Open Access

Genetic diversity of rotavirus genome segment 6 (encoding VP6) in Pretoria, South Africa

  • Martin M Nyaga1Email author,
  • Mathew D Esona1, 2,
  • Khuzwayo C Jere1, 3,
  • Ina Peenze1,
  • Mapaseka L Seheri1 and
  • M Jeffrey Mphahlele1
SpringerPlus20143:179

https://doi.org/10.1186/2193-1801-3-179

Received: 6 December 2013

Accepted: 27 March 2014

Published: 5 April 2014

Abstract

Background

Rotavirus viral protein 6 (VP6), encoded by genome segment (GS) 6, is the primary target for rotavirus diagnosis by serological and some molecular techniques. Selected full length nucleotide sequences of GS 6 of rotavirus strains from South Africa were sequenced and analysed to determine genetic diversity and variations within the circulating rotaviruses.

Findings

The VP6 amplicons were sequenced using the Sanger ABI 3130xl. Phylogenetic and pairwise analysis revealed that the VP6 genes of the study strains belonged to two different VP6 [I] genotypes. Five sequences were assigned genotype I1 and seven as genotype I2. Comparison of the group specific antigenic regions of the South African strains to the reference strains, shows that the South African VP6 sequences belonging to the VP6 genotype I2 were highly conserved, with only two amino acids changes at positions 239 (T›N) and 261(I›V). On the other hand, South African VP6 sequences belonging to I1 genotypes revealed several amino acid variations mostly within the antigenic region III.

Conclusions

Rotavirus strains with I1 and I2 genotype are predominantly circulating within the South African communities of which the later seems to be more conserved within the antigenic regions. The observed genetic variations observed within GS 6 of rotaviruses analysed in the current study are unlikely to impact negatively on the performance of the current VP6-based detection methods. Nevertheless, investigators should continually consider this diversity and adapt the primer design for the detection and characterization of the VP6 gene accordingly.

Keywords

RotavirusesViral protein 6Genetic diversitySouth AfricaPretoria

Findings

Introduction

Rotaviruses form a genus rotavirus within the Reoviridae family (Estes and Kapikian 2007). Group A rotaviruses (RVA) are the major cause of severe dehydrating diarrhoea. Every year RVA infects approximately 114 million children that leads to 453,000 childhood deaths of which most occur in Africa and Asia (Tate et al. 2012). In South Africa, approximately 17,644 to 25,630 children are hospitalised of which 2,882 die annually due to rotavirus diarrhoea (WHO, 2012). An estimated 224 to 318 children die due to rotavirus disease in Pretoria and neighboring Brits areas of South Africa (Mapaseka et al. 2010).

RVA are non-enveloped tripled-layered enteric viruses that contain 11 double-stranded RNA GSs. The genome is encased within the VP2 (encoded by GS 2), inner VP6 (encoded by GS 6) capsid shell, and the outer glycosylated VP7 (encoded by GS 9) capsid that contains VP4 (encoded by GS 4). VP6 constitutes more than half of the mass of the rotavirus particle (Estes and Kapikian 2007) and contains rotavirus group antigens that are used to sub-classify RVA into subgroups (SG) I, II, I + II, and “non-I, non-II”) based on their reactivity to monoclonal antibodies (Greenberg et al. 1983).

Molecular methods have an edge in RVA diagnosis and classification over serological methods due to problems associated with the availability of monoclonal antibodies raised against specific rotavirus antigens. Of late, 16 genotypes (I1-I16) have been determined using a nucleotide sequence-based whole genome classification system (Matthijnssens et al. 2011). There is a correlation between the serological and genotype classifications where SG 1 and II corresponds to genotype 2 and 1, respectively (Iturriza-Gómara et al. 2002; Kerin et al. 2007; Matthijnssens et al. 2011).

