Infection, Genetics and Evolution 30 (2015) 27–36 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier .com/locate /meegid Molecular epidemiology of noroviruses detected in Nepalese children with acute diarrhea between 2005 and 2011: Increase and predominance of minor genotype GII.13 http://dx.doi.org/10.1016/j.meegid.2014.12.003 1567-1348/� 2014 Elsevier B.V. All rights reserved. ⇑ Corresponding author at: Department of Hygiene and Molecular Epidemiology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel.: +81 95 819 7061; fax: +81 95 819 7064. E-mail address:
[email protected] (O. Nakagomi). 1 Present address: Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam. Thi Nguyen Hoa-Tran a,1, Toyoko Nakagomi a,b, Daisuke Sano c, Jeevan B. Sherchand d, Basu D. Pandey e, Nigel A. Cunliffe b, Osamu Nakagomi a,b,⇑ aDepartment of Hygiene and Molecular Epidemiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan bDepartment of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, UK cDivision of Environmental Engineering, Faculty of Engineering, Hokkaido University, Sapporo, Japan d Tropical Disease Research and Prevention Center, Tribhuvan University, Kathmandu, Nepal e Sukra Raj Tropical and Infectious Disease Hospital, Kathmandu, Nepal a r t i c l e i n f o Article history: Received 10 September 2014 Received in revised form 24 November 2014 Accepted 2 December 2014 Available online 9 December 2014 Keywords: Gastroenteritis Norovirus GII.13 GII.P16/GII.13 Histo-blood group antigen Recombination a b s t r a c t Noroviruses, an important cause of acute gastroenteritis, possess a highly divergent genome which was classified into five genogroups and dozens of genotypes. However, changes in genotype distribution over time were poorly understood, particularly in developing countries. We therefore conducted a molecular epidemiological study which characterized the norovirus strains detected in 4437 Nepalese children with acute diarrhea between November 2005 and January 2011 to gain insight into how their genotypes chan- ged over time. Of the 356 samples positive for noroviruses, 277 (78%) were successfully genotyped into 22 capsid genotypes; GII.4 (n = 113), GII.3 (n = 38) and GII.13 (n = 37) were the majority. Interestingly, GII.13 accounted for only 1.7% (4/230) between 2005 and 2008 (period 1) but increased substantially to 26.2% (33/126) between 2009 and 2011 (period 2). Phylogenetic analysis of the VP1 nucleotide sequences of 35 GII.13 strains indicated that they clustered into two lineages named NPL2008 and NPL2009 to which two period 1 strains and 33 period 2 strains belonged, respectively. Lineage NPL2009 contained GII.13 strains that were detected in a large-scale gastroenteritis outbreak in Germany in 2012. Both Nepalese and German VP1 sequences carried two substitutions, H378N and V394Q, in the putative histo-blood group antigen (HBGA)-binding sites. As to the polymerase genotypes of Nepalese strains, the period 1 strains possessed GII.Pm, but the period 2 strains possessed GII.P13, GII.P16, and GII.P21. Together with recent reports on the predominance of GII.P13/GII.13 and GII.P16/GII.13 in India and GII.P16/GII.13 in European countries, this study predicts that genotype GII.13 which was previously regarded as a minor genotype has a potential to become an epidemiologically important genotype. � 2014 Elsevier B.V. All rights reserved. 1. Introduction Noroviruses, a member of the Caliciviridae family, are a common and important etiological agent of sporadic acute gastroenteritis in children worldwide (Parashar et al., 2009). The norovirus genome comprises a single-stranded, positive-sense RNA encompassing three open reading frames (ORFs), ORF1, 2, and 3. ORF 1 encodes non-structural viral proteins including the RNA-dependent RNA polymerase (RdRp) (Green, 2013); ORF 2 and 3 encode the major capsid protein (VP1) and the minor capsid protein (VP2), respec- tively. VP1 is divided into the shell (S) and the protruding (P) domains; the P domain is further divided into the P1-1, P1-2, and P2 sub-domains (Prasad et al., 1999); the P2 sub-domain is located at the outer surface and carries potential antigenic sites (Prasad et al., 1999); the P2 and P1-2 sub-domains carry determinants for histo-blood group antigen (HBGA) binding (Cao et al., 2007) that are regarded as receptors or co-receptors for noroviruses (Tan and Jiang, 2005). Accumulation of point mutations and recombination occurring between the ORF1 and ORF2 are two mainly driving forces of evolution of noroviruses (Green, 2013). http://crossmark.crossref.org/dialog/?doi=10.1016/j.meegid.2014.12.003&domain=pdf http://dx.doi.org/10.1016/j.meegid.2014.12.003 mailto:
