Full genomic analysis of Indian G1P[8] rotavirus strains

Infection, Genetics and Evolution 11 (2011) 504–511

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Full genomic analysis of Indian G1P[8] rotavirus strains Ritu Arora, Shobha D. Chitambar * Department of Rotavirus, Enteric viruses Group, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune 411 001, Maharashtra, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 November 2010 Received in revised form 27 December 2010 Accepted 4 January 2011 Available online 20 January 2011

Rotavirus G1P[8] strains are the most predominant cause of rotavirus diarrhea, worldwide and are an important component of currently licensed RotaTeq and Rotarix vaccines. Despite a significant contribution of these strains in causing diarrhea in Indian children, none of them has been characterized completely, to date. This issue was addressed in the present study by sequencing and phylogenetic analysis of complete genomes of 3 Indian rotavirus strains (06361, 0613158 and 061060) of G1P[8] specificity. Genotype of G1P[8] I1R1C1M1A1N1T1E1H1 respectively, for the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 genes was assigned to all of the three strains. The sequence analysis of structural and nonstructural genes indicated genetic relatedness (94–99.5%) with recently circulating strains and divergence (2.4–15.6%) with old prototype strains. Phylogenetic analysis revealed that new strains (Western Indian rotavirus strains and recently isolated strains – Dhaka16-03 (G1P[8]), Dhaka25-02 (G12P[8]), Matlab13-03 (G12P[6]), B3458 (G9P[8]), Matlab36-03 (G11P[8]), and B4633-03 (G12P[8]) and old prototype strains (KU and Wa) clustered in the same lineages of VP1, VP2, VP3, NSP2 and NSP4 genes however, grouped separately in VP6, NSP1 and NSP5 genes with 10–11%, 15.6–16.7% and 6.3–7.5% nucleotide sequence divergence, respectively. These results suggest that the rotavirus VP6, NSP1 and NSP5 genes of Wa-like rotaviruses are more prone to temporal mutations. Both structural and nonstructural genes of the Western Indian rotavirus strains shared nucleotide and amino acid substitutions with the Bangladeshi strain, Dhaka16-03 (G1P[8]) in the year 2003. This study documents for the first time the phylogenetic and evolutionary relationships of Indian G1P[8] rotavirus strains with the rotavirus strains from other parts of world and provides data useful for the evaluation of rotavirus vaccine programs. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Rotavirus G1P[8] Full-genome analysis India

1. Introduction Group A rotaviruses, belonging to the family Reoviridae, are regarded as a major cause of acute diarrhea in humans and animals, worldwide. The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA), ranging from 0.6 to 3.3 kb that encode six structural proteins (VP1–4, VP6, VP7) and six nonstructural proteins (NSP1–6) (Estes and Kapikian, 2007). The dsRNA is enclosed in a capsid formed by three concentric layers of protein. The innermost capsid layer, VP2 contains an internal polymerase complex that is composed of VP1 (viral RNAdependent RNA polymerase) and VP3 (viral capping enzyme) (Estes and Kapikian, 2007). The middle layer of the virion, consisting of VP6 trimers, defines the rotavirus group and subgroup (SG) specificities. The outermost layer of the virion is formed by two proteins, VP7 and VP4, which are known to be responsible for the attachment of the viral particle to specific cellular receptors and the penetration of the virion into the cell’s

* Corresponding author. Tel.: +91 020 26127301; fax: +91 020 26122669. E-mail address: [email protected] (S.D. Chitambar). 1567-1348/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.01.005

cytoplasm (Kaljot et al., 1988). According to the classification system of VP7 gene (G type) and VP4 gene (P type) rotaviruses have been differentiated into 25 G and 33 P types (Matthijnssens et al., 2008a; Schuman et al., 2009; Ursu et al., 2009; Collins et al., 2010; Esona et al., 2010a) with identification of >45 distinct G–P combinations (Grimwood and Kirkwood, 2008). At present, 12 G and 15 P genotypes are known to infect human beings (Ba´nyai et al., 2009). The nonstructural proteins (NSP1–NSP3, NSP5, and NSP6) interact with viral RNA and are involved in replication (Estes and Kapikian, 2007). NSP3 has been identified to play a role in the extraintestinal spread of rotavirus in neonatal mice (Mossel and Ramig, 2002) while NSP4, the viral enterotoxin, glycoprotein in nature, has been described to have multiple functions in rotavirus morphogenesis and pathogenesis (Au et al., 1989; Tian et al., 1995; Taylor et al., 1996; Ball et al., 1996). Recently, a classification system based on the nucleotide cut-off values for the ORF of each of the 11 gene segments of rotaviruses has been proposed by the rotavirus classification working group (RCWG) (Matthijnssens et al., 2008a). The data accumulated from different geographical locations indicate considerable diversity among circulating rotavirus strains

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

(Matthijnssens et al., 2008a). However, five genotype combinations, G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] are known to be responsible for 90% of infections, worldwide. Among these, G1P[8] strains are predominant (50–65%) in humans of both developed and developing countries (Castello et al., 2004; Gentsch et al., 2005; Santos and Hoshino, 2005; Desselberger et al., 2006; Grimwood and Kirkwood, 2008; Lennon et al., 2008; Sdiri-Loulizi et al., 2008; Min et al., 2004; Moea et al., 2009; Aung et al., 2010). A significant contribution of these strains in causing acute gastroenteritis has been reported in a multi center hospital-based surveillance of rotavirus disease and strains among Indian children (Kang et al., 2009). In India, reports are available on the phylogenetic analysis of several important rotavirus genes, however, data on whole genome has been documented only for a few unusual strains (Varghese et al., 2004, 2006; Mukherjee et al., 2009; Ramani et al., 2009). Although both currently licensed rotavirus vaccines (Rotarix and RotaTeq) contains strains with G1 and P[8] genotypes, data on the full genome analysis of G1P[8] strains is limited to only one Bangladeshi strain, Dhaka16-03 (Rahman et al., 2010). The present study was performed to characterize complete genomes of Indian rotavirus strains of G1P[8] specificity. The data obtained in the study was analyzed and explored to determine the relationship of Indian G1P[8] strains with rotavirus strains from other parts of the world.

