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1 Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium
2 Laboratory of Virology, ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh
Correspondence
Jelle Matthijnssens
jelle.matthijnssens{at}uz.kuleuven.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the sequences determined in this work are EF554082–EF554092.
Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). RVs belong to the family Reoviridae, and possess a genome of 11 segments of double stranded RNA, encoding six structural (VP) and six non-structural proteins. Based on the protease-sensitive VP4 and the glycosylated VP7 proteins forming the outer capsid of the rotavirus particles, a widely used dual classification system exists, dividing VP7 into 19 G genotypes and VP4 into 27 P genotypes (Estes & Kapikian, 2007; Khamrin et al., 2007; Martella et al., 2006, 2007; Matthijnssens et al., 2008a; Rahman et al., 2005; Rao et al., 2000; Steyer et al., 2007). Recently, the remaining rotavirus genes encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, NSP4 and NSP5 have also been divided into respectively 4 R genotypes, 5 C genotypes, 6 M genotypes, 11 I genotypes, 14 A genotypes, 5 N genotypes, 7 T genotypes, 11 E genotypes, and 6 H genotypes, based on specific nucleotide sequence cut-off identity values for each gene segment (Matthijnssens et al., 2008a).
At least 11 G and 11 P genotypes have been isolated from humans, but only G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] are currently of epidemiological importance worldwide (Estes & Kapikian, 2007; Matthijnssens et al., 2008b). G6 RVs are the most common rotavirus genotype among cattle and they are mainly found in combination with P[5], P[1] and P[11] (Matthijnssens et al., 2008b). A limited number of human rotaviruses with the G6 specificity have been described in literature. Human G6 RVs in combination with P[14] have been isolated in Italy (PA169, 111/05-27), Australia (MG6, MG6.01, aG6.01, ASG6.02), Hungary (Hun5) and Belgium (B10925-97) (Bányai et al., 2003; Gerna et al., 1992; Palombo & Bishop, 1995, Matthijnssens et al., unpublished data). A few G6P[9] human rotavirus strains have been found in the United States (Se584), Italy (PA151) and Hungary (Hun1-4, Hun6-8) (Bányai et al., 2003; Gerna et al., 1992; Griffin et al., 2002). A recent multiplex RT-PCR-based study identified G6 human rotaviruses in France, Italy, and Spain (Van Damme et al., 2007). So far, G6 in combination with P[6] has been described only once in the literature. This strain, B1711, was isolated in Belgium from a child who had just returned from a holiday in Mali in 2002 (Rahman et al., 2003). From a second human G6P[6] rotavirus strain, R353, isolated in France, only the partial VP7 gene sequence is available in GenBank (accession no. DQ122400
[GenBank]
).
In recent years, an increasing number of papers have reported the sequencing of multiple complete rotavirus genomes in order to study rotavirus interspecies transmission, reassortments and evolutionary relationships between human and animal rotavirus strains (Ito et al., 2001; Matthijnssens et al., 2006a, b, 2008a; Rahman et al., 2007; Small et al., 2007). To unravel the evolutionary history of the previously described unusual strain B1711, we sequenced its entire genomic complement and conducted phylogenetic and SimPlot analyses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
RT-PCR.
Viral RNA was extracted using the QIAamp Viral RNA Mini kit (Qiagen) according to the manufacturer's instructions. The extracted RNA was denatured at 95 °C for 3 min and reverse transcription followed by polymerase chain reaction (RT-PCR) were carried out using the Qiagen OneStep RT-PCR kit (Qiagen). Used primers are shown in Supplementary Table S1, available with the online version of this paper. The RT-PCR reaction was carried out with an initial reverse transcription step at 45 °C for 30 min, followed by PCR activation at 95 °C for 15 min, 30 cycles of amplification and a final extension of 10 min at 70 °C by using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). For the smaller gene segments VP6, NSP1, NSP2, NSP3, NSP4 and NSP5 the amplification cycle conditions were as follows: 45 s at 94 °C, 45 s at 47 °C and 3 min at 72 °C. For the larger segments encoding VP1, VP2, VP3 and VP4, the cycle conditions were 45 s at 94 °C, 45 s at 47 °C and 6 min at 70 °C.
Nucleotide sequencing.