[email protected] http://dx.doi.org/10.1016/j.meegid.2014.12.003 http://www.sciencedirect.com/science/journal/15671348 http://www.elsevier.com/locate/meegid 28 T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 Noroviruses are classified into five genogroups, and genogroups GI, GII, and GIV are associated with human infection. Genogroup is further divided into genotypes and variants. Genotypes are defined based on either the VP1 amino acid (aa) sequence divergence (hereafter referred to as the capsid genotype) (Zheng et al., 2006) or the nucleotide sequence divergence of RdRp (hereafter referred to as the polymerase genotype) (Bull et al., 2007). Thus far, there are 9 and 22 capsid genotypes reported for GI and GII, respectively, and 14 and 26 polymerase genotypes reported for GI and GII, respectively (Kroneman et al., 2011, 2013). To avoid confusion and distinguish the polymerase genotypes from the capsid geno- types, the polymerase genotypes are proposed to be designed by a capital P, followed by the genotype designation (e.g., the capsid genotype GII.4 and the polymerase genotype GII.P4) (Kroneman et al., 2013). Among capsid genotypes, GII.4 is the most common genotype, accounting for about 70% of sporadic infections in children (Hoa Tran et al., 2013) and 55–86% of outbreaks (Siebenga et al., 2007; Lindesmith et al., 2008). Other major capsid genotypes include GII.3, GII.2, and GII.6 that are circulating globally (Hoa Tran et al., 2013). Minor genotypes, including genotypes GII.12 and GII.13 have shown much more restricted circulation both in frequency and geographical locations (Hoa Tran et al., 2013). Recently, how- ever, there have been an increasing number of reports describing the emergence and predominance of GII.12 and GII.13 (Chan-It et al., 2012; Iritani et al., 2012; Vega and Vinjé, 2011; Giammanco et al., 2012; Nataraju et al., 2011a; Medici et al., 2014a,b). Thus, continued monitoring of the circulation of norovi- rus genotypes is crucial for developing and designing efficacious vaccines as well as other preventive measures. However, changes in genotype distribution over a long time period are poorly 31.7% 27.2% 0 10 20 30 40 50 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 2005 2006 2007 2 N um be r of p os iti ve s pe ci m en s Monthly distribution of num Monthly detection rate of 0 2 4 6 8 10 12 14 16 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 2005 2006 2007 20 N um be r o f sp ec im en s GII.4 GII.3 A B Fig. 1. Molecular epidemiology of norovirus infection in Nepal between November 200 detection rate of noroviruses in each month during the study period. (B) The stacked are GII.13 in each month during the study period. described in developing countries where the burden of norovirus diarrhea is high. Nepal is a country belonging to the South-East Asia region where there is high child and adult mortality due to diarrhea (Parashar et al., 2009); however, few data are available about molecular epidemiology of noroviruses among diarrheal children in this land-locked country. We conducted a cross-sectional study of norovirus diarrhea in children aged Table 1 Distribution of capsid genotypes of noroviruses circulating in Nepal between November 2005 and January 2011. Years 2005 2006 2007 2008 2009 2010 2011 Total in inpatients Total in outpatients Grand total GII.4 2 31 20 24 31 2 3 67 46 113 GII.3 2 15 11 7 3 23 15 38 GII.13 2 2 21 12 15 22 37 GII.6 1 1 6 4 12 13 11 24 GI.3 1 1 7 3 6 6 12 GII.7 1 3 6 4 6 10 GII.10 1 5 4 2 6 GI.5 1 2 2 3 2 5 GI.7 2 2 1 4 1 5 GII.2 2 3 3 2 5 GII.14 2 1 1 3 1 4 GII.21 1 2 3 3 GII.22 1 1 1 1 2 3 GI.4 1 1 2 2 GII.16 1 1 2 2 GII.9 1 1 2 2 GI.6 + GII.16 1 1 1 GII.1 1 1 1 GII.17 1 1 1 GII.20 1 1 1 GII.8 1 1 1 GII.unknown 2 28 28 5 7 2 51 21 72 GI.unknown 1 5 1 2 5 7 GIV.1 1 1 1 Grand total 10 69 75 76 78 45 3 208 148 356 T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 29 USA). To identify norovirus genogroups, purified RNA preparations were subjected to real-time reverse-transcription