1 full-genome characterization of g8p[8] rotavirus emerged among

1

Full-genome characterization of G8P[8] rotavirus emerged among children with diarrhea in 1

Croatia, in 2006 2

3

Roberto Delogu1‡, Alessandra Lo Presti2‡, Franco Maria Ruggeri3#, Eleonora Cella2, Marta Giovanetti2, 4

Massimo Ciccozzi2, Suncanica Ljubin Sternak4, Suzana Bukovski-Simonoski5, Amarela Lukic-Grlic6, 5

Giovanni Ianiro1, Lucia Fiore1. 6

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1National Center for Immunobiologicals Research and Evaluation, 2Deptartment of Infectious, 8

Parasitic and Immune-Mediated Diseases, and 3Department of Veterinary Public Health and Food 9

Safety, Istituto Superiore di Sanità, Rome, Italy; 4Croatian National Institute of Public Health, and 10

School of Medicine University of Zagreb, Zagreb, Croatia; 5University Hospital for Infectious Diseases 11

“Dr Fran Mihaljevic”, Zagreb, Croatia; 6Zagreb University Children’s Hospital, and School of 12

Medicine University of Zagreb, Zagreb, Croatia. 13

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‡RDL and ALP have equally contributed to the work 15

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Running title: Full-genome sequencing of G8P[8] rotavirus in Croatia 17

18

#Corresponding author: 19

Franco M Ruggeri, PhD 20

Dept. Veterinary Public Health & Food Safety 21

Istituto Superiore di Sanità, 22

Viale Regina Elena, 299, 00161 Rome, Italy 23

Phone: +39 06 4990 2980 24

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00396-13 JCM Accepts, published online ahead of print on 20 February 2013

on April 3, 2018 by guest

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Fax: +39 06 4938 7101 25

E-mail: [email protected] 26

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Abstract 28

A 2006 epidemic G8P[8] rotavirus from Croatia was whole-genome sequenced, showing a Wa-like 29

genotype constellation. Its VP7 clustered with DS1-like G8 African rotaviruses, and a G8P[4] German 30

strain. Remaining genes clustered with contemporary Belgian G1P[8] rotaviruses, suggesting 31

reassortment between human G8 and G1P[8] rotaviruses, in Croatia or other European countries. 32

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Rotavirus A (RVA) causes acute gastroenteritis in man and animals globally, killing 450,000 children 34

per year, mainly in developing countries (1). 35

RVA genome includes 11 double-stranded RNA segments, encoding six viral (VP1-4,6,7) and six non-36

structural proteins (NSP1-6) (2). Rotavirus outer layer protein VP7 and spike protein VP4 are 37

neutralization antigens, determining virus genotypes G (glycoprotein, VP7) and P (protease-sensitive, 38

VP4). At least 27 G and 37 P genotypes have been identified among human, animal and avian RVAs 39

(3-5). 40

Five genotypes (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) represent over 90% of global human 41

RVAs (6), that may explain the efficacy of both monovalent (P[8], G1) Rotarix and pentavalent (P[8], 42

G1-4) RotaTeq vaccines against rotavirus diarrhea worldwide (7). 43

Serotypes other than G1-4, and strains evolving by mutation or genomic reassortment between human 44

and/or animal RVAs represent an emerging threat for children (8-10). Millions of people are exposed to 45

animal rotaviruses worldwide, and several uncommon human genotypes (G6, G8, G12, P3[9], P5A[3]) 46

closely resemble animal RVA strains (11). 47

Molecular epidemiology and viral typing can help monitor emergence of novel RVAs, confirming 48

current vaccine efficacy against unconventional genotypes. 49

Reassortment events can involve any dsRNA segments, and G and P typing may be insufficient to 50

investigate RVA origin and inter-species transmission. Genotyping based on sequencing all 11 genome 51

segments (3, 4) is very helpful, distinguishing 9 VP1 (R), 9 VP2 (C), 8 VP3 (M), 35 VP4 (P), 16 VP6 52

