Infection, Genetics and Evolution 12 (2012) 1492–1500

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Short communication

Full genome characterization of a porcine-like human G9P[6] rotavirusstrain isolated from an infant in Belgium

Mark Zeller, Elisabeth Heylen, Sarah De Coster, Marc Van Ranst, Jelle Matthijnssens ⇑Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium

a r t i c l e i n f o

Article history:Received 18 August 2011Received in revised form 1 March 2012Accepted 2 March 2012Available online 10 March 2012

Keywords:Group A rotavirusInterspecies transmissionFull genome characterizationGene rearrangementG9P[6]Pig

1567-1348/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.meegid.2012.03.002

⇑ Corresponding author. Address: Laboratory ofVirology, Department of Microbiology and ImmunoloResearch, Minderbroedersstraat 10, B-3000 Leuven, Bfax: +32 16 332131.

E-mail address: jelle.matthijnssens@uz.kuleuven.a

a b s t r a c t

Interspecies transmissions of group A rotavirus (RVA) strains among animals and humans are thought totake place frequently. During a RVA surveillance study in Belgium we isolated an unusual G9P[6] RVAstrain, RVA/human-wt/BEL/BE2001/2009/G9P[6], from a 1 month old boy, which did not cluster withother G9 or P[6] strains isolated in Belgium. In this study we sequenced and characterized the completegenome of this unusual G9P[6] strain BE2001. Phylogenetic analyses of all 11 genes revealed a uniquegenotype constellation: G9-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1. The VP6 and NSP1 genotypes I5 andA8 are genotypes commonly found in porcine RVA strains, while the VP7 and VP4 genes clustered onlydistantly to human lineages of G9 and P[6], respectively. The VP1, VP2, VP3, NSP2, NSP4 and NSP5 genesall belonged to Wa-like genotypes, but clustered more closely to porcine strains than to human strains.NSP3 belonged to the rare T7 genotype. Thus far, T7 genotypes have only been detected in one porcine-like human strain (RVA/human-tc/CHN/R479/2004/G4P[6]), one bovine-like human strain (RVA/human-xx/IND/mani-265/2007/G10P[6]) and one bovine RVA strain (RVA/cow-tc/GBR/UK/1973/G6P7[5]).Sequence analysis of the BE2001 NSP5 gene segment revealed a 300 nucleotide duplication in the 30

end non-coding region. BE2001 is most likely a direct interspecies transmission between a pig and ahuman. Inquiry with the patient’s physician revealed that the father of the patient had been workingon a pig farm in the week the patient became ill, providing a plausible route of transmission.

� 2012 Published by Elsevier B.V.

1. Introduction

Group A rotavirus (RVA) is a member of the Reoviridae familyand cause gastroenteritis in the young of humans and many animalspecies (Estes and Kapikian, 2007). RVAs have a segmented dsRNAgenome which comprises 11 segments. Except for the shortest seg-ment, which encodes NSP5 and NSP6, all segments are monocis-tronic. Traditionally, RVAs are classified according to their twoouter capsid proteins: VP7 and VP4, in which VP7 determines theG-genotype and VP4 determines the P-genotype. Recently a newnomenclature system was proposed encompassing all 11 segmentsallowing a better insight in genetic relationships among RVAs andproviding a framework in which unusual genotype combinationscan be more easily detected (Matthijnssens et al., 2008a,b, 2011).

In humans, six RVA genotypes are responsible for the majorityof disease: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8](Matthijnssens et al., 2008c). Full genome analyses of humanRVA strains revealed that there are two major genogroups: human

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Clinical and Epidemiologicalgy, Rega Institute for Medicalelgium. Tel.: +32 16 332166;

c.be (J. Matthijnssens).

P[8] strains are generally associated with a Wa-like backbonemeaning that they possess a Gx-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 genotype constellation. Human P[4] strains on the other handare most often associated with a DS-1-like backbone and possessa G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotype constellation(Matthijnssens et al., 2008a). Inter-genogroup reassortment is rel-atively rare and is likely constrained by evolutionary adaptation asboth genogroups have different evolutionary origins: Human Wa-like genotypes most likely share a common ancestor with porcineRVA strains, while DS-1-like strains most probably share a com-mon ancestor with bovine RVA strains (Matthijnssens et al.,2008a). Since human and animal RVA strains partially share similargenotype constellations, phylogenetic analyses are necessary todistinguish between the two as very often human and animal RVAswith the same genotype are found in different intra-genotypicclusters (Martella et al., 2006; Mukherjee et al., 2010b).