PCR assays using a LightCycler480 II System (Roche Diagnostics GmbH, Mannheim, Germany) with the primers and probes designed based on the con- sensus sequence of the ORF1/ORF2 junction (Kageyama et al., 2003). For norovirus-positive samples, cDNA was synthesized from the extracted RNA by using random hexamers (SuperScript III First- Strand Synthesis System, Invitrogen Corporation, CA, USA). To determine the capsid genotypes, PCR (Go Taq Green Master Mix, Promega Corporation, CA, USA) was conducted to amplify the region C (Kojima et al., 2002) and the resulting PCR products were sequenced (BigDye Terminator Cycle Sequencing Ready Reaction Kit, version 3.1, Applied Biosystems, CA, USA) with an automated 3730 DNA sequencer (Applied Biosystems). For samples genotyped as GII.13, the entire capsid gene (ORF2) and a partial polymerase gene (about 800 nucleotides located at the 30 end of ORF1) were amplified, and were sequenced. The primers used were synthe- sized according to previously published papers (Chhabra et al., 2010; Kageyama et al., 2003; Kojima et al., 2002) as well as by our own design (Primer 3 v. 0.4.0). 2.3. Sequence and phylogenetic analysis Nucleotide sequences were assembled by using the Lasergene 8 software package (DNAstar, Inc. Madison, WI, USA). Nucleotide and amino acid sequence alignments were carried out by ClustalX. Phy- logenetic trees were constructed by the maximum likelihood method after selecting the optimal model to get the lowest Bayes- ian information criterion (BIC) or corrected Akaike information cri- terion (AICc), and the bootstrap probabilities were calculated after 1000 replicate trials. The genetic distances of amino acid sequences were calculated by the uncorrected method (Zheng et al., 2006). Evolutionary analyses were conducted using the MEGA v.6.0 pack- age (Tamura et al., 2013). The putative positions of recombination were identified by SimPlot v.3.5.1 (Lole et al., 1999). The reference strains with the capsid genotype GII.13 used in the study were Goulburn Valley G5175 B/1983/AUS (DQ379714), Fayetteville/ 1998/US (AY113106), Kashiwa47/JP (AB078334), Maizuru/ 000324/2000/JP (EF547405), Pune/PC25/2006/IND (EU921354), GII.13/VA173/2010/USA (JN899242), Oranienburg1190/2012/DE (KC832471), Berlin1162/2012/DE (KC832470), Luckenwalde1378/ 2012/DE (KC832473), and Berlin1195/2012/DE (KC832 472) of which the entire capsid sequences were available in GenBank. 2.4. Homology modeling Homology modeling, using crystal structures of the P domain of GII.4/VA387 (Protein Data Bank identification (PDB ID), 2obs) (Cao et al., 2007) and GII.4/THC005 (PDB ID, 3sej) (Shanker et al., 2011) as templates, was employed to predict the HBGA-binding sites 1 and 2 of Nepalese norovirus strains. Specifically, SWISS-MODEL workspace (http://swissmodel.expasy.org/workspace/) (Arnold et al., 2006; Schwede et al., 2003) was utilized. The visualization for the constructed models was conducted on the molecular mod- eling software NOC 3.01 (http://noch.sourceforge.net/). 2.5. Accession numbers The GenBank (DDBJ) accession numbers for the nucleotide sequences of Nepalese GII.13 strains are from AB809971 to AB810014. 3. Results 3.1. Detection of noroviruses Noroviruses were detected in 356 (8%) of 4437 samples col- lected between November 2005 and January 2011. During the same period rotaviruses were detected 1191 (27%) samples and there were 27 samples in which both rotavirus and norovirus were detected (data not shown). The detection rate of norovirus was not significantly different between inpatients and outpatients; norovi- ruses were detected in 208 (7.8%) of 2678 inpatients and in 148 (8.4%) of 1759 outpatients (p = 0.47). While norovirus infections were detected in all age groups up to five years of age, they occurred most frequently in children aged from 6 to 23-months, and the infections occurring in children aged http://swissmodel.expasy.org/workspace/ http://noch.sourceforge.net/ 11N4758 11N4706 10N4688 10N4551 10N4587 09N3822 09N3716 09N3729 09N3724 09N3708 09N3694 09N3689 09N3685 09N3679 09N3676 09N3637 09N3636 09N3633 09N3629 09N3599 09N3587 09N3578 09N3522 09N3462 09N3413 09N3354 09N3323 GU445325 N Orleans1805/2009/USA 09N3608 09N3712 09N3680 09N3757 09N3414 Nepalese GII.4 New_Orleans_2009 (2009-2011_N=32) JX459908 Sydney/NSW0514/2012/AU AB445395 Apeldoorn317/2007/NL 