505

2.4. Nucleotide sequencing and phylogenetic analyses The PCR amplicons were purified using a QIAquick PCR purification kit (Qiagen, Germany) according to the manufacturer’s instructions and sequences in both directions were determined by the dideoxy nucleotide chain terminator method using a Bigdye Terminator cycle sequencing reaction kit V3.1 (Applied Biosystems, USA). The sequence data were collected from an automated sequencer ABI 3130 XL (Applied Biosystems, USA). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA4.1 (Tamura et al., 2007). Genetic distances were calculated by using the Kimura2 parameter model at the nucleotide level and phylogenetic trees were constructed by using neighbor-joining method with 1000 bootstrap replicates. 2.5. Sequence submission The nucleotide sequences obtained in this study were submitted to GenBank under the accession numbers – HQ609552–HQ609554 (VP1), HQ609555–HQ609557 (VP2), HQ609558–HQ609560 (VP3), EU984107, HM467806 and HM467807 (VP4), HQ609561– HQ609563 (VP6), FJ948838, FJ948854 and FJ948855 (VP7), HQ609564–HQ609566 (NSP1), HQ609567–HQ609569 (NSP2), HQ609570–HQ609572 (NSP3), HQ609573–HQ609575 (NSP4) and HQ609576–HQ609578 (NSP5).

2. Materials and methods 3. Results 2.1. Specimen selection A previous study conducted for rotavirus surveillance had identified G1P[8] as a predominant genotype combination infecting pediatric population in Pune, western India (Kang et al., 2009). Three rotavirus strains (06361, 0613158 and 061060) from the year 2006 representing this combination were selected for complete genome characterization. These strains were also subjected to isolation in MA104 cell culture according to the protocol described earlier (Urasawa et al., 1981). 2.2. ELISA, electron microscopy and RNA PAGE Antigen capture enzyme-linked immuno sorbent assay (ELISA) was performed on the supernatants of infected cell cultures at passage level 5, using a Group A Rotavirus detection kit (IDEIATM Rotavirus, DAKO, USA) according to the manufacturer’s protocol. To determine rotavirus morphology samples were examined by negative staining in an electron microscope (Palmer and Martin, 1988). RNA migration patterns were analyzed on 7.5% polyacrylamide gel stained with silver nitrate (Theil et al., 1981). 2.3. Viral RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) Viral RNA was extracted from 30% stool suspension using Trizol, LS reagent (Invitrogen, USA) according to the manufacturer’s instructions. RT-PCR was performed for amplification of all structural and nonstructural genes using the primers published earlier (Gouvea et al., 1990; Matthijnssens et al., 2006a; Chitambar et al., 2009). Briefly, the extracted RNA was denatured at 97 8C for 5 min, and RT-PCR was carried out by using a QIAGEN Onestep RTPCR kit (Qiagen, Germany). This involved an initial reverse transcription step of 30 min at 45 8C, followed by PCR activation at 95 8C for 15 min, 40 cycles of amplification (1 min at 94 8C, 1 min at 45 8C/50 8C, and 2.5 min at 70 8C), with a final extension of 7 min at 70 8C. PCR products were electrophoresed in 2% agarose gels containing ethidium bromide (0.5 mg/ml) and visualized under UV transilluminator.

RNA segments extracted from three stool specimens (06361, 0613158 and 061060) showed long migration pattern (4-2-3-2) in polyacrylamide gel electrophoresis (PAGE), specific to group A rotaviruses. All three specimens showed absence of amplification for enteric adenovirus, astrovirus, norovirus and enterovirus in PCR/RT-PCR. The presence of virus-like particles, 70 nm in diameter was confirmed in these specimens by electron microscopy (data not shown). Full genomic sequence analysis revealed that all the three rotavirus strains have G1-P[8]-I1-R1-C1-M1-A1N1-T1-E1-H1 genotype which is related to Wa-like human rotavirus. 3.1. Sequence analyses of structural genes The complete sequences of gene segment 1 (encoding VP1) of the Western Indian rotavirus strains were compared with the corresponding sequences of VP1 genotypes (R1–R4) available in GenBank. The strains showed 86–99% nucleotide identity with human strains of R1 genotype and clustered in R1a lineage (Fig. 1a). The sequence identity was found to be 95.5–99.1% with Bangladeshi strains, Dhaka16-03, B4633-03, B3458 and Dhaka2502 of genotype R1a lineage and 86–88% with KU, YM and Gottfried strains of R1b and R1c lineages, respectively. Percent nucleotide sequence identities with other (R2–R4) genotypes were in the range of 71–80% (
[()TD$FIG]

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

506

B4633-03 0613158 Dhaka12-03 06361 99 Dhaka25-02 100 061060 75 B3458 ST3 M1a Matlab13-03 10095 100 Dhaka16-03 KU M1 A64 99 70 WI-61 100 L26 Hochi 85 Wa 100 YM 100 M1b OSU Matlab36-02 M1e 100 Dhaka6 100 MCS13/07 M1c RMC321 100 M1d HP140 M6 L338 T152 100 AU-1 100 M3 B4106 30/96 100 80 SA11 SA11 5N 100 M5 SA11 5S SA11 H96 RF 94 100 UK 69M DS-1 100 100 S2 M2 TB-Chen 100 100 DRC86 DRC88 N26-02 100 100 RV161-00 PO-13 M4 100

a. VP1

72

Dhaka16-03 Dhaka25-02 06361 B4633-03 B3458 100 R1a 061060 100 Matlab13-03 R1 Dhaka12-03 100 Matlab36-02 99 Wa 0613158 100 100 R1b KU YM R1c Gottfried 93 T152 R3 AU-1 100 B4106 99 100 UK 80 RF SA11 L26 100 100 S2 R2 100 TB-Chen 72 DS-1 100 DRC88 100 DRC86 RV161-00 100 100 N26-02 R4 PO-13

b. VP2

c. VP3

100 N26-02 100 RV161-00

DRC86 DRC88 TB-Chen S2 100 100 C2 DS-1 L26 100 100 RF UK B4106 86 100 30/96 88 100 T152 C3 AU-1 SA11/5S 99 C5 100 SA11/5N 100 HP140 C1b 75 RMC321 C1c IAL28 Wa 100 87 KU Matlab36-02 99 0613158 99 99 Matlab13-03 100 Dhaka25-02 98 C1a B4633-03 B3458 87 97 Dhaka12-03 100 061060 Dhaka16-03 100 06361 99 C4 PO-13 99

100

76

0.02

C1

0.05

0.05

d.VP4

e. VP6

100 Dhaka1203 95 Matlab13-03

06361 061060 Dhaka16-03 98 Dhaka25-02 RMC437 90 I1 Matlab36-02 B3458 99 100 99 CMH202/01 RMC100 B4633-03 100 0613158 Wa 84 KU 99 L-338 95 I6 HO-5 100 HI-23 100 I11 Ch-1 OSU 100 I5 YM 100 I7 EO AU-1 I3 93 CMH222 I8 88 TUCH I9 76 LP14 I10 100 TBchen 74 S2 100 89 DS-1 L26 SA11 I2 B4106 84 UK 100 RV161-00 100 DRC86 100 100 DRC88 PO13 I4 Ty-1 100

f. VP7

70

99

98

0.01

27B3 061060 ISO114 Dhaka25-02 CAU 202 ISO93 B4633-03 Matlab36-02 Dhaka16-03 Hun9 P[8]-3 DH402 OP351 ISO22 ISO124 06361 78 99 PA25/03 0613158 Kagawa/90-544 Strain90-551 99 Kagawa/90-513 KU P[8]-2 F45 95 OP511 89 OP354 P[8]-4 47B3 99 ISO116 Wa P[8]-1 Odelia 99 Hochi 74 DS-1 Outgroup L26 99