GS 6 and its encoded VP6 play a major role in RVA detection. VP6 is the primary target antigen for RVA routine diagnostic serological techniques such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunochromatography (Greenberg et al. 1983; Estes and Kapikian 2007). On the other hand, broadly reactive molecular diagnostic assays such as Reverse Transcriptase-Polymerase Chain Reaction (RT)-PCR protocols targets the ends or internal conserved regions of GS 6 nucleotide sequences by employing hybridization methods which uses sequence specific primers and/or probes (Iturriza-Gómara et al. 2002; Lin et al. 2008). The first molecular assays that were developed in the early 1990s utilised GS 6 nucleotide sequences of rotavirus strains circulating from early 1970s to late 1980s (Grinde et al. 1995). More recently, RT-PCR assays based on GS6 have also been developed using old reference and a few new human rotavirus GS 6 sequences. This resulted in a more reliable scheme for conveying various VP6 genogroups of human rotaviruses (Lin et al. 2008; Matthijnssens et al. 2012).

The availability of many complete GS 6 sequences in the GenBank database will ease the prior challenges of improving, developing and validating rotavirus characterisation methods that utilises VP6 encoding GS (Lin et al. 2008). It is important to continuously evaluate the nucleotide sequences for GS 6 of strains circulating particularly in African countries owing to the wide rotavirus G and P genotype variations that have been consistently reported (Mwenda et al. 2010; Kiulia et al. 2014; Seheri et al. 2014; Tsolenyanu et al. 2014). Such studies would inform in advance, potential evolutionary variations that would eventually affect efficiency of VP6 based rotavirus characterisation methods. In this report, the genetic variation of the GS 6 of rotavirus strains collected recently in Pretoria, South Africa, were characterised and compared against those of strains reported from other parts of the world.

Materials and methods

Ethical consideration and sample selection

The project was approved by the Medunsa Research Ethics Committee (MREC/P/168/2008; MREC/P/108/2013; PG). Eleven human stool specimens were selected randomly from previously screened ELISA rotavirus-positive samples from children presenting with diarrhoea at Lancet private pathology laboratories, Pretoria in 2008. These samples included two commonly characterised G1P[8], six uncommon G and P genotype combinations (G1P[4] and G2P[6]), and three G9P[8]/G9P[6] strains that have emerged in the last two decades. One rotavirus ELISA rotavirus-positive porcine sample obtained from the archive of the Medical Research Council-Diarrhoeal Pathogens Research Unit (MRC-DPRU) laboratory that could not be assigned G and P genotypes was also analysed. Nucleotide sequences for human and animal rotavirus strains containing G2, G8, G9 and G12 were selected from our previous studies (Jere et al. 201120122014; Nyaga et al. 2013) to cover frequently characterised strains around this region.

VP7, VP4 and VP6 RT-PCR and genotyping of rotavirus strains

Rotavirus dsRNA was extracted from 10% fecal supernatant using the QIAamp viral extraction kit by following the manufacturer’s instructions (Qiagen, Valencia, CA). Synthesis of cDNA and genotyping of the GS 4 and 9 were performed using RT-PCR as described previously (Góuvea et al. 1990; Gentsch et al. 1992; Iturriza-Gómara et al. 2001; Iturriza-Gómara et al. 2004). In-house designed primer set VP6F (nt 1–20, 5’GGCTTTAAAACGAAGTCTTC3’) and VP6R (nt 1336–1356, 5’TGTAGTGAGAGGATGTGACC 3’) was used to amplify the entire GS 6 (1356 bp). In brief, RNA was denatured at 95°C for 5 min followed by reverse transcription at 50°C for 30 min. Thirty PCR cycles of 95°C for 30 sec, 50°C for 30 sec and 72°C for 45 sec, followed by an elongation step at 72°C for 7 min was performed. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) by following the manufacturer’s instructions. The PCR amplicon were run on a 1% Tris-acetate- ethylenediaminetetraacetic acid- agarose gel stained with 1% ethidium bromide. The GS 6 cDNA amplicons were sequenced using the Sanger ABI 3130xl at Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa. RotaC [http://rotac.regatools.be/] (Maes et al. 2009) was used to assign genotypes to GS 6.