Human G9 strains have recently emerged globally and are gen-erally found in combination with P[8] and a Wa-like genotype con-stellation (Matthijnssens et al., 2010). As all these recent G9P[8]strains cluster in G9 lineage 3, they have very high similaritiesamong each other and are likely the result of a single reassortmentevent between a human Wa-like strain and a G9 strain with un-known origin in the past (Matthijnssens et al., 2010). Occasionally,human G9 strains have also been isolated in combination with

M. Zeller et al. / Infection, Genetics and Evolution 12 (2012) 1492–1500 1493

other VP4 genotypes, including P[4], P[5], P[6], P[9], P[10], P[11],and P[19] (Das et al., 2004; Matthijnssens et al., 2009; Potgieteret al., 2009; Rippinger et al., 2010; Santos and Hoshino, 2005). Be-sides humans, pigs are the only other host for G9 RVA strainsknown to date. Although few porcine G9 strains have been charac-terized compared with human G9 strains, they represent a highergenetic diversity than human G9 strains and have been found incombination with P[6], P[7], P[13] and P[23] (Collins et al., 2010;Paul et al., 1988; Teodoroff et al., 2005).

Human P[6] strains on the other hand are associated with a verylarge diversity of VP7 genotypes, including G1–G6, G8–G12 (Abdel-Haq et al., 2011; Armah et al., 2010; Banyai et al., 2009a; Clarket al., 2011; Kabue et al., 2010; Li et al., 2008; Matthijnssenset al., 2006b, 2008d; Rippinger et al., 2010). The reason for thisdiversity could be the seemingly lack of preference for Wa-like orDS-1-like backgrounds as P[6] strains have been found in combina-tion with both genotype constellations (Potgieter et al., 2009;Rippinger et al., 2010, Heylen, personal communication). Untilnow the (near) complete genome sequence of only two otherG9P[6] RVAs are known: RVA/human-wt/RSA/GR10924/1999/G9P[6] and RVA/human-xx/IND/mcs-13/2007/G9P[6]. GR10924,was isolated in South Africa and possesses a predominantly bovineDS-1-like background while mcs-13 was isolated in India and has apredominantly porcine Wa-like background (Mukherjee et al.,2009; Potgieter et al., 2009).

During routine RVA surveillance in Belgium in 2009 we identi-fied a G9P[6] strain, designated as BE2001, isolated from a 1 monthold boy that was hospitalized with acute diarrhea. The patient’sparents were living in a rural area in the province of East Flanders.Initial sequence analyses of BE2001 of approximately 800 nt longfragments of VP7 and VP4 showed that both genes had relativelylow similarity with known G9 and P[6] strains in GenBank anddid not belong to the common human G9 and P[6] lineages foundin G9 and P[6]. Since previously both G9 as P[6] strains have beenreported in pigs it seemed of interest to sequence all 11 segmentsto gain more insight in the origin of strain BE2001.

2. Methods

Viral RNA was extracted using the QIAamp Viral RNA mini-kit(Qiagen/Westburg, Leusden, The Netherlands) according to themanufacturer’s instructions. Subsequently RNA extracts weredenatured at 95 �C for 2 min and RT-PCR was carried out usingthe Qiagen One Step RT-PCR kit (Qiagen/Westburg). Primers usedfor all 11 segments can be found in the Supplementary Informa-tion. The RT-PCR was carried out with an initial RT step at 50 �Cfor 30 min; PCR activation was at 95 �C for 15 min, followed by35 cycles of amplification (30 s at 94 �C, 30 s at 45 �C, and 2 or6 min at 72 �C for the seven shortest segments and the four longestsegments, respectively), with a final extension of 10 min at 72 �Cusing the Biometra T3000 thermocycler (Biometra, Westburg BV,Netherlands). Subsequently all 11 amplicons were pooled and se-quenced on the 454 Roche GS-FLX sequencing platform. The se-quence reads obtained from the 454™ Roche GS-FLX weretrimmed for primers and de novo assembled using MIRA 3.0.4.(Chevreux et al., 1999). Sites with insufficient sequence read cover-age were resequenced using traditional Sanger sequencing. The ob-tained consensus sequences were aligned with a reference set ofRVA genomes and manually edited for insertions and deletions athomopolymer regions. Phylogenetic trees were constructed usingthe neighbor-joining method with the kimura-2-parameter inMEGA 5.04 (Tamura et al., 2011).