The PCR amplicons were purified with the QIAquick PCR purification kit (Qiagen) and sequenced using the dideoxynucleotide chain-termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) with an ABI PRISM 3100 automated sequencer (Applied Biosystems). The sequencing was performed with the same forward and reverse primers as for the RT-PCR. Primer walking sequencing was performed to cover the complete genome on both strands.
Determination of the 5' and 3' terminal sequences.
To obtain the complete nucleotide sequence of strain B1711, the 5' and 3' terminal sequences of the 11 gene segments were determined using a modified version of the single-primer amplification method (Lambden et al., 1992). Briefly, after RNA extraction a modified amino-linked oligonucleotide (TGP-Linker: 5'-PO4-TTCCTTATGCAGCTGATCACTCTGTGTCA-spacer-NH2-3') was ligated to the 3' end of both strands of the viral dsRNA with T4 RNA Ligase (Promega, Leiden, the Netherlands). RT-PCR with primers TGP-3Out: 5'-TGACACAGAGTGATCAGC-3' (complementary to TGP-Linker) and appropriate gene specific primers (based on the known internal sequences of each segment) was carried out. The following thermal cycling conditions were used: an initial reverse transcription step at 45 °C for 30 min, PCR activation at 95 °C for 15 min, 45 min during which the temperature was gradually lowered from 83 °C to 60 °C (to allow the newly transcribed complementary DNA strands to anneal), 10 min at 72 °C (to allow the DNA polymerase to repair the partial duplexes), 40 cycles of amplification (45 s at 94 °C, 45 s at 45 °C, 1 min at 70 °C), and a final extension of 10 min at 70 °C. These amplified products were purified and sequenced as described above.
RNA and protein sequence analysis.
The chromatogram sequencing files were analysed using Chromas 2.23 (Technelysium), and contigs were generated using SeqMan II (DNASTAR). Nucleotide and protein sequence similarity searches were performed using the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD, USA) BLAST (Basic Local Alignment Search Tool) server on GenBank database release 143.0 (Altschul et al., 1990) and nucleotide identities were calculated using the P-distance. Multiple sequence alignments were calculated using CLUSTAL_X 1.81 (Thompson et al., 1997). The sequence alignment was manually edited in the GeneDoc version 2.6.002 alignment editor (Nicholas et al., 1997).
Phylogenetic analysis.
Phylogenetic and molecular evolutionary analyses were conducted using the MEGA version 2.1 software (Kumar et al., 2001), based on the nucleotide sequences of the different RV gene segments available in GenBank. Genetic distances at the nucleotide level were calculated using the Kimura two-parameter method. The dendrograms were constructed using the neighbour-joining method and bootstrap analysis.
Sequence submission.
The complete nucleotide sequence data of the 11 gene segments of strain B1711 have been deposited in GenBank under the accession numbers EF554082
[GenBank]
–EF554092
[GenBank]
for the genes encoding VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively.
SimPlot analysis.
Simplot analysis was carried out using the Kimura two-parameter method, a window size of 400 bp and a step size of 50 bp with the concatenated genome sequence of strain B1711 as query, and with the concatenated human rotavirus genomes of strains DRC86 and TB-Chen, and the bovine rotavirus strain UK(tc).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and 2, Supplementary Figs S1–S3). For the VP1-, VP2-, VP6-, NSP1-, NSP2-, NSP3-, NSP4- and NSP5-encoding gene segments of strain B1711, a close phylogenetic clustering was found with human strains belonging to the DS-1-like genogroup, respectively in the R2, C2, I2, A2, N2, T2, E2 and H2 genotypes (Figs 1 and 2, Supplementary Figs S1 and S3). Although all eight above-mentioned gene segments belong to the DS-1-like genogroup, there is a varying degree of genetic identity between B1711 and other recently isolated DS-1-like rotavirus strains. For example, there is a very close relationship between B1711 and recently isolated strains from China (TB-Chen), Bangladesh (RV176-00, RV161-00, N26-02) and the Democratic Republic of Congo (DRC88, DRC86), for the gene segments VP2 (96–98 % nucleotide identity), NSP1 (96–98 %), NSP3 (97–99 %) and NSP5 (98–100 %), whereas the identity of VP1 between strains B1711 and the above-mentioned strains is considerably lower (89–92 %). For VP6, NSP2 and NSP4 gene segments of B1711, there is a mixed picture, with high identities to some of the above-mentioned strains (VP6, 99 % nucleotide identity between B1711 and RV161-00; NSP2, 98 % between B1711 and DRC86; NSP4, 96 % between B1711 and TB-Chen), and low identities to other strains (VP6, 87 % nucleotide identity between B1711 and TB-Chen; NSP2, 87 % between B1711 and TB-Chen; NSP4, 82 % between B1711 and RV176-00).