94

95

99

97 92 99 99

CMH222/G3 CH3/G14 SA11/G3 EW/G16 Ecu534/G20 CMP178/G5 YM/G11 DRC88/G8 BA201/G9 Hg18/G15 UK/G6 RF/G6 99 NCDV/G6 99 61A/G10 B223/G10 99 AzuK-1/G21 T152/G12 EQ/G13 HU/G4 KU/G1 99 Wa/G1 0613158 99 K18/G1 99 vn355/G1 Thai-804 G1 99 061060 06361 Thai-1604 99 Dhaka8-02 76 Dhaka16-03 DS-1/G2 TB-Chen/G2 99 Ch2/G7 Phea17655-Hun-08/G23 Ty-1/G17 PO-13/G18 Ch-06V0661/ G19 Tu-03V0002E10/G22

0.05

0.02

Fig. 1. Phylogenetic analysis of complete nucleotide sequences of (a) VP1, (b) VP2, (c) VP3, (d) VP4, (e) VP6 and (f) VP7 genes of Indian G1P[8] rotavirus strains. The strains of the present study are indicated with ^.

2000s with 94.1–99.1% nucleotide identity, while identity with other lineages (M1b, M1c, M1d and M1e) was 86.7–88.5% (Fig. 1c). Nucleotide sequence identities with other (M2–M6) genotypes were in the range of 62–78%. The potential trypsin cleavage sites of arginine described at positions 241 and 247 in VP4 gene (Arias et al., 1996) were conserved in all strains of the present study (data not shown).

These strains showed the highest (89.6–98.1%) nucleotide sequence identity with P[8] genotype and clustered with strains of P[8]-3 lineage (Hun9-like lineage) of the four (P[8]-1–P[8]-4) lineages of P[8] genotype indicating 98.1–99.5% nucleotide identity (Fig. 1d). Identities with other lineages (P[8]-1, P[8]-2 and P[8]-4) were 85.3–94.6%. The amino acid residues 120 (N), 135 (D) and 195 (G), specific for the P[8]-3 lineage (Phan et al., 2007)

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

507

Table 1 Amino acid substitutions in VP7 protein of G1P[8] rotavirus strains of the present study. Strain name

KU Dhaka 16-03 06361 0613158 061060

Amino acid position and substitution 19

28

29

33

36

41

49

50

55

57

65

66

68

72

74

75

94

106

147

217

266

268

281

291

L . . F .

R . . Q .

I . . M .

I . . L .

F . . S .

T F F S F

R K K . K

A . T . .

L . . I .

L I . I I

T . . A .

V . . A .

T S S A S

Q R R . .

E . . G G

V . . I I

N S S . S

F . L . .

S N N N N

M T T . T

S A A . A

V I I . I

T I I . I

K R R . R

were displayed by all three strains. The nucleotide sequence identity with other (P[1]–P[7] and P[9]–P[27]) P genotypes ranged from 59.8% to 87.1%. In comparison to prototype strain KU, all strains of the present study showed amino acid substitutions at positions 19 (H-Y), 75 (K-T), 106 (V-I), 120 (T-N), 150 (E-D), 195 (DG), 266 (C-Y), 385 (N-D), 560 (I-V), 580 (I-V) and 738 (I-T). Phylogenetically, the strains of the present study clustered with the strains of I1 genotype of VP6 gene indicating 89.1–98.9% nucleotide sequence identity (Fig. 1e) and identified to be closer to Bangladeshi strains Dhaka16-03, Dhaka25-02 and B4633-03. The nucleotide identity with other (I2-I11) genotypes ranged from 70.6% to 84.6%. Nucleotide sequence analysis of the VP7 gene of the Western Indian rotavirus strains revealed their clustering in G1 genotype with 91.9–99% identity (Fig. 1f). Nucleotide identities with other (G2–G23) genotypes were detected to be less (62.7–77.2%) than the cutoff value (80%). Strains 06361, 0613158 and 061060 were classified in IA, IIB and IB lineages of G1 genotype, respectively, as reported earlier (Arora et al., 2009). In comparison with prototype strains KU and Wa, amino acid substitutions in Indian strains were found in antigenic regions A (N94S, D97E), B (S147N) and C (M217T). Along with these, several strain specific substitutions were also detected (Table 1). 3.2. Sequence analyses of nonstructural genes The nucleotide sequence identity of NSP1 gene of Western Indian rotavirus strains was highest (80.1–98.9%) with the strains of A1 genotype (Fig. 2a) and lowest (42.1–78.1%) with those from other (A2–A13) genotypes. Phylogenetically, these strains clustered with A1b lineage strains, Dhaka16-03, Dhaka25-02, Matlab13-03, B4633-03 and B3458 indicating 97.1–98.9% nucleotide sequence identity. With strains from other lineages (A1a and A1c) identity was in the range of 79.4–84.4%. Four conserved cysteine residues at positions 6, 8, 85 and 285 in the NSP2 gene involved in disulfide bonds were detected in all of the Western Indian rotavirus strains (Patton et al., 1993). Nucleotide identity was highest (89.3–99.3%) with N1 genotype and lowest (62.3–84.5%) with other (N2–N5) genotypes. Phylogenetically, these strains clustered with N1a lineage strains, Dhaka16-03, Dhaka12-03, Matlab13-03, RMC100, Wa, and KU indicating 89.5–99.3% nucleotide identity, while identity with strains of N1b lineage was 86.6–87.8% (Fig. 2b). The NSP3 gene has been described to have conserved leucine residues at positions 275, 281, 288, 295 and 302 (Mattion et al., 1992). However, in the Western Indian rotavirus strains, leucine was identified only at position 281. Amino acids at positions 275, 288, 295 and 302 were replaced by amino acid residues isoleucine, aspartic acid, methionine and glutamine, respectively. The strains grouped specifically with T1 genotype of NSP3 gene (Fig. 2c) with 88.5–99.6% nucleotide identity however, showed lower identity (56.2–84.1%) with other (T2–T6) genotypes. NSP4 genes of the Western Indian rotavirus strains showed the presence of conserved N-linked glycosylation sites at positions 8 and 18, and cysteine residues at positions 63 and 71. Phylogeneti-