3. Results

3.1. Genotype constellation

The complete sequence of the open reading frame from strainBE2001 was determined and subsequently analyzed with theRVA genotype tool, RotaC (http://rotac.regatools.be/) (Maes et al.,2009). The genotype for each of the 11 segments of strainBE2001 was G9-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1 (Table 1).

Although most segments (encoding VP1, VP2, VP3, NSP2, NSP4and NSP5) belonged to the Wa-like genotype 1, other genotypessuch as I5 (VP6), A8, (NSP1) and T7 (NSP3) are less often found inhumans. In fact BE2001 is the first RVA described which possessesa genotype constellation including I5, A8 and T7. The gene constel-lation of BE2001 was compared with two other human G9P[6]strains (mcs-13 and GR10924), a human G9P[8] strain from Bel-gium (RVA/human-wt/BEL/B3458/2003/G9P[8]) and several animalstrains, including porcine and bovine strains (Table 1). The geno-type constellation of strain BE2001 was most closely related tothe constellation of human strain RVA/human-tc/CHN/R479/2004/G4P[6] and mcs-13, sharing all genotypes except VP7 and NSP1,and VP6 and NSP3, respectively. The human G9P[8] strain B3458has a complete Wa-like background and hence has different geno-types for VP6, NSP1 and NSP3. Besides VP7, VP4 and NSP3, mostporcine strains possess a similar genotype constellation as strainBE2001 although some porcine strains have been found with otherVP6 (I1 instead of I5) and NSP1 (A1 instead of A8) genotypes thanBE2001 (Table 1). Besides strain BE2001, T7 genotypes have beenfound in a bovine strain (RVA/cow-tc/GBR/UK/1973/G6P7[5]), a bo-vine-like human strain (RVA/human-xx/IND/mani-265/2007/G10P[6]), and a porcine-like human strain (R479).

3.2. Phylogenetic analyses

Because for most Wa-like gene segments, porcine and humanRVAs cluster separately in different lineages, we constructed phy-logenetic trees for each of the 11 segments of strain BE2001 to-gether with human, porcine and bovine RVA strains. For VP7,BE2001 was not located in G9 lineage 3, the lineage were most hu-man G9 strains are clustering including both G9P[6] strains, mcs-13 and GR10924. Instead, BE2001 clustered relatively distantlyfrom G9 lineage 3 strains and clustered among porcine strainsRVA/Pig-tc/USA/A2/198x/G9P9[7] and RVA/Pig-wt/JPN/Hokkaido-14/2001/G9P[23] (Hoshino et al., 2005; Teodoroff et al., 2005). Allother porcine strains analyzed in this study possess a differentVP7 genotype: G3-G5, G11 or G12 (Fig. 1).

For VP4, several human G9P[6] strains cluster very closely to-gether, while R479, BE2001 and porcine strain RVA/Pig-tc/USA/Gottfried/1983/G4P[6] were found more distantly from the humanG9P[6] cluster (Fig. 1). The VP6 gene of BE2001 clustered in geno-type I5 together with several porcine strains and human strainR479. However, there is a significant genetic diversity among theI5 genotype strains and BE2001 clustered only distantly to strainR479 and porcine strains. The three other viral structural proteins,VP1, VP2 and VP3, were all Wa-like, but clustered more closely toporcine than to human RVA strains. BE2001 NSP1 clustered ingenotype A8 and was most closely related with porcine strainGottfried (Fig. 2). BE2001 NSP2, NSP4 and NSP5 were all Wa-likegenotypes and clustered more closely to porcine or humanporcine-like Wa-like strains (Fig. 3). The NSP3 gene segment ofBE2001 was assigned to genotype T7. The T7 genotype has thusfar only been found in three other strains: human strains R479and mani-265, and bovine strain UK. However, T7 genotypes are

Table 1Comparison of strain BE2001 with reference strains Wa and DS-1 and other bovine, human and porcine RVA strains. Genotypes are color coded green (human Wa-like), dark green (porcine Wa-like), red (DS-1 like) and blue (G9 and P[6]). Gene segments that are only partially sequenced are indicated with an asterisk.

VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5

RVA/Human-tc/USA/Wa/1974/G1P1A[8] G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-tc/USA/DS-1/1976/G2P1B[4] G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/BEL/BE2001/2009/G9P[6] G9 P[6] I5 R1 C1 M1 A8 N1 T7 E1 H1

RVA/Human-tc/CHN/R479/2004/G4P[6] G4 P[6] I5 R1 C1 M1 A1 N1 T7 E1 H1

RVA/Pig-tc/USA/Gottfried/1983/G4P[6] G4 P[6] I1 R1 C1 M1 A8 N1 T1 E1 H1

RVA/Pig-tc/MEX/YM/1983/G11P9[7] G11 P[7] I5 R1 C1 M1 A8 N1 T1 E1 H1

RVA/Pig-wt/IND/RU172/2002/G12P[7] G12 P[7]* I5 R1 C1* M1 A1 N1 T1 E1 H1

RVA/Pig-tc/USA/OSU/1977/G5P9[7] G5 P[7] I5 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Pig-tc/VEN/A253/1988/G11P9[7] G11 P[7] I5 R1 C2 M1 A1 N1 T1 E1 H1

RVA/Pig-tc/VEN/A131/1988/G3P9[7] G3 P[7] I5 R1 C2 M1 A1 N1 T1 E1 H1

RVA/Human-xx/IND/mcs-13/2007/G9P[6] G9 P[6]* I1 R1* C1* M1* A8 N1 T1 E1 H1

RVA/Human-wt/BEL/B3458/2003/G9P[8] G9 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/RSA/GR10924/1999/G9P[6] G9 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-xx/IND/mani-265/2007/G10P[6] G10 P[6]* I2 R2* C2* M2* A3 N2 T7 E2 H2

RVA/Cow-tc/GBR/UK/1973/G6P7[5] G6 P[5] I2 R2 C2 M2 A3 N2 T7 E2 H3

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Fig. 1. Phylogenetic trees of the full length VP7, VP4 and VP6 ORF sequences. Phylogenetic trees were constructed using the neighbor-joining method with the kimura-2-parameter. Bootstrap values (1000 replicates) above 70% are shown. Strains were color coded green (porcine), magenta (bovine) and dark blue (human) and PO-13 was usedas an outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Phylogenetic trees of the full length VP1, VP2, VP3 and NSP1 ORF sequences. Phylogenetic trees were constructed using the neighbor-joining method with the kimura-2-parameter. Bootstrap values (1000 replicates) above 70% are shown. Strains were color coded green (porcine), magenta (bovine) and dark blue (human) and PO-13 or E403was used as an outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1496 M. Zeller et al. / Infection, Genetics and Evolution 12 (2012) 1492–1500

Fig. 3. Phylogenetic trees of the full length NSP2, NSP3, NSP4 and NSP5 ORF sequences. Phylogenetic trees were constructed using the neighbor-joining method with thekimura-2-parameter. Bootstrap values (1000 replicates) above 70% are shown. Strains were color coded green (porcine), magenta (bovine) and dark blue (human) and PO-13was used as an outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

M. Zeller et al. / Infection, Genetics and Evolution 12 (2012) 1492–1500 1497

Fig. 4. Analysis of the rearranged NSP5 gene of BE2001. Schematic representation of the duplication event: comparison between porcine strain Gottfried and BE2001 (upperpanel). Sequence alignment of the coding and duplicated sequence of BE2001 NSP5. Identities are shown in gray (lower panel).

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rare and all four known T7 genotypes are only distantly related toeach other (Fig. 3).

3.3. Rearrangement of NSP5

A comparison between the NSP5 gene segment of porcine RVAstrains Gottfried and BE2001 revealed a 300 nucleotide duplica-tion, starting outside the ORF at nucleotide 326 and comprisingthe 30 part of the BE2001 NSP5 ORF and 10 nucleotides in thenon-coding region (Fig. 4). Over time, many extra mutations haveaccumulated in the duplicated sequence, resulting in only 88.3%similarity with the coding sequence. The coding and duplicated se-quences are compared in the lower panel of Fig. 4.