Phylogenetic method

Sequences were aligned using the MUSCLE program within MEGA version 5 (Tamura et al. 2011). Once aligned, the JModelTest 2 program (Posada 2008) was used to identify the optimal evolutionary model that best fitted the sequence datasets. Using corrected Akaike Information Criterion (AICc) the following model; TN93 + G + I was found to best fit the sequence data for the VP6 gene. Using these models, maximum likelihood trees were constructed using MEGA version 5 with 500 bootstrap replicates to estimate branch support. Nucleotide and amino acid distance matrixes were prepared using the p-distance algorithm in MEGA version 5 (Tamura et al. 2011).

Results

The complete sequences of the GS 6 of each selected strain contained 1356 nt and a single ORF from nt 24 to 1214 resulting in deduced protein of 397 amino acid. These were submitted to the GenBank under accession numbers KC618421-KC618432.

GS 6 of the study strains were assigned either I1 or I2 genotypes as indicated in Additional file 1: Table S1. All the South Africa strains with genotype combination of G1P[8] or G9P[8] were assigned VP6 genotype I1 and they shared a nucleotide (amino acid) identity in the range of 82.8-99.6% (91.7-99%), while those with G2P[6] or G1P[4] or G9P[6], were closely related to strains belonging to genotype I2 and shared a nucleotide (amino acid) identity in the range of 79.2-100% (91.2-100%). The South African VP6 genotype I1 strains analysed in this study shared a very high nucleotide (amino acid) identity in the range of 81.1-99.7% (91.4-99.7%) with both reference human (such as RVA/Human-tc/USA/Wa/1974/G1P[8]) and animal (such as RVA/Pig-tc/USA/OSU/1977/G5P9[7]) genotype I1 VP6 sequences obtained from the GenBank database. On the other hand, South African strains assigned as VP6 genotype I2 also shared a nucleotide (amino acid) identity in the range of 86.6-99% (96.2-99.7%) to both reference human (such as RVA/Human-tc/USA/DS-1/1976/G2P[4]) and animal (such as RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]) strains in the same genotype (Additional file 1: Table S1). Phylogenetic analysis divides the South African strains into two separate clusters (Figure 1). Consistent with distance analyses, South African strains in genotype I1 formed a phylogenetic cluster with human rotavirus SGII (Wa-like or genotype I1) strains, while the remaining in genotype I2 clustered with DS-1-like or I2 genotype (SGI) GS 6.
Figure 1

Maximum likelihood phylogenetic trees built in MEGA version 5 with bootstrap statistics as support, show the genetic relationships of nucleotide sequences of VP6 of human and porcine strains from South Africa with known human and animal rotavirus VP6 sequences from the GenBank database. The tree was drawn to scale. Only bootstrap values of 80% and greater are shown. Bars represent 0.2 substitutions per nucleotide position. South African study strains are indicated by filled circle.

Comparison of the amino acid sequences of VP6 proteins of South African strains determined in this study and those from our previous studies, couple with some human and animal rotavirus reference strains reveals several regions that are completely or highly conserved among the South African GS 6 sequences compared to their respective genotypes (Additional file 2). However, South African strains belonging to the I1 or I2 genotypes were closely related to Wa-like or DS-1-like strains, respectively (Additional file 1: Table S1 and Figure 1). Comparison of the group specific antigenic regions of the South African strains to the reference strain RF, shows that the South African VP6 sequences belonging to the VP6 genotype I2 were highly conserved, with only two amino acids changes at positions 239 (T›N) and 261(I›V). On the other hand, South African VP6 sequences belonging to I1 genotypes revealed eight amino acid changes at positions 39(I›V), 45(E›D), 60(N›T), 239 (T›N), 248(Y›F), 252(V›I), 261(I›V) and 396 (V›I). Also, the Subgroup I (SGI) and Subgroup II (SGII) residues were highly conserved among South African strains belonging to I2 genotype, while amino acid changes were observed in SGI residues at positions 172 (A›M), 305 (A›N) and 310 (N›Q) of South African strain in I1 genotype.