cally these strains were grouped into the E1 genotype of NSP4 gene and clustered with the human strains (Dhaka16-03, Dhaka12-03, Matlab13-03, B3458, Wa, and KU) of the lineage E1a (Fig. 2d) with 86.7–96.3% nucleotide sequence identity. With strains from other lineages (E1b, E1c and E1d) identity was in the range of 86.3–90.9%. Lower nucleotide sequence identity (48.2–83%) was detected with other (E2–E11) genotypes. Sequence analysis of the enterotoxin region (amino acid position 114–135) of NSP4 genes showed substitution at position 129 (S-R). The strain specific changes were also detected at positions 131 (H-Y) and 133 (N-S) for 06361and 061060 strains, respectively. The serine residues at positions 153, 155, 163, and 165 and cysteine residues at positions 171 and 174 are known to be conserved in the NSP5 gene (Eichwald et al., 2002). In the Western Indian rotavirus strains serine residues were detected at their respective positions, however, cysteine residues were replaced by lysine. The highest (93–99.2%) nucleotide sequence identity of these strains identified with Dhaka16-03, Matlab13-03, KU and RMC321 placed them in H1 genotype of NSP5 gene (Fig. 2e). This was further confirmed by the phylogenetic analysis which showed clustering of these strains with H1a lineage strains Dhaka 16-03, Matlab13-03, RMC100, Dhaka12-03 and Dhaka25-02 with 97.5– 99.4% nucleotide identity. Identity with strains of other lineages (H1b and H1c) was noted to be 93.3–95.6%. The other (H2–H4) genotypes showed lower (63–89%) nucleotide identities. 3.3. Nucleotide and amino acid substitutions in the culture adapted strains All structural and nonstructural genes of culture adapted strains 06361-CA, 0613158-CA and 061060-CA from passage 10 were also amplified to find out the nucleotide and amino acid substitutions in cell culture passaged and adapted rotavirus strains. Variable number of nucleotide and amino acid substitutions was observed in all of the three culture-adapted strains (Supplementary data Table 1). These included mainly strain specific substitutions. Interestingly, VP4 genes of all of the three culture-adapted strains displayed substitutions in comparison to their wild type counterparts. However, no substitutions were detected in NSP2, NSP4 and NSP5 genes of any of these strains. 4. Discussion The segmented genome of rotaviruses is the driving force for their diversity in nature. Full-length genome characterization of rotaviruses is important to identify the origin of a strain and its genetic relatedness to other circulating strains (Matthijnssens et al., 2008a). The classification scheme proposed by RCWG has been reported to be useful for identification of reassortants (Matthijnssens et al., 2008a,b). This scheme favored nucleotide sequences over deduced amino acid sequences, as the results obtained with nucleotide sequences were more reliable than those of amino acid sequences (Matthijnssens et al., 2008a,b). Further differentiation of rotavirus genotypes in lineages has been also proposed (Wang et al., 2010).

[()TD$FIG]

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

508

94 Dhaka16-03

Matlab36-02 Dhaka25-02 B3458 061060 85 Matlab13-03 A1b 83 06361 10074 B4633-03 100 0613158 ST3 A1 99 M37 97 OSU KU 88 116E 99 A1a I321 100 Wa 100 Hochii 100 RMC321 A1c RMC/G7 100 100 YM A8 Gottfried 100 DS1 69M 100 TB-Chen 100 A2 97 N26-02 100 DRC88 84 100 DRC86 B223 A13 99 Hun5 100 A11 100 MG6 AU-1 76 RF A3 100 B641 100 T152 A12 EW A7 100 SA11 4F A5 SA11 H96 100 FI23 100 A10 100 H2 99 L338 A6 RRV 100 30/96 100 100 B4106 A9 C-11 99 Alabama 100 BAP-2 72 PO-13 A4

a. NSP1

0.05

c. NSP3

b. NSP2

99

98 Matlab36-02 72 Dhaka16-03 99 B3458

81 N26-02

Matlab13-03 97 Dhaka25-02

0.05

Dhaka16-03 06361 Matlab36-02 98 B4633-03 Dhaka12-03 99 99 Matlab13-03 78 Dhaka25-02 E1a B3458 100 92 061060 Wa 76 0613158 56 E1 YO 99 KU 99 44 GR1186/86 E1b 100 GR856/86 HP113 92 E1c 100 HP140 OSU G00841 96 91 100 RMC/G7 E1d RMC321 82 100 ST3 CMP034 E9 A G4 120 100 47 97 BAP-2 99 BAP 100 ALA E5 B4106 30/96 84 100 NCDV 94 88 RF SA11 40 100 DRC88 100 DRC86 100 E2 UK DS-1 93 KUN 92 98 TBChen 82 S2 98 RV176-00 E6 100 N26-02 99 CU-1 AU-1 E3 95 CMH222 94 PP-1 RV52/96 E8 100 RV198/95 100 EC EW E7 100 EHP 100 Ch-1 E10 PO-13 E4 100 Ty-3 99 E11 AvRV-1 100

d. NSP4

06361 061060 B4633-03 75 Dhaka12-03 0613158 wi61 I321 T1 RMC100 ST3 Wa 100 99 IGV-P Dhaka25-02 100 KU RMC321 78 GO KJ44 100 100 OSU 99 T7 UK B4106 100 30/96 MP409 T6 100 RF 100 100 NCDV SA11 5S 100 SA11 H96 T5 SA11 5N RRV 80 99 T3 T152 AU-1 69M 84 DS-1 84 L26 100 90 TB-Chen 100 S2 90 DRC86 90 DRC88 T2 Dhaka107-99 IS2 90 Dhaka116-00 Matlab13-03 N26-02 72 RV161-00 70 NR1 PO-13 T4 100

0.05

Dhaka16-03 Matlab36-02 06361 96 B4633-03 100 Dhaka12-03 061060 N1a 100 70 RMC321 Wa RMC100 0613158 100 B3458 93 L26 100 OSU 76 99 KU TUCH N1b 98 IAL28 100 uk TB-Chen 100 DS1 100 99 DRC88 DRC86 100 IS2 N2 NR1 100 NCDV RF 30/96 100 B4106 100 T152 100 N3 AU-1 SA11 5N 83 SA11 5S N5 100 SA11 H96 PO-13 N4

0.05

87 96 90

N1

e. NSP5 Dhaka16-03 Matlab36-02 B4633-03 B3458 RMC100 06361 061060 H1a 85 Matlab13-03 71 Dhaka12-03 Dhaka25-02 84 L26 0613158 YM OSU 100 75 99 KJ44 KU 96 Wa H1b IAL28 87 RMC321 95 HP113 H1c 98 100 HP140 T152 99 H6 RRV SA11 H5 B4106 100 30/96 UK 97 H3 RF AU-1 84 512A 98 69M v115 95 v61 89 94 B37 DS-1 99 99 DRC88 100 H2 DRC86 TB-Chen 89 KUN IS2 NR1 N26-02 PO-13 H4 100

H1

0.05

Fig. 2. Phylogenetic analysis of complete nucleotide sequences of (a) NSP1, (b) NSP2, (c) NSP3, (d) NSP4 and (e) NSP5 genes of Indian G1P[8] rotavirus strains. The strains of the present study are indicated with ^.