(I), 27 VP7 (G), 16 NSP1 (A), 9 NSP2 (N), 12 NSP3 (T), 14 NSP4 (E), and 11 NSP5 (H) genotypes. 53

Bovine-like G8 rotavirus was first reported in an Indonesian child (12), and further G8 RVA human 54

cases occurred worldwide, including industrialized countries (13-21). Using full-genome sequencing, 55

G8P[8] African RVAs were shown to involve reassortment between at least four human, swine, and 56

bovine strains (22). 57


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This paper reports the whole genome characterization of a G8P[8] human rotavirus strain emerged in 58

Croatia in 2006 (23). 59

Cases showed severe gastroenteritis, and included <5 year-old children from Croatian hospitals in 60

2005-2006. Stools were screened by Rotalex, and RVA characterized by RT-PCR (23). Agarose gel 61

purified amplicons were sequenced using RT-PCR primers (8, 18, 24, 25), assembling consensus 62

sequences for all gene segments (18, 22, 26). 63

Twelve datasets were built, including all NCBI sequences (http://www.ncbi.nlm.nih.gov/pubmed), 64

showing >89% similarity with query sequences (between 116 for VP2 and 127 for NSP4). 65

Sequences were aligned using ClustalX software (27), and edited by Bioedit software. The ModelTest 66

v3.0 (28) with hierarchical likelihood ratio test was used to select the best-fit models for sequence data 67

analysis. 68

Bayesian phylogenetic trees were constructed for VP7, VP4 and NSP4 datasets (29), using GTR+I+G 69

nucleotide substitution model for VP7, and HKY+G model for VP4/NSP4. 70

Markov Chain Monte Carlo searches were made (50x106 generations, tree-sampling every 5000 71

generation, and 10% burn-in fraction. Clade statistical support followed >0.90 posterior probability). 72

Maximum-likelihood (ML) phylogenetic trees of NSP1-3, NSP5-6, VP1-3 and VP6 were generated 73

with PAUP* 4.0 package (30), using GTR+I+G for VP6, NSP1, VP1; HKY+G model for NSP6/NSP5; 74

TrN+I+G for NSP2, NSP3, VP2 and VP3 datasets. Statistical robustness and reliability of branching 75

order within trees was confirmed by bootstrap analysis (1000 replicates), considering bootstrap >70% 76

as clades support. 77

Sequences were deposited in GenBank database (accession numbers JQ988894-JQ988904). 78

79

Initial sequencing of genes encoding, VP4 and NSP4 for 10/31 G8P[8] RVAs showed nearly complete 80

nucleotide identity suggesting circulation of a single G8P[8] strain in Croatia in 2005-6. The consensus 81


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Croatia 2006 (CR2006) VP7 gene showed >98% nucleotide identity with other human G8 GenBank 82

strains, particularly from neighboring Slovenia, 2006, and Africa, 2000-2004. CR2006 VP4 and NSP4 83

recalled human G3P[8] strains reported in Italy, 2005 (31). To investigate Croatian G8P[8] origin, full-84

genome sequencing was performed, using RotaC (http://rotac.regatools.be) (32). CR2006 presented a 85

genotype G8-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. 86

Bayesian analysis identified CR2006 VP7 in a statistically supported cluster with African (Malawi, 87

Tunisia, Cameroon, Ethiopia), and European strains (Slovenia [Sl-885/06], Germany [GER1H-09]) 88

(Fig 1A). In VP4 and NSP4 trees (Fig 1B and 2), CR2006 also clustered with European and African 89

RVAs, including Hu/Slo/2006/G8P[8]/Sl-885/06, Hu/Italy/2005/G3P[8], 90

Hu/Ethiopia/2004/G8P[8]/ARN, Hu/Tunisia/2000-2004/G8P[8]/ARN. 91

CR2006 clustered with Wa-like genotype constellation Hu/Bel/2005-2009/G1P[8], 92

Hu/Ger/G3G12P[8]/GER126-08, and other global RVAs in NSP1-NSP6, VP1-VP3, and VP6 93