4. Discussion

To our knowledge the genotype combination of strain RVA/hu-man-wt/BEL/BE2001/2009/G9P[6] (G9-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1) has not been described before. G9P[6] RVAs are widespread and have been found in many countries, such as Bangladesh,India, the Republic of South Africa, the UK, the USA and Vietnam(Abdel-Haq et al., 2011; Iturriza-Gomara et al., 2000; Mukherjeeet al., 2010a; Nguyen et al., 2007; Potgieter et al., 2009; Unicombet al., 1999). Partial sequence analyses of VP7 and VP4 indicate thatall these were typical human G9P[6] strains, and most of them havea long electropherotype indicating a Wa-like genotype constella-tion. Strain BE2001 also possesses a Wa-like genome, but did notcluster among human or porcine G9 and P[6] strains. In addition,besides NSP3 all the other gene segments of strain BE2001 sharedtheir genotypes with RVA strains commonly found in pigs: I5, R1,C1, M1, A8, N1, E1 and H1. The NSP3 T7 genotype has been isolatedin two other human RVA strains and one bovine strain. Both humanstrains were found with a different backbone: R479 has a typicallyporcine genotype constellation, while mani-265 possessed a geno-type constellation mainly found in bovine RVA strains (Mukherjeeet al., 2010b; Wang et al., 2010). A larger genetic diversity wasfound among T7 genotypes of porcine-like strains than amongthose of bovine-like strains, which might be an indication that T7genotypes have evolved for a longer time in pigs than in cows. How-ever, the evolutionary origins of NSP3 T7s are currently difficult toinfer due to the limited number of T7 genotypes and the fact thatthey have been found in complex genotype constellations.

For most gene segments, BE2001 is only distantly related to hu-man strains circulating in Belgium (B3458 and B4633). Except forthe NSP2 gene of strain B3458, which clustered together with por-cine strains in genotype N1, both B3458 and B4633 strains clus-

tered in human lineages. Therefore it seems highly improbablethat strain BE2001 is the result of a reassortment event betweenporcine and human strains, but more likely a direct interspeciestransmission from pigs to humans. To investigate this we con-tacted the patient’s physician to inquire if there was any contactbetween pigs and the patient within the days before the onset ofthe symptoms. Indeed, it turned out that the father of the patientworked on a pig farm in the week before the patient became ill.Unfortunately we were not able to take feces samples from thesepigs as the farm was already closed down by the time we contactedthe physician. Although we cannot formally rule out other routesof transmission, it is very likely, given the porcine characteristicsof strain BE2001, that these pigs are the source of infection. Formost segments BE2001 has still a considerable genetic distanceto known porcine RVA strains and this could be explained by thefact that available porcine RVA sequences in Genbank are mostlyfrom strains isolated in Asia and South-America and not in Belgiumor Europe. Also in our phylogenetic analysis an apparent clusteringof porcine RVA strains by region of isolation was present, indicat-ing a geographical division. Furthermore, in contrast to the humanRVA population only little is known about porcine RVA populationin Belgium and therefore the full genetic diversity of porcine RVAsis still largely to be discovered.

Rearrangements in the RVA genome have been most frequentlyreported in the NSP5 segment of animal RVA strains (Banyai et al.,2009d; Matthijnssens et al., 2006a). However, in contrary to thefrequent detection of NSP5 rearrangements in lapine RVAs, onlytwo porcine RVAs with a rearranged NSP5 have been reported todate (Bellinzoni et al., 1987; Mattion et al., 1988). In porcine-likehuman RVA strains, like BE2001, NSP5 rearrangements have beenfound only once: RVA/human-wt/THA/Mc345/1989/G9P[6](Kojima et al., 1996). All these rearrangements were present down-stream of the 30 end of the NSP5 gene, resulting in normal NSP5translation. There was only 88.3% similarity between the dupli-cated and the coding region, indicating that this strain must havebeen circulating for a prolonged period of time in pigs beforeinfecting the infant.

RVA surveillance networks are an important tool for the detec-tion of RVA interspecies transmissions, as has been shown by manyother studies (Banyai et al., 2009b,c, 2010; Martella et al., 2009;Matthijnssens et al., 2006a). The increased understanding of theRVA genetic variability in animals has greatly aided to determinethe origins of interspecies transmissions. However, continuingRVA surveillance in humans and animals is required to elucidatethe origins of rare genotypes, such as the T7 genotype found inthe present study.

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Acknowledgements

M.Z. was supported by the Institute for the Promotion of Inno-vation through Science and Technology in Flanders (IWT Vlaander-en). J.M. was supported by an FWO (‘Fonds voor WetenschappelijkOnderzoek’) post-doctoral fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2012.03.002.

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