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

It is to be noted that all over the world, emphasis has been given mainly to full-length genome sequencing of uncommon or novel strains. Complete genomes of only old prototype strains and a few recently isolated strains of common (G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]) genotypes have been characterized, worldwide (Heiman et al., 2008; Matthijnssens et al., 2008a,c; McDonald et al., 2009; Rahman et al., 2010). G1P[8] rotavirus strains are responsible for 50–65% of infections and are, thus, identified to be a predominant cause of rotavirus disease. Sequence analyses of NSP4 and VP6 genes of these strains are widely reported (Le et al., 2010; Khamrin et al., 2010; Benati et al., 2010). However, a complete genome sequence is available only for the Bangladeshi G1P[8] rotavirus strain, Dhaka 1603 (Rahman et al., 2010). Very recently, a rotavirus vaccine candidate CDC-9 has been adapted to Vero cells and all genes of its wild type and culture adapted strains were compared. However, the nucleotide sequence data is not available in GenBank for comparative analysis (Esona et al., 2010b). In India, rotavirus infections have been identified with common (G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]), uncommon (G1P[6], G2P[8], G9P[4], G12P[8] etc.) and mixed (G2,G9P[4], G3,G9P[6], G4,G10P[4], G1,G2,G9P[4] etc.) genotypes (Kang et al., 2005, 2009). However, analysis of full length genomes of only three uncommon rotavirus strains of G9P[19], G9P[6] and G10P[11] specificities has been documented (Varghese et al., 2004, 2006; Mukherjee et al., 2009; Ramani et al., 2009). Among the circulating rotavirus strains G1P[8] strains have been found to contribute significantly to rotavirus diarrhea in all age groups (Kang et al., 2009; Tatte et al., 2010a). Molecular characterization of such strains is limited to only VP4, VP6, VP7 and NSP4 genes (Arora et al., 2009; Zade et al., 2009; Tatte et al., 2010a,b). It is essential to understand completely a rotavirus genotype prevalent in the region for its evolutionary variations and assessment of the vaccine program. The study presented here reports for the first time the complete genome analysis of three Indian rotavirus strains of G1P[8] specificity. The complete genetic analysis of Indian strains demonstrated that the strains belonged to human Wa-like genogroup of rotaviruses, and thus, indicated that the acute gastroenteritis patients were infected with a single strain of rotavirus. It has been reported that on the basis of nucleotide and amino acid cutoff values, P[4] and P[8] genotypes of VP4 genes could not be separated due to their close identity (Matthijnssens et al., 2008a). The P[8] strains of the present study also showed close genetic relatedness with other P[8] (85.3–99.5%) and P[4] (83.7– 85.1%) genotypes. Phylogenetic analysis of the VP4 gene sequences has shown the existence of four distinct lineages within P[8] genotypes, globally (Gouvea et al., 1999; Cunliffe et al., 2001; Arista et al., 2005, 2006; Ansaldi et al., 2007; Espinola et al., 2008). Phylogenetic analysis of P[8] genotypes of the Indian strains revealed clustering of all three strains in P[8]-3 lineage. These findings are in agreement with the studies reporting predominance of P[8]-3 lineage in the 2000s (Arista et al., 2006; Ansaldi et al., 2007; Araujo et al., 2007; Espinola et al., 2008). It is known that the VP7 protein of rotavirus has nine variable regions (VR1–VR9) and that four (VR5, VR7, VR8 and VR9) of these constitute A (aa 86–101), B (aa 142–152), C (aa 208–221) and F (aa 235–242) regions of antigenic importance (Ciarlet et al., 1997). Amino acid substitutions in these antigenic regions, especially at positions 94, 96, 147, 148, 190, 208, 211, 213, 217, 238 and 291 with or without glycosylation change are known to alter the antigenicity of viruses and are responsible for escape from host immunity (Ahmed et al., 2007; Trinh et al., 2007). Hence, the substitutions detected at 94, 147 and 217 positions may be important to generate altered antigenicity in the Western Indian rotavirus strains. Although these strains differed from the prototype strains in their amino acid sequence (Table 1), the substitutions displayed by these

509

strains matched with Dhaka 16-03 isolated in 2003 from Bangladesh. Classification of these strains in different lineages/ sublineages has been described earlier (Arora et al., 2009). Western Indian rotavirus strains were suggested to have subgroup II specificity because they possessed subgroup II specific amino acids at the antigenic sites in VP6 (Phe248, Asn305, Ala306, Gln310 and Gln315) (data not shown). Previous studies have identified the genetic linkage (SGI-NSP4 genotype A, recently referred to as I2-E2 genotypes respectively and SGII-NSP4 genotype B, recently referred to as I1-E1 genotypes respectively) between rotavirus VP6 and NSP4 genes (Iturriza-Gomara et al., 2003). Recent studies have shown that G1P[8] strains were linked with I1 and E1 genotypes of VP6 and NSP4 genes respectively (Benati et al., 2010; Khamrin et al., 2010). Results obtained in the present study were in agreement with these findings. Amino acid substitutions in the VP4, VP6, NSP1 and NSP5 genes have been reported for the cell culture adapted G1P[8] strain, CDC9 (Esona et al., 2010b). The present study documents for the first time the substitutions in the VP1, VP2, VP3, VP7 and NSP3 genes along with those in VP4, VP6 and NSP1 genes during cell culture adaptation. The maximum number of amino acid substitutions was observed in the VP4 gene which is known to be involved in rotavirus cell entry and penetration. Nucleotide and amino acid substitutions detected in the culture adapted G1P[8] rotavirus strains of the present study needs to be evaluated further for their antigenicity, attenuation and structural conformation. The changes have been described to occur in different gene segments of the currently circulating rotavirus strains over time resulting in the increase in genetic distance from the prototype strains (Rahman et al., 2010). The strains of the present study were closer to recently isolated strains of different G and P specificities from Bangladesh and Belgium and distantly related to the old prototype strains (KU and Wa), suggesting possibility of a common genetic backbone for the strains prevailing in western India, Bangladesh and Belgium (Rahman et al., 2007; Freeman et al., 2009; Matthijnssens et al., 2009). It is noteworthy that the Western Indian rotavirus strains and recently isolated strains (Dhaka16-03 (G1P[8]), Dhaka25-02 (G12P[8]), Matlab13-03 (G12P[6]), B3458 (G9P[8]), Matlab36-03 (G11P[8]), and B4633-03 (G12P[8]) clustered within R1a, C1a, M1a, N1a and E1a lineages, respectively of VP1, VP2, VP3, NSP2 and NSP4 genes along with old prototype strains (KU and Wa) but formed separate clusters from the same prototype strains with VP6, NSP1 and NSP5 gene sequences (Figs. 1 and 2). In the VP6 gene, the nucleotide divergence of Western Indian rotavirus strains from Wa and KU strains was noted to be 10–11% while it was only 1.4–3.9% with rotavirus strains isolated in 2000s (Dhaka16-03, Dhaka25-02, Matlab13-03, B3458, Matlab36-03 and B4633-03). The NSP1 gene known to be a most divergent gene in rotaviruses grouped old strains in the A1a lineage but clustered with new strains (including Western Indian rotavirus strains) in the A1b lineage with 15.6–16.7% nucleotide divergence from prototype strains. Similarly lineage H1a of NSP5 gene clustered with new strains (including Western Indian rotavirus strains) carrying 6.3–7.5% nucleotide divergence from the KU and Wa strains grouped in H1b lineage. These data indicate that although the Wa-like backbone is the most successful backbone of rotaviruses VP6, NSP1 and NSP5 genes are more susceptible towards evolutionary changes. Continuous monitoring of such strains for their complete genetic frame would identify their variations and evolution and help improve formulation of rotavirus vaccines. Acknowledgements The study was financially supported by National Institute of Virology, (Indian council of Medical Research, Govt of India) Pune.