(Supplementary Fig S1-9). 94

G8P[8] rotavirus circulated abnormally in Croatia in 2006, reaching 15.8% of children hospitalized 95

with diarrhea (23). Full-genome sequencing of these viruses was performed to investigate links 96

between their emergence and genome characters. 97

The G8 rotavirus genotype, normally combined with P[1],[5],[11] or [21] types (11, 33, 34), is typical 98

of bovine strains, but has also been sporadically detected in humans since 1978 (35) associated with 99

different P-types (13, 16-18, 36-38). Human G8 strains normally exhibit P[4] or P[6] both in developed 100

countries and in Africa, where they are particularly prevalent (15-17, 26). Recently, full-genome 101

sequencing of a human G8P[8] rotavirus in Democratic Republic of Congo (DRC) in 2003 (18, 22) 102

suggested this strain to originate from reassortment between a bovine G8P[6] ancestor with three or 103

more different rotaviruses, including human Wa- and DS1-like strains. This strain exhibited a DS-1-104

like genotype constellation. 105


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Differently, Croatian CR2006 G8P[8] presented the Wa-like constellation (3) G8-P[8]-I1-R1-C1-M1-106

A1-N1-T1-E1-H1, all genes showing close evolutionary relationship to prototype G1P[8] human strain 107

Wa, except the VP7 gene, related to African and Slovenian human G8 strains. Rotaviruses are usually 108

species-specific, and gene reassortment with human strains appears necessary for zoonotic adaptation 109

(26, 39-41). Characterization of epidemic CR2006 further supports this view. As VP7 plays a major 110

role in the immune response (42, 43), CR2006 emergence and epidemic spread may have involved 111

reassortment with common rotaviruses pre-existing in Croatia. 112

CR2006 was more frequent in younger children (23) than any other rotavirus genotypes co-circulating 113

in Croatia, 2006. This could be fortuitous, or suggest that maternal antibodies protected younger 114

children from CR2006 less than from other serotypes. Maternal immunity fades quickly (44, 45), and it 115

is conceivable that older Croatian children in 2006 were similarly unprotected hence susceptible to G8 116

or any other common rotavirus genotypes. 117

Phylogenetic analyses revealed that, except for the VP7 gene, CR2006 was more closely related to 118

humans G1P[8] strains identified between 2005-2009 in Belgium. Therefore, CR2006 origin likely 119

involved reassortment of a Wa-like rotavirus circulating in one or more countries of Europe, including 120

Belgium and possibly Croatia, with a co-circulating G8 virus. In 2006, a similar G8P[8] strain was 121

reported and partially sequenced in Slovenia (20), whereas no G8P[8] rotaviruses were detected in 122

other European countries. It is possible that the reassortment event leading to CR2006 may have 123

occurred in or near Croatia. Low VP7 correlation between CR2006 and G8 DS1-like rotaviruses 124

characterized in African DRC (18) indicates independent evolution of these G8 reassortants. 125

Conversely, CR2006 shared a similar gene 9 with human G8 strains from Northern/Central Africa in 126

the early 2000s, suggesting that the G8 gene followed immigration routes from Africa. A closely 127

similar VP7 was found in a G8P[4] human strain identified in Germany two years later (21), suggesting 128

that the CR2006 G8 gene participated to additional reassortments with European DS1-like rotaviruses. 129


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The German strain was strictly correlated with the DRC G8 rotaviruses reported earlier, except for VP4 130

(18), and may represent a further evolution of G8P[8] rotaviruses imported from Africa. The VP7 of all 131

European G8 strains differed markedly from bovine G8 rotaviruses, contrary to a recent human-bovine 132

reassortment event within Europe. 133

Rotavirus genetic evolution is certainly influenced by immune pressure (46), that may favor G8 and 134

other uncommon rotavirus G-types spreading (19, 20, 47), as occurred for G9 strains originated from 135

swine (48, 49). International transmission of genotypes is facilitated by globalization and travelling, 136

urging worldwide rotavirus strain surveillance to anticipate emergence of novel strains and 137

animal/human reassortants, and monitor effectiveness of current human rotavirus vaccines. 138