07N1729 EU078417 Minerva.OSD-CS/2006/USA 06N743 Nepalese GII.4 Den_Haag_2006b (2006-2007+N=2) 09N3445 07N1898 08N2347 08N2387 07N1781 07N1727 07N1655 07N1677 08N2227 07N1915 07N1759 07N1777 08N2777 08N2778 08N2132 EU921388 Pune/PC51/2007/India 07N1761 08N2825 08N2721 08N2798 08N2893 08N2796 09N2997 08N2868 08N2734 08N2736 08N2731 08N2576 08N2738 08N2739 08N2741 08N2742 08N2761 08N2790 08N2867 Nepalese GII.4 Osaka_2007 (2007-2009_N=34) AB220922 Sakai/04-179/2005/JP 06N420 05N27 06N380 Nepalese GII.4 Asia_2003 (2005-2006_N=3) AY502023 Farm Hills/2002/USA DQ364459 Lanzhou/35666/2002/China AJ583672 Ast6139/01/Sp 06N181 06N215 DQ078794 Hunter 284E/2004/AU AY883096 2004/NL Nepalese GII.4 Hunter_2004 (2006_N=2) EF126963 Yerseke38/2006/NL 06N385 06N656 06N710 06N700 06N638 06N660 06N496 06N740 06N419 06N654 06N706 06N721 06N730 06N726 06N709 Nepalese GII.4 Yerseke_2006a (2006_N=15) AF427117 NLV/Erfurt/007/00/DE AJ004864 Grimsby/1995/UK AB294779 Kaiso/030556/2003/JP FJ537135 CHDC2094/1974/US AY032605 MD145-12/1987/US X86557 Lordsdale/93/UK 88 80 99 99 81 81 97 98 99 86 85 95 99 85 99 99 0.05 Fig. 2. A phylogenetic tree for the P2 region of 88 Nepalese norovirus strains possessing the capsid genotype GII.4. This maximum likelihood tree was constructed by using the MEGA 6 software package. The genetic distances were computed according to the Kimura 2-parameter model while a discrete Gamma distribution was used to model evolutionary rate differences among sites. Bootstrap values were obtained after 1000 replicate trials. The bootstrap values lower than 70% were omitted. 30 T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 less than 2 years accounted for 84% of the total norovirus gastroen- teritis cases. Although norovirus diarrhea occurred year-round, the detection of noroviruses peaked in October and November of each year (Fig. 1A). 3.2. The capsid genotypes of noroviruses in Nepal Of 356 norovirus-positive specimens, 277 (78%) were success- fully genotyped into 22 capsid genotypes (Table 1). Of those, 16 capsid genotypes belonged to genogroup II, four genotypes geno- group I, and one genotype genogroup IV (Table 1). The most prevalent genotype was GII.4 (n = 113; 32%) that was detected almost year-round (Fig. 1B), followed by globally circulating geno- types GII.3 (n = 38; 11%), GII.6 (n = 24; 7%), and a minor genotype GII.13 (n = 37; 11%) (Table 1). There was no apparent difference in the genotypes detected between inpatients and outpatients (Table 1). 3.3. Periodic shift of GII.4 variants in Nepal On the basis of genetic analysis of the P2 region, 88 of 113 GII.4 isolates were successfully identified into 6 GII.4 variants; of those 10N4439 10N4441 10N4358 10N4254 10N4555 10N4487 10N4488 10N4489 09N3796 10N3852 09N3816 10N3922 09N3688 09N3721 09N3546 09N3564 09N3609 09N3617 09N3751 10N3917 09N3683 09N3798 09N3779 Berlin1195/2012/DE(KC832472) Luckenwalde1378/2012/DE(KC832473) Oranienburg1190/2012/DE(KC832471) Berlin1162/2012/DE(KC832470) 09N3645 10N4598 09N3698 09N3758 Sublineage NPL2009.2 09N3120 09N3223 09N3145 09N3289 09N3180 09N3206 Sublineage NPL2009.1 VA173/2010/USA (JN899242) Lineage NPL2009 Goulburn Valley G5175 B/1983/AUS (DQ379714) Pune/PC25/2006/India (EU921354) Kashiwa47 (AB078334) Fayetteville/1998/US (AY113106) Maizuru/000324/2000/JP/2468 (EF547405) Hy-718/KOR (KC662537) 08N2045 08N2250 Lineage NPL2008 99 77 99 97 100 94 100 77 100 77 76 99 77 86 100 0.02 Fig. 3. A phylogenetic tree for the VP1 gene of 35 Nepalese norovirus strains with the capsid genotype GII.13 drawn in reference to all the GII.13 strains for which the entire capsid sequences were available in GenBank. Although the VP1 gene of prototype strain GII.4/Lordsdale/93/UK was used as an outgroup, it is not shown in the tree. This maximum likelihood tree was constructed by using the MEGA 6 software package. The genetic distances were computed according to the Kimura 2-parameter model, bootstrap values were obtained after 1000 replicate trials. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable. The bootstrap values lower than 70% were omitted. Of note was that the sequence of strain 09N3289 lacked 421 nucleotides at the 50 end of the ORF2. T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 31 the prevalent variants were GII.4 Yerseke_2006a (n = 15), GII.4 Osaka_2007 (n = 34), and GII.4 New_Orleans_2009 