuki, Y. Aboudy, I. Shif, I. Silberstein, and T. Nakagomi Hemagglutination by a human rotavirus isolate as evidence for transmission of animal rotaviruses to humans. J Clin Microbiol 30: Nakagomi, O., and T. Nakagomi Genomic relationships among rotaviruses recovered from various animal species as revealed by RNA-RNA hybridization assays. Res Vet Sci 73:

18 Nakagomi, O., T. Nakagomi, K. Akatani, and N. Ikegami Identification of rotavirus genogroups by RNA-RNA hybridization. Mol Cell Probes 3: Nakagomi, O., T. Nakagomi, Y. Hoshino, J. Flores, and A. Z. Kapikian Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses. J Clin Microbiol 25: Nakagomi, O., A. Ohshima, Y. Aboudy, I. Shif, M. Mochizuki, T. Nakagomi, and T. Gotlieb-Stematsky Molecular identification by RNA-RNA hybridization of a human rotavirus that is closely related to rotaviruses of feline and canine origin. J Clin Microbiol 28: Nakagomi, T., K. Akatani, N. Ikegami, N. Katsushima, and O. Nakagomi Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes. J Clin Microbiol 26: Nakagomi, T., and O. Nakagomi Human rotavirus HCR3 possesses a genomic RNA constellation indistinguishable from that of feline and canine rotaviruses. Arch Virol 145: Palombo, E. A Genetic analysis of Group A rotaviruses: evidence for interspecies transmission of rotavirus genes. Virus Genes 24: Palombo, E. A., and R. F. Bishop Genetic and antigenic characterization of a serotype human rotavirus isolated in Melbourne, Australia. J Med Virol 47: Park, S. I., J. Matthijnssens, L. J. Saif, H. J. Kim, J. G. Park, M. M. Alfajaro, D. S. Kim, K. Y. Son, D. K. Yang, B. H. Hyun, M. I. Kang, and K. O. Cho Reassortment among bovine, porcine and human rotavirus strains results in G8P[7] and P[7] strains isolated from cattle in South Korea. Vet Microbiol 152: Ramani, S., M. Iturriza-Gomara, A. K. Jana, K. A. Kuruvilla, J. J. Gray, D. W. Brown, and G. Kang Whole genome characterization of reassortant G10P[11] strain (N155) from a neonate with symptomatic rotavirus infection: identification of genes of human and animal rotavirus origin. J Clin Virol 45: Reidy, N., G. Lennon, S. Fanning, E. Power, and H. O'Shea Molecular characterisation and analysis of bovine rotavirus strains circulating in Ireland Vet Microbiol 117: Santos, N., and Y. Hoshino Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol 15: Santos, N., E. M. Volotao, C. C. Soares, G. S. Campos, S. I. Sardi, and Y. Hoshino Predominance of rotavirus genotype G9 during the 1999, 2000, and 2002 seasons among hospitalized children in the city of Salvador, Bahia, Brazil: implications for future vaccine strategies. J Clin Microbiol 43: Steyer, A., M. Bajzelj, M. Iturriza-Gomara, Z. Mladenova, N. Korsun, and M. Poljsak-Prijatelj Molecular analysis of human group A rotavirus G10P[14] genotype in Slovenia. J Clin Virol 49:121-5.

19 Suzuki, Y., T. Sanekata, M. Sato, K. Tajima, Y. Matsuda, and O. Nakagomi Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture. J Clin Microbiol 31: Swiatek, D. L., E. A. Palombo, A. Lee, M. J. Coventry, M. L. Britz, and C. D. Kirkwood Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and Vet Microbiol 140: Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: Tsugawa, T., and Y. Hoshino Whole genome sequence and phylogenetic analyses reveal human rotavirus G3P[3] strains Ro1845 and HCR3A are examples of direct virion transmission of canine/feline rotaviruses to humans. Virology 380: Varshney, B., M. R. Jagannath, R. R. Vethanayagam, S. Kodhandharaman, H. V. Jagannath, K. Gowda, D. K. Singh, and C. D. Rao Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India. Arch Virol 147: Vesikari, T., D. O. Matson, P. Dennehy, P. Van Damme, M. Santosham, Z. Rodriguez, M. J. Dallas, J. F. Heyse, M. G. Goveia, S. B. Black, H. R. Shinefield, C. D. Christie, S. Ylitalo, R. F. Itzler, M. L. Coia, M. T. Onorato, B. A. Adeyi, G. S. Marshall, L. Gothefors, D. Campens, A. Karvonen, J. P. Watt, K. L. O'Brien, M. J. DiNubile, H. F. Clark, J. W. Boslego, P. A. Offit, and P. M. Heaton Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 354: Wyatt, R. G., H. B. Greenberg, W. D. James, A. L. Pittman, A. R. Kalica, J. Flores, R. M. Chanock, and A. Z. Kapikian Definition of human rotavirus serotypes by plaque reduction assay. Infect Immun 37: Yamamoto, D., M. Kawaguchiya, S. Ghosh, M. Ichikawa, K. Numazaki, and N. Kobayashi Detection and full genomic analysis of P[9] human rotavirus in Japan. Virus Genes 43: Zeller, M., E. Heylen, S. De Coster, M. Van Ranst, and J. Matthijnssens Full genome characterization of a porcine-like human G9P[6] rotavirus strain isolated from an infant in Belgium. Infect Genet Evol.