139


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Acknowledgments 140

Grant sponsor: Ministry of Health, Italy (to LF CCM ‘‘Epidemiologia molecolare di rotavirus in eta` 141

pediatrica in Italia. Creazione di una rete di sorveglianza per monitorare la diffusione e l’evoluzione di 142

genotipi virali’’ and to FMR Strategic Program: ‘‘Preparedness and response to emerging zoonoses and 143

exotic viral infection through an integrated medical and veterinary approach’’); Grant sponsor: 144

ISS/NIH (to FMR ‘‘Molecular and antigenic evolution of rotavirus strains of human and animal 145

origin’’); Grant sponsor: EuroRotaNet (http://www.eurorota.net). 146

147


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Legends to the Figures 299

300

FIG. 1. Bayesian phylogenetic analysis of Human Rotavirus VP7 and VP4 nucleotide sequences. The 301

trees were rooted by using the midpoint rooting method. Branch lengths were estimated with the best 302

fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant 303

statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle 304

for simplicity, and its size is an indication of the number of sequences it represents. The number of the 305

strain for each place of isolation is indicated in parenthes. RV strain isolated in Croatia is in boldface 306

type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. 307

The number of substitutions per site is indicated by the scale bar. 308

(A) Phylogenetic tree based on VP7 gene nucleotide sequence. (B) Detail of the phylogenetic 309

relationship between G8 genotype RV strains. 310

(C) Phylogenetic tree based on VP4 gene nucleotide sequence. (D) Detail of the phylogenetic 311

relationship between CR2006 and other RV strains. 312

313

FIG. 2. Bayesian phylogenetic analysis of Human Rotavirus NSP4 nucleotide sequences. The tree was 314

rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting 315

nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant 316

statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle 317

for simplicity, and its size is an indication of the number of sequences it represents. The number of the 318

strain for each place of isolation is indicated in parenthes. Phylogenetic clusters containing CR2006 are 319

boxed and the strain is in boldface type. For each strain, the following data are given: species of 320

origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale 321

bar. 322


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VP7

A.

G8

G1

G12G3

G10

G2

G4

0.2

Hu / Jap / 1982 / G3P9 / AU-1 La / Italy / 1996 / G3P14 / 30/96

Bo / India / 2007 / G8P14 (3)

Hu / Philip / G12P4 / L26

Hu / Africa / 2004 / G8P6 / SA4948JHB/04

Hu / Australia / 2002 / G8P14 (2)

Hu / G1P8 / KU

Bo / USA / 1996 / G8 / Cody I801

Hu / India / G9 / RMC321

Hu / China / 1996 / G2P4 / TB-ChenBo / G6 / UK - NCDV

Bo / Kor / G8 (28)

Bo / USA / 2003 / G8 (4)

Hu / Malawi / 2000 / G8P8 / MW4103

Hu / India / G8P1 / MP409

Bo / USA / 2007 / G8

Hu / Finland / HAL 1166Hu / Australia / G8 (3)

Hu / Congo / 2003 / G8P8 / DRC88

Po / Kor / 2006 (15)

Bo / Thailand / G10

Ov / Spain / G8P14 / OVR762

Hu / USA / 1976 / G2P4 / DS-1

Lama / Arg / 1998 / G8P14 (4)

Hu / Cameroon / 2000 / G8 / ARN (4)

Hu / Nigerian / 2003 / G8 (2)

Po / USA / 1976 / G4P6 / Gottfried

Po / G5P7 / OSU

Bo / Jap / G8 (5)

Hu / UK / 1985 / G8 (2)

Hu / USA / 1974 / G1P8 / Wa

Si / Kenia / 1999 / G8P6 / Si/KY1646

Hu / Egypt / G8P14 / EGY1850

Bo / Nigeria / 2003 / G8 / NGRBg8

Hu / Tunisia / 2002-04 / G8 (3)