510

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511

The authors thank Dr. A.C. Mishra, Director, NIV for his constant support, and Dr. A. Basu for facilitating the electron microscopic studies.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2011.01.005. References Ahmed, K., Nakagomi, T., Nakagom, O., 2007. Molecular identification of a novel G1 VP7 gene carried by a human rotavirus with a super-short RNA pattern. Virus Genes 35, 141–145. Ansaldi, F., Pastorino, B., Valle, L., Durando, P., Sticchi, L., Tucci, P., Biasci, P., Lai, P., Gasparini, R., Icardi, G., 2007. Molecular characterization of a new variant of rotavirus P[8]G9 predominant in a sentinel-based survey in Central Italy. J. Clin. Microbiol. 45, 1011–1015. Araujo, I.T., Assis, R.M., Fialho, A.M., Mascarenhas, J.D., Heinemann, M.B., Leite, J.P., 2007. Brazilian P[8],G1, P[8],G5, P[8],G9, and P[4],G2 rotavirus strains: nucleotide sequence and phylogenetic analysis. J. Med. Virol. 79, 995–1001. Arias, C.F., Romero, P., Alvarez, V., Lopez, S., 1996. Trypsin activation pathway of rotavirus infectivity. J. Virol. 70, 5832–5839. Arista, S., Giammanco, G.M., De Grazia, S., Ramirez, S., Blundo, C., Colombo, C., Cascia, A., Martella, V., 2006. Heterogeneity and temporal dynamics of evolution G1 human rotaviruses in a settled population. J. Virol. 80, 10724–10733. Arista, S., Giammanco, G.M., De Grazia, S., Colomba, C., Martella, V., 2005. Genetic variability among serotype G4 Italian human rotaviruses. J. Clin. Microbiol. 43, 1420–1425. Arora, R., Chhabra, P., Chitambar, S.D., 2009. Genetic diversity of genotype G1 rotaviruses co-circulating in Western India. Virus Res. 146, 36–40. Au, K.S., Chan, W.K., Burns, J.W., Estes, M.K., 1989. Receptor activity of rotavirus nonstructural glycoprotein NS28. J. Virol. 63, 4553–4562. Aung Meiji-Soe, Aung Tin-Sabai, Oo Khin-Yi, Win Ne, Ghosh, S., Yamamoto, D., Kobayashi, N., 2010. Phylogenetic analysis of human rotavirus in Myanmar: detection of Indian–Bangladeshi G1/G2 lineages, Chinese G3 lineage and OP354-like P[8] lineage (P[8]b subtype). S. Asian J. Trop. Med. Public Health 41 . Ba´nyai, K., Esona, M.D., Kerin, T.K., Hull, J.J., Mijatovic, S., Va´sconez, N., Torres, C., de Filippis, A.M., Gentsch, J.R., 2009. Molecular characterization of a rare, humanporcine reassortant rotavirus strain, G11P[6], from Ecuador. Arch. Virol. 154, 1823–1829. Ball, J.M., Tian, P., Zeng, C.Q., Morris, A.P., Estes, M.K., 1996. Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272, 101–104. Benati, F.J., Maranhao, A.G., Lima, R.S., da Silava, R.C., Santos, N., 2010. Multiple gene characterization of rotavirus strains: evidence of genetic linkage among the VP7 VP4 VP6 and NSP4 encoding genes. J. Med. Virol. 82, 1797–1802. Castello, A.A., Arvay, M.L., Glass, R.I., Gentsch, J., 2004. Rotavirus strain surveillance in Latin America: a review of the last nine years. Pediatr. Infect. Dis. J. 23, S168– 172. Chitambar, S.D., Arora, R., Chabbra, P., 2009. Molecular characterization of a rare G1P[19] rotavirus strain from India: an evidence of reassortment between human and porcine rotavirus strains. J. Med. Microbiol. 58, 1611–1615. Ciarlet, M., Hoshino, Y., Liprandi, F., 1997. Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum? J. Virol. 71, 8213–8220. Collins, P.J., Martella, V., Buonavoglia, C., O’Shea, H., 2010. Identification of a G2-like porcine rotavirus bearing a novel VP4 type P[32]. Vet. Res. 41, 73. Cunliffe, N.A., Gondwe, J.S., Graham, S.M., Thindwa, B.D., Dove, W., Broadhead, R.L., Molyneux, M.E, Hart, C.A., 2001. Rotavirus strain diversity in Blantyre, Malawi, from 1997 to 1999. J. Clin. Microbiol. 39, 836–843. Desselberger, U., Wolleswinkel-van den Bosch, J., Mrukowicz, J., Rodrigo, C., Giaquinto, C., Vesikari, T., 2006. Rotavirus types in Europe and their significance for vaccination. Pediatr. Infect. Dis. J. 25, S30–41. Eichwald, C.F., Fabbretti, V.E., Burrone, O.R., 2002. Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. J. Virol. 76, 3461–3470. Esona, M.D., Foytich, K., Wang, Y., Shin, G., Wei, G., Gentsch, J.R., Glass, R.I., Jiang, B., 2010. Molecular characterization of human rotavirus vaccine strain CDC-9 during sequential passages in Vero cells. Hum. Vaccine 6, 247–253. Esona, M.D., Mijatovic-Rustempasic, S., Conrardy, C., Tong, s., Kuzmin, I.V., Agwanda, B., Breiman, R.F., Bangai, K., Niezgoda, M., Rupprecht, c.E., Gentsch, J.R., Bown, M.D., 2010. Reassortant group A rotavirus from straw-colored fruit bat (Edidolon helvum). Emerg. Infect. Dis. 16, 1844–1852. Espinola, E.E., Amarilla, A., Arbiza, J., Parra, G.I., 2008. Sequence and phylogenetic analysis of the VP4 gene of human rotaviruses in Paraguay. Arch. Virol. 153, 1067–1073. Estes, M.K., Kapikian, A.Z., 2007. Rotaviruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M., Griffin, D.E., Lamb, R.A., Martin, M.A., Roizman, B., Straus, S.E. (Eds.), Fields Virology. 5th ed. Lippincott, Williams and Wilkins, Philadelphia, PA, pp. 1917–1974.