Discussion and conclusions

The GS 6 in this study showed significant amounts of genetic variation among strains within the two genotypes. The fact that strains within the I2 genotype were more conserved with less amino acid changes than the I1 strains was supported by the differences in the nucleotide and deduced amino acid identities. For instance, the nucleotide changes for genotype I1 diverged by nt (aa) 3.1 (0.5) and 18.0 (8.6) between themselves and when compared to reference sequences from the GenBank, respectively. Those of I2 diverged by a wider margin, nt (aa) 17.2 (8.3) and 18.0 (8.6) between themselves and when compared to reference sequences from the GenBank, respectively. This could mean that in Pretoria, South Africa, genotype I1 is more prone to genetic changes than genotype I2 and could potentially result in detection inconsistencies in future. The effect of the observed genetic variations on the host disease presentation warrants further understanding.

In these analyses, the GS 6 of previously analysed local circulating strains were also included (Jere et al. 201120122014; Nyaga et al. 2013). Like any other rotavirus GS, the VP6 encoding GS 6 from these strains could be swiftly evolving at different rates due to antigenic drifts caused by point mutations and was clearly shown by the clustering of strain RVA/Human-wt/ZAF/MRC-DPRU3259/2008/G1P[4] with I2 strains. The G1s have previously been reported as only I1 strains (Ghosh and Kobayashi 2011). The nucleotide and amino acid changes reported here were consisted with other studies (Johne et al. 2011; Matthijnssens et al. 2012). Changes occurring at the nucleotide level of VP6-encoding gene over time could cause amino acid sequence variations resulting in changes in VP6 epitopes. This could potentially affect the efficiency of the rotavirus detection methods (Kerin et al. 2007).

The current VP6 detection techniques should work efficiently as the changes observed in the antigenic regions of these South African strains seems not to vary significantly. The limitation of this study is the small sample size albeit including various distinct strains, hence a conclusively determination on how rare or common these changes are occurring in Pretoria, South Africa, cannot be made. Although the sample size was small, the findings were consistent with those reported elsewhere (Kerin et al. 2007; Johne et al. 2011; Matthijnssens et al. 2012). The results suggest that rotavirus investigators should continually consider this diversity and update the primer required for the detection and characterization of the VP6 gene accordingly.

Abbreviations

VP: 

Viral protein

GS: 

Genome segment

RVA: 

Group A rotaviruses

WHO: 

World Health Organization

RNA: 

Ribonucleic acid

SG: 

Subgroup

ELISA: 

Enzyme linked immunosorbent assay

RT-PCR: 

Reverse transcriptase polymerase chain reaction

MREC: 

Medunsa Research Ethics Committee

MRC: 

Medical research council

DPRU: 

Diarrhoeal Pathogens Research Unit

cDNA: 

complementary Deoxyribonucleic acid

kb: 

Kilobase

AICs: 

Akaike Information Criterion

nt: 

Nucleotide

aa: 

Amino acid.

Declarations

Acknowledgements

We thank the Medical Research Council of South Africa and Poliomyelitis Research Foundation for financial support (Grant no: 09/43; 13/62 [PhD]), Lancet laboratories for providing the samples and all staff of the MRC/DPRU for their support.