20 Figure legends. Fig 1. Electropherogram of Ro8059 and bovine rotaviruses form India, Japan and the US. Double stranded RNA extracted from the rotavirus strains indicated above each lane was resolved on SDS-PAGE stained with ethidium bromide and photographed under ultraviolet light. Fig. 2. Genogroup analysis of Ro8059. Double stranded RNA from the strains indicated above each lane was denatured and hybridized in solution to the [ 32 P]-labeled probe prepared from Ro8059 or NCDV as indicated below the lanes. The resulting hybrids were resolved by SDS-PAGE, stained with ethidium bromide, and photographed under ultraviolet light (A) and then autoradiographed (B) Fig 3. Phylogenetic analysis for segments VP7, VP6 and NSP4 of Ro8059. Phylogenetic trees were constructed for genomic segments (A) VP7, (B) VP6 and (C) NSP4. (Phylogenetic trees for the remaining segments, VP4, VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 are presented in supplement Fig S1 A-H, respectively). The phylogenetic analysis included nucleotide sequences of rotavirus strains analyzed in this study (strain names in red and indicated by black dots) along with representative sequences obtained from the DNA databases, including three bovine rotavirus strains (strains names in red) and seven animal-like human rotavirus strains (strains name in blue) which were used for comparison of nucleotide identities with Ro8059 in the Table 1. The trees were constructed using the neighbor-joining method included in the MEGA 5 software package with bootstrap probabilities after 1,000 replicate trials, and rooted with sequences of different

21 genotypes. The genetic distance is indicated at the bottom. Percent bootstrap support is indicated by the value at each node when the value was 70% or larger. For the sake of convenience in annotation, the numbers for P genotypes are without square brackets. For the sake of space and clearer presentation, only representative sequences from the DNA databases were included in the figure although the actual analysis were performed by employing all available sequences in the DNA databases. Downloaded from on November 3, 2018 by guest

22 Table Title Table 1.Genome constellation and nucleotide sequence identities (%) of the 11 gene segments of Ro8059 to those of selected human and animal rotavirus strains. Downloaded from on November 3, 2018 by guest

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28 VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5 RVA/Human-tc/ISR/Ro8059/1995/P[1] - P[1] A3 - - T RVA/Cow-tc/JPN/BRV101/ /P[1] 99.9 P[1] A T RVA/Cow-tc/JPN/BRV105/1983/P[1] 99.8 P[1] A T RVA/Cow-tc/JPN/BRV106/1983/P[1] 99.9 P[1] A T RVA/Cow-tc/IND/C31/39/ /P[1] 99.8 P[1] A T RVA/Cow-tc/USA/NCDV/1967/P[1] 98.9 P[1] A T RVA/Cow-tc/VEN/BRV033/1990/P6[1] 90.1 P[1] A T RVA/Cow-tc/USA/WC3/1981/P[5] 97.2 P[5] A T RVA/Goat-tc/BGD/GO34/1999/P[1] 83.5 P[1] A T RVA/Human-wt/BEL/B1711/2002/P[6] 81.4 P[6] A T H RVA/Human-wt/JPN/KF17/2010/P[9] 81.4 P[9] A T E RVA/Human-wt/HUN/Hun5/1997/P[14] 81.8 P[14] A T RVA/Human-wt/ITA/ /2005/P[14] 84.2 P[14] A T RVA/Human-wt/BEL/B /1997/P[14] 84.2 P[14] A T RVA/Human-tc/ITA/PA169/1988/P[14] 83.4 P[14] A T RVA/Human-tc/Kenya/B12/1987/G8P[1] G P[1] A T RVA/Human-tc/JPN/KUN/1980/G2P[4] G P[4] A T H RVA/Human-tc/USA/Wa/1974/G1P1A[8] G P[8] 68.3 I R C M A N T E H RVA/Human-tc/JPN/AU-1/1982/G3P3[9] G P[9] 68.1 I R C M A N T E Grey *Same genotype as Ro8059. White *Genotype different from Ro8059. Human Rotavirus strains Animal-like human rotavirus strains Caprine Strain name and origin Genotypes* and % homology to R08059 Bovine Query Sequence on November 3, 2018 by guest Downloaded from