Freeman, M.M., Kerin, T., Hull, J., Teel, E., Esona, M., Parashar, U., Glass, R.I., Gentsch, J.R., 2009. Phylogenetic analysis of novel G12 rotaviruses in the United States: a molecular search for the origin of a new strain. J. Med. Virol. 81, 736–746. Gentsch, J.R., Laird, A.R., Bielfelt, B., Griffin, D.D., Banyai, K., Ramachandran, M., Jain, V., Cunliffe, N.A., Nakagomi, O., Kirkwood, C.D., Fischer, T.K., Parashar, U.D., Bresee, J.S., Jiang, B., Glass, R.I., 2005. Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J. Infect. Dis. 192, S146–159. Gouvea, V., Glass, R.I., Woods, P., Taniguchi, K., Clark, H.F., Forrester, B., Fang, Z.Y., 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28, 276–282. Gouvea, V., Lima, R.C., Linhares, R.E., Clark, H.F., Nosawa, C.M., Santos, N., 1999. Identification of two lineages (WA-like and F45-like) within the major rotavirus genotype P[8]. Virus Res. 59, 141–147. Grimwood, K., Kirkwood, C.D., 2008. Human rotavirus vaccines: too early for the strain to tell. Lancet 371, 1144–1145. Heiman, E.M., McDonald, S.M., Barro, M., Taraporewala, Z.F., Bar-Magen, T., Patton, J.T., 2008. Group A human rotavirus genomics: evidence that gene constellations are influenced by viral protein interactions. J. Virol. 82, 11106–11116. Iturriza-Gomara, M., Anderton, E., Kang, G., Gallimore, C., Phillips, W., Desselberger, U., Gray, J., 2003. Evidence for genetic linkage between the gene segments encoding NSP4 and VP6 proteins in common and reassortant human rotavirus strains. J. Clin. Microbiol. 41, 3566–3573. Kaljot, K.T., Shaw, R.D., Rubin, D.H., Greenberg, H.B., 1988. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis. J. Virol. 62, 1136–1144. Kang, G., Arora, R., Chitambar, S.D., Deshpande, J., Gupte, M.D., Kulkarni, M., Naik, T.N., Mukherji, D., Venkatasubramaniam, S., Gentsch, J.R., Glass, R.I., Parashar, U.D., 2009. Multicenter, hospital-based surveillance of rotavirus disease and strains among Indian children aged <5 years. J. Infect. Dis. 200, 147–153. Kang, G., Kelkar, S.D., Chitambar, S.D., Ray, P., Naik, T.N., 2005. Epidemiological profile of rotaviral infection in India: challaenges for the 21st century. J. Infect. Dis. 192, S120–S126. Khamrin, P., Maneekarn, N., Malasao, R., Nguyen, T.A., Ishida, S., Okitsu, S., Ushijima, H., 2010. Genotypic linkages of VP4, VP6, VP7, NSP4 NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand. Infect. Genet. Evol. 10, 467–472. Le, V.P., Chung, Y.C., Kim, K., Chung, S.I., Lim, I., Kim, W., 2010. Genetic variation of prevalent G1P[8] human rotaviruses in South Korea. J. Med. Virol. 82, 886–896. Lennon, G., Reidy, N., Cryan, B., Fanning, S., O’ Shea, H., 2008. Changing profile of rotavirus in Ireland: predominance of P[8] and emergence of P[6] and P[9] in mixed infections. J. Med. Virol. 80, 524–530. Matthijnssens, J., Ciarlet, M., Heiman, E., Arijs, I., Delbeke, T., McDonald, S.M., Palombo, E.A., Go´mara, M.I., Maes, P., Patton, J.T., Rahman, M., Ranst, M.V., 2008. Full genome-based classification of rotaviruses reveals a common origin between human Wa-like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains. J. Virol. 82, 3204–3219. Matthijnssens, J., Ciarlet, M., Rahman, M., Attoui, H., Ba´nyai, K., Estes, M.K., Gentsch, J.R., Iturriza-Go´mara, M., Kirkwood, C.D., Martella, V., Mertens, P.P., Nakagomi, O., Patton, J.T., Ruggeri, F.M., Saif, L.J., Santos, N., Steyer, A., Taniguchi, K., Desselberger, U., Van Ranst, M., 2008. Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments. Arch. Virol. 153, 1621–1629. Matthijnssens, J., Potgieter, C.A., Ciarlet, M., Parreno, V., Martella, V., Banyai, K., Garaicoechea, L., Palombo, E.A., Novo, L., Zeller, M., Arista, S., Gerna, G., Rahman, M., Van Ranst, M., 2009. 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, 2917–2929. Matthijnssens, J., Rahman, M., Ciarlet, M., Van Ranst, M., 2008. Emerging human rotavirus genotypes. In: Palombo, A.E. (Ed.), Viruses in the Environment. Research Signpost, Trivandrum, India. Matthijnssens, J., Rahman, M., Martella, V., Xuelei, Y., De Vos, S., De Leener, K., Ciarlet, M., Buonavoglia, C., Van Ranst, M., 2006. Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission. J. Virol. 80, 3801–3810. Mattion, N.M., Cohen, J., Aponte, C., Estes, M.K., 1992. Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34. Virology 190, 68–83. McDonald, S.M., Matthijnssens, J., McAllen, J.K., Hine, E., Overton, L., Wang, S., Lemey, P., Zeller, M., Van Ranst, M., Spiro, D.J., Patton, J.T., 2009. Evolutionary dynamics of human rotaviruses: balancing reassortment with preferred genome constellations. Plos Pathog. 5, e1000634. Min, B.S., Noh, Y.J., Shin, J.H., Baek, S.Y., Kim, J.O., Min, K.I., Ryu, S.R., Kim, B.G., Lee, S.H., Min, H.K., Ahn, B.Y., Park, S.N., 2004. Surveillance study (2000–2001) of Gand P-type human rotaviruses circulating in South Korea. J. Clin. Microbiol. 42, 4297–4299. Moea, K., Thua, H.M., Ooa, W.M., Ayea, K.M., Shwea, T.T., Mara, W., Kirkwood, C.D., 2009. Genotyping of rotavirus isolates collected from children less than 5 years of age admitted for diarrhoea at the Yangon Children’s Hospital. Myanmar Vaccine 27S, F89–F92. Mossel, E.C., Ramig, R.F., 2002. Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse. J. Virol. 76, 6502– 6509. Mukherjee, A., Dutta, D., Ghosh, S., Bagchi, P., Chattopadhyay, S., Nagashima, S., Kobayashi, N., Dutta, P., Krishnan, T., Naik, T.N., Chawla-Sarkar, M., 2009. Full