Authors’ Affiliations

(1)
Medical Research Council/Diarrhoeal Pathogens Research Unit, Department of Virology, Medunsa Campus, University of Limpopo/NHLS Dr George Mukhari Tertiary Laboratory
(2)
Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, NCIRD, CDC
(3)
Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool

References

  1. Estes M, Kapikian A: Rotaviruses. In Fields Virology. 5th edition. Edited by: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizmzn B. Philadelphia: Kluver Health/Lippincott, Williams&Wilkins; 2007:1917-1974.Google Scholar
  2. Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, Das BK, Bhan MK: Identification of group A rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol 1992, 30: 1365-1373.Google Scholar
  3. Ghosh S, Kobayashi N: Whole-genomic analysis of rotavirus strains: current status and future prospects. Future Microbiol. 2011, 6: 1049-1065. 10.2217/fmb.11.90View ArticleGoogle Scholar
  4. Góuvea V, Glass RI, Woods P, Taniguchi K, Clark HF, Forrester B, Fang ZY: Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol 1990, 28(2):276-282.Google Scholar
  5. Greenberg HB, Valdesuso J, Van Wyke K, Midthun K, Walsh M, McAuliffe V, Wyatt RG, Kalica AR, Flores J, Hoshino Y: Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus. J Virol 1983, 47: 267-275.Google Scholar
  6. Grinde B, Jonassen TO, Ushijima H: Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction. J Virol Methods 1995, 55: 327-338. 10.1016/0166-0934(95)00070-XView ArticleGoogle Scholar
  7. Iturriza-Gómara M, Isherwood B, Desselberger U, Gray J: Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999. J Virol 2001, 75: 3696-3705. 10.1128/JVI.75.8.3696-3705.2001View ArticleGoogle Scholar
  8. Iturriza-Gómara M, Wong C, Blome S, Desselberger U, Gray J: Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation. J Virol 2002, 76: 6596-6601. 10.1128/JVI.76.13.6596-6601.2002View ArticleGoogle Scholar
  9. Iturriza-Gómara M, Kang G, Gray J: Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol 2004, 31: 259-265. 10.1016/j.jcv.2004.04.009View ArticleGoogle Scholar
  10. Jere KC, Mlera L, O'Neill HG, Potgieter AC, Page NA, Seheri ML, Van Dijk AA: Whole genome analyses of African G2, G8, G9, and G12 rotavirus strains using sequence-independent amplification and 454® pyrosequencing. J Med Virol. 2011, 83: 2018-2042. 10.1002/jmv.22207View ArticleGoogle Scholar
  11. Jere KC, Mlera L, O'Neill HG, Peenze I, Van Dijk AA: Whole genome sequence analyses of three African bovine rotaviruses reveal that they emerged through multiple reassortment events between rotaviruses from different mammalian species. Vet Microbiol. 2012, 159: 245-250. 10.1016/j.vetmic.2012.03.040View ArticleGoogle Scholar
  12. Jere KC, Esona MD, Ali YH3, Peenze I, Roy S, Bowen MD, Saeed IK, Khalafalla AI, Nyaga MM, Mphahlele J, Steele D, Seheri ML: Novel NSP1 genotype characterised in an African camel G8P[11] rotavirus strain. Infect Genet Evol 2014, 21: 58-66.View ArticleGoogle Scholar
  13. Johne R, Otto P, Roth B, Löhren U, Belnap D, Reetz J, Trojnar E: Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology 2011, 412: 384-391. 10.1016/j.virol.2011.01.031View ArticleGoogle Scholar
  14. Kerin TK, Kane EM, Glass RI, Gentsch JR: Characterization of VP6 genes from rotavirus stains collected in the United States from 1996–2002. Virus Genes 2007, 35: 489-495. 10.1007/s11262-007-0119-7View ArticleGoogle Scholar