Hu / Switz / G8 / U25.96

Hu / Croatia / 2006 / G8P8 / CR2006

Hu / Congo / 2003 / G8P6 / DRC86

Hu / Malawi / 2000-01 / G8 (7)

Si / Sa11 / G3Hu / Bangl / 2000-03 / G12P6 (2)

Hu / Africa / G8 / QEH14262

Bo / Thailand / G8 / A5Hu / Jap / 1994-05 / G8 (2)

Hu / Egypt / G8P14 / EGY2295

Hu / 69M / G8P10

Hu / India / G10P11 / I321

Hu / Ger / 2009 / G8P4 / GER1H-09

Hu / Ethiopia / 2004 / G8 / ARN6810

Si / USA / PTRV

Hu / Kenia / 2002 / G8 (3)

Hu / Brazil / G8P4 / R291

Hu / UK / 678

Hu / Slo / 2006 / G8P8 / SI-885/06

1

0,97

1

1

1

1

1

1

0,951

1

0,991

1

1

0,94

0,91

1

0,91

1

1

1

1

Hu / Cameroon / 2000 / G8 / ARN6780


Hu / Tunisia / 2000 / G8 / ARN6862

Hu / Malawi / 2000 / G8P8 / MW4103

Hu / Tunisia / 2004 / G8 / ARN6736

Hu / Ger / 2009 / G8P4 / GER1H-09



Hu / Croatia / 2006 / G8P8 / CR2006

Hu / Tunisia / 2002 / G8 / ARN6854

Hu / Slo / 2006 / G8P8 / SI-885/06

Hu / Cameroon / 2000 / G8 / ARN67871

0,94

B.

FIG. 1. Bayesian phylogenetic trees of Human Rotavirus VP7 and VP4 nucleotide sequences. The trees were rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle for simplicity, and its size is an indication of the number of sequences it represents. The number of the strain for each place of isolation is indicated in parenthes. RV strain isolated in Croatia is in boldface type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale bar. (A) Phylogenetic tree based on VP7 gene nucleotide sequence. (B) Detail of the phylogenetic relationship between G8 genotype RV strains. (C) Phylogenetic tree based on VP4 gene nucleotide sequence.

0.3

Hu / Ger / 2009 / G8P4 / GER1H-09

Hu / India / G9 / RMC321

Hu / Bel / 2003 / G12P8 / B4633-03

Hu / China / 2007 / G3P8 (4)

Hu / Croatia / 2006 / G8P8 / CR2006Hu / Slo / 2006 / G8P8 / SI-885/06

Hu / Bangl / / G12P8 / Dhaka25-02

Hu / India / 2006 / G1P8 (3)

Hu / Bel / 2009 / G1P8 (8)

Hu / USA / 1974 / G1P8 / Wa

Hu / Jap / P8 (2)

Hu / USA / 2010 / G9P11 / 116E/AG

Hu / Italy / 2005 / G9P8 / BIA2

Hu / Bel / 2008 / G1P8 (6)

Hu / Italy / 2006 / G9P8 (2)

Hu / Russia / 2004-09 / P8 (16)

Hu / Russia / 2004-07 / P8 (2)

Hu / Russia / 2008 / G4P8 (3)

Hu / Bangl / 2003 / G12P6 (2)

Hu / China / 2007 / G3P8 (4)

Hu / Congo / 2003 / G8P8 / DRC88

Hu / Africa / 2000-04 / G8 / ARN (5)

Hu / Russia / 2009 / G4P8 (4)

Hu / Thailand / 2009 / P8 (7)

La / Italy / 1996 / G3P14 / 30/96

Hu / China / 2007 / P8 (6)

Hu / USA / 2007 / G1P8

Hu / Bangl / 2005 / P8 (9)

Hu / China / 1996 / G2P4 / TB-ChenHu / USA / 1976 / G2P4 / DS-1

Hu / India / 2006 / P8 / mani-133/06

Hu / Congo / 2003 / G8P6 / DRC86

Hu / GBR / 1975 / G4P6 / ST3

Hu / Bel / 2006 / G1P8 (7)Hu / Russia / 2006 / G9P8 / RUS-Nov06-1289

Po / USA / 1976 / G4P6 / Gottfried

Hu / USA / G1P8 / US6668

Hu / Ger / 2008 / G3G12P8 / GER126-08

Hu / Malawi / 1997 / P8 (4)

Hu / Italy / 2005 / G9P8 (2)

0,93

0,99

1

0,95

1

1

0,93

1

1

0,98

1

1

1

0,99

VP4

C.