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) (Supplementary Fig. S1). P[6] is a very common genotype on the African continent (Santos & Hoshino, 2005). The VP3-encoding gene segment belonged to the M2 genotype and clustered most closely with PA169, which is believed to have a bovine origin (Gerna et al., 1992). Several other bovine rotavirus strains also clustered closely with B1711, suggesting a possible bovine origin of the third genome segment of strain B1711 (Fig. 1
). The VP7 gene segment showed G6 specificity, a genotype which is also believed to be of bovine origin. This is reflected by the clustering of strain B1711 with bovine G6 strains, rare human G6P[9] strains and the French G6P[6] strain R353, as was shown previously (Rahman et al., 2003) (Supplementary Fig. S2).
The previous observations were confirmed in the SimPlot analysis, where the concatenated genome of strain B1711 was compared with the human DS-1-like strains DRC86 (G8P[6]) and TB-Chen (G2P[4]), and the bovine strain UK(tc) (G6P[5]) (Fig. 3). As could be expected, the identities between B1711 and the human DS-1-like strains was highest for VP1, VP2, VP4, VP6 and NSP1-5, whereas the VP3- and VP7-encoding gene segments of B1711 were more closely related with the bovine strain UK(tc). These data suggest that one or multiple reassortments between bovine and human DS-1-like P[6] rotavirus strain(s) resulted in the emergence of strain B1711.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Matthijnssens et al., 2008b), this reassortment event is not unlikely. Although the isolation of a rare human G6P[6] rotavirus strain might seem like an isolated event, a second G6P[6] human rotavirus strain, R353, was isolated in France. Unfortunately, epidemiological data on this strain have not been published, and only the partial VP7 gene segment, which clusters closely together with strain B1711 (Supplementary Fig. S3), is available in GenBank. Since the original sample was not available, no further analysis could be conducted on this strain, to elucidate the possible relationship with strain B1711. However, the isolation of these G6P[6] strains in different geographical regions – Mali (or Belgium) and France – might suggest that the G6P[6] strains may today be more widely distributed than believed.
A role of VP3 in host range restriction has been proposed by Hoshino and colleagues, who performed a pathogenicity study with human–porcine reassortant rotaviruses in the piglet model (Hoshino et al., 1995). It is not absolutely surprising that the introduction of bovine rotavirus genes (VP7 and VP3) into a human DS-1-like rotavirus results in a viable rotavirus particle able to cause disease in a human host, since DS-l-like human rotaviruses and bovine rotaviruses are genetically rather closely related and might have a common origin, as was proposed previously (Matthijnssens et al., 2008a).
The large geographical distribution of human G6 rotavirus strains is illustrated by the fact that they are being detected all over the world, in Europe, Australia, the USA, Africa and India (Matthijnssens et al., 2008b). Although the RotaTeq vaccine (Merck) was initially designed against the human G1 to G4 genotypes, it was constructed by making mono-reassortants between the bovine rotavirus strain WC3 (G6P7[5]) and human rotavirus strains with G1, G2, G3, G4 and P[8] specificity, leading to the following strains which are included in the vaccine formulation: G1P[5], G2P[5], G3P[5], G4P[5] and G6P[8] (Heaton & Ciarlet, 2007). Although the G6 moiety included in this vaccine was more an artefact than a goal in itself, this vaccine might confer some level of protection against the emerging G6 strains. Although the Rotarix vaccine (GSK) does not contain either the G6 or P[6] genotype, it has been shown to confer a certain level of heterotypic protection (Vesikari et al., 2007). However, studies conducted in settings where the G6 and/or P[6] genotype are more prevalent are needed before the efficacy of both vaccines against these genotypes can be assessed.
Altogether, our data indicate that B1711 was a reassortant strain, with human P[6], DS-1-like rotavirus strain(s) and a bovine G6 rotavirus as parental strains. This kind of analysis would largely benefit from the availability of more complete genome sequences.
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ACKNOWLEDGEMENTS |
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Received 7 May 2008;
accepted 13 June 2008.
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