  15. Kiulia NM, Nyaga MM, Seheri ML, Wolfaardt M, Van Zyl WB, Esona MD, Irimu G, Inoti M, Gatinu BW, Njenga PK, Taylor MB, Nyachieo A: Rotavirus G and P types circulating in the eastern region of Kenya: predominance of G9 and emergence of G12 genotypes. Pediatr Infect Dis J. 2014, 1: 85-88.View ArticleGoogle Scholar
  16. Lin YP, Kao CL, Chang SY, Taniguchi K, Hung PY, Lin HC, Huang LM, Huang HH, Yang JY, Lee CN: Determination of human rotavirus VP6 genogroups I and II by reverse transcription-PCR. Clin Microbiol 2008, 46: 3330-3337. 10.1128/JCM.00432-08View ArticleGoogle Scholar
  17. Maes P, Matthijnssens J, Rahman M, Van Ranst M: RotaC: a web-based tool for the complete genome classification of group A rotaviruses. BMC Microbial 2009, 9: 238. 238 10.1186/1471-2180-9-238View ArticleGoogle Scholar
  18. Mapaseka SL, Dewar JB, van der Merwe L, Geyer A, Tumbo J, Zweygarth M, Bos P, Esona MD, Steele AD, Sommerfelt H: Prospective hospital-based surveillance to estimate rotavirus disease burden in the Gauteng and North West Province of South Africa during 2003–2005. J Infect Dis 2010, 202: 131-138. 10.1086/653558View ArticleGoogle Scholar
  19. Matthijnssens J, Ciarlet M, McDonald SM, Attoui H, Bányai K, Brister JR, Buesa J, Esona MD, Estes MK, Gentsch JR, Iturriza-Gómara M, Johne R, Kirkwood CD, Martella V, Mertens PP, Nakagomi O, Parreño V, Rahman M, Ruggeri FM, Saif LJ, Santos N, Steyer A, Taniguchi K, Patton JT, Desselberger U, Van Ranst M: Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol 2011, 156: 1397-1413. 10.1007/s00705-011-1006-zView ArticleGoogle Scholar
  20. Matthijnssens J, Otto PH, Ciarlet M, Desselberger U, Van Ranst M, Johne R: VP6-sequence-based cutoff values as a criterion for rotavirus species demarcation. Arch Virol 2012, 157: 1177-1182. 10.1007/s00705-012-1273-3View ArticleGoogle Scholar
  21. Mwenda JM, Ntoto KM, Abebe A, Enweronu-Laryea C, Amina I, Mchomvu J, Kisakye A, Mpabalwani EM, Pazvakavambwa I, Armah GE, Seheri LM, Kiulia NM, Page N, Widdowson MA, Steele AD: Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network. J Infect Dis. 2010, 202: 5-11. 10.1086/653557View ArticleGoogle Scholar
  22. Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol 2008, 25: 1253-1256. 10.1093/molbev/msn083View ArticleGoogle Scholar
  23. Nyaga MM, Jere KC, Peenze I, Mlera L, Van Dijk AA, Seheri ML, Mphahlele MJ: Sequence analysis of the whole genomes of five African human G9 rotavirus strains. Infect Genet Evol. 2013, 16: 62-77.View ArticleGoogle Scholar
  24. Seheri M, Nemarude L, Peenze I, Netshifhefhe L, Nyaga MM, Ngobeni HG, Maphalala G, Maake LL, Steele AD, Mwenda JM, Mphahlele JM: Update of rotavirus strains circulating in Africa from 2007 through 2011. Pediatr Infect Dis J. 2014, 1: 76-84.View ArticleGoogle Scholar
  25. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28: 2731-2739. 10.1093/molbev/msr121View ArticleGoogle Scholar
  26. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, Parashar UD: The WHO-coordinated global Rotavirus Surveillance Network. 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis 2012, 12: 136-141. 10.1016/S1473-3099(11)70253-5View ArticleGoogle Scholar
  27. Tsolenyanu E, Seheri M, Dagnra A, Djadou E, Tigossou S, Nyaga M, Adjeoda E, Armah G, Mwenda JM, Atakouma Y: Surveillance for rotavirus gastroenteritis in children less than 5 years of age in Togo. Pediatr Infect Dis J. 2014, 1: 14-18.View ArticleGoogle Scholar
  28. WHO 2012.http://www.who.int/immunization/monitoring_surveillance/burden/estimates/rotavirus/en/

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