R. Arora, S.D. Chitambar / Infection, Genetics and Evolution 11 (2011) 504–511 genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission. Arch. Virol. 154, 733–746. Palmer, E.L., Martin, M.L., 1988. Electron microscopy in viral diagnosis, vol. 77. CRC Press Inc.. Patton, J.T., Salter-Cid, L., Kalbach, A., Mansell, E.A., Kattoura, M., 1993. Nucleotide and amino acid sequence analysis of the rotavirus nonstructural RNA-binding protein NS35. Virology 192, 438–446. Phan, T.G., Okitsu, S., Maneekarn, N., Ushijima H, 2007. Genetic heterogeneity, evolution and recombination in emerging G9 rotaviruses. Infect. Genet. Evol. 7, 656–663. Rahman, M., Matthijnssens, J., Saiada, F., Hassan, Z., Heylen, A., Azim, T., Ranst, M.V., 2010. Complete genomic analysis of a Bangladeshi G1P[8] rotavirus strain detected in 2003 reveals a close evolutionary relationship with contemporary human Wa-like strains. Infect. Genet. Evol. 10, 746–754. Rahman, M., Matthijnssens, J., Yang, X., Delbeke, T., Arijs, I., Taniguchi, K., IturrizaGo´mara, M., Iftekharuddin, N., Azim, T., Van Ranst, M., 2007. Evolutionary history and global spread of the emerging G12 human rotaviruses. J. Virol. 81, 2382–2390. Ramani, S., Iturriza-Gomara, M., Jana, A.K., Kuruvilla, K.A., Gray, J.J., Brown, D.W., Kang, G., 2009. 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, 237–244. Santos, N., Hoshino, Y., 2005. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev. Med. Virol. 15, 29–56. Schuman, T., Hotzel, H., Otto, P., Johne, R., 2009. Evidence of interspecies transmission and reassortment among avian group A rotaviruses. Virology 386, 334– 343. Sdiri-Loulizi, K., Gharbi-Khelifi, H., de Rougemont, A., Chouchane, S., Sakly, N., Ambert-Balay, K., Hassine, M., Guediche, M.N., Aouni, M., Pothier, P., 2008. Acute infantile gastroenteritis associated with human enteric viruses in Tunisia. J. Clin. Microbiol. 46, 1349–1355. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4. 0. Mol. Biol. Evol. 24, 1596–1599. Tatte, V.S., Gentsch, J.R., Chitambar, S.D., 2010a. Characterization of group A rotavirus infections in adolescents and adults from Pune India: 1993–1996 and 2004–2007. J. Med. Virol. 82, 519–527.

511

Tatte, V.S., Rawal, K.N., Chitambar, S.D., 2010b. Sequence and phylogenetic analysis of the VP6 and NSP4 genes of human rotavirus strains: evidence of discordance in their genetic linkage. Infect. Genet. Evol. 10, 940–949. Taylor, J.A., O’Brien, J.A., Yeager, M., 1996. The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains. EMBO J. 15, 4469–4476. Theil, K.W., McCloskey, C.M., Saif, L.J., Redman, D.R., Bohl, E.H., Hancock, D.D., Kohler, E.M., Moorhead, P.D., 1981. Rapid, simple method of preparing rotaviral double-standard ribonucleic acid for analysis by polyacrylamide gel electrophoresis. J. Clin. Microbiol. 14, 273–280. Tian, P., Estes, M.K., Hu, Y., Ball, J.M., Zeng, C.Q., Schilling, W.P., 1995. The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J. Virol. 69, 5763–5772. Trinh, Q.D., Nguyen, T.A., Phan, T.G., Khamrin, P., Yan, H., Le Hoang, P., Maneekaran, N., Yan, L., Yagyu, F., Okitsu, S., Ushijima, H., 2007. Sequence analysis of the VP7 gene of human rotavirus G1 isolated in Japan, China, Thailand and Vietnam in the context of changing distribution of rotavirus G-types. J. Med. Virol. 79, 1009–1016. Urasawa, T., Urasawa, S., Taniguchi, K., 1981. Sequential passages of human rotavirus in MA-104 cells. Microbiol. Immunol. 25, 1025–1035. Ursu, K., Kisfali, P., Rigo, D., Ivanics, E., Erdelyi, K., Dan, A., Melegh, B., Martella, V., Banyai, K., 2009. Molecular analysis of the VP7 gene of pheasant rotaviruses identifies a new genotype, designated G23. Arch. Virol. 154, 1365–1369. Varghese, V., Das, S., Singh, N.B., Kojima, K., Bhattacharya, S.K., Krishnan, T., Kobayashi, N., Naik, T.N., 2004. Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4. Arch. Virol. 149, 155–172. Varghese, V., Ghosh, S., Das, S., Bhattacharya, S.K., Krishnan, T., Karmakar, P., Kobayashi, N., Naik, T.N., 2006. Characterization of VP1 VP2 and VP3 gene segments of a human rotavirus closely related to porcine strains. Virus Genes 32, 241–247. Wang, Yuan-Hong., Kobayashi, N., Nagashima, S., Zhou, X., Ghosh, S., Peng, Jin-Song, Hu, Q., Zhou, Dun-Jin., Yang, Zhan-Qiu., 2010. Full Genomic Analysis of a porcine–bovine reassortant G4P[6] Rotavirus Strain R479 Isolated From an Infant in China. J. Med. Virol. 82, 1094–1102. Zade, J.K., Chhabra, P., Chitambar, S.D., 2009. Characterization of VP7 and VP4 genes of rotavirus strains: 1990–1994 and 2000–2002. Epidemiol. Infect. 137, 936–942.