P[8]

P[4]

P[11]

P[6]

P[14]


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0.3

Hu / Jap / G3 (2)

Hu / Tunisia / 2000-04 / G8 / ARN (3)

Hu / G3P2 / RV3

Hu / Jap / G3 / ITO

Hu / Bangl / 2000-03 / G12P6 (4)

Hu / Bel / 2009 / G1P8 (2)

Hu / G1P8 / KU

La / Italy / 1996 / G3P14 / 30/96

Hu / GBR / 1975 / G4 / ST3

Hu / Italy / 2006 / G3P6 / 128/07-34

Hu / South Africa / 1986 / G4P6 (4)

Hu / Australia / G4 (2)

Hu / Italy / 2005 / G3P8 / PA116

Hu / Russia / G1P8 / N.N.12813

Hu / USA / 2008 / G1P8

Hu / China / G3 (4)

Hu / Ger / 2008 / G3G12P8 / GER126-08

Hu / Russia / 2008-09 / G9P8 (2)

Hu / China / G1 (2)

Hu / Russia / 2004-09 / P8 (6)

Hu / USA / 2005-08 / G1P8 (3)

Hu / USA / G1P8 (3)

Hu / Slo / 2006 / G8P8 / SI-885/06


Hu / Bel / 2006 / G1P8 (2)

Hu / China / 1996 / G2P4 / TB-ChenHu / Congo / 2003 / G8 / DRC (2)


Hu / Russia / 2007 / G4P8 / Omsk07-78

Hu / USA / 1976 / G2P4 / DS-1

Hu / Italy / 2005 / G3P8 / PA106

Hu / Italy / 2005 / G3P8 / PA110

Hu / Jap / G3 (2)

Hu / USA / G1P8 (7)

Hu / China / 1994 / G4 (2)Hu / Jap / 1990 / G4 (2)

Hu / Jap / G3 (4)

Hu / Italy / 2005 / G3P8 / PA49

Hu / Italy / 2005 / G3P8 / PA57

Hu / Ger / 2009 / G8P4 / GER1H-09

Hu / India / G9/ RMC321

Hu / Bel / 2004-08 / G1P8 (4)

Po / OSU / G5P7

Hu / Bel / 2006-08 / G1P8 (3)

Hu / Russia / 2009 / G1P8 / D83Hu / India / 2006 / G1P8 / AM06-I


Hu / Bel / 2007-09 / G1P8 (4)


Hu / Jap /1982 / G3P9 / AU-1

Hu / Bel / 2005-06 / G1P8 (2)

Hu / Croatia / 2006 / G8P8 / CR2006

Hu / Italy / 2005 / G3P8 / PA105

Hu / Bel / 2007 / G1P8

Hu / India / G10 / I321Bo / Kor / CBN-1

Hu / USA / G1P8 / Wa (6)

Hu / Jap / G3-G4 (2)

1

1

11

1

1

1

1

1

0,96

1

1

1

1 1

1

1

1

1

1

1

1NSP4

E1

E2

E3E5

FIG. 2. Bayesian phylogenetic tree of Human Rotavirus NSP4 nucleotide sequence. The tree was rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle for simplicity, and its size is an indication of the number of sequences it represents. The number of the strain for each place of isolation is indicated in parenthes. Phylogenetic clusters containing CR2006 are boxed and the strain is in boldface type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale bar.


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1 full-genome characterization of g8p[8] rotavirus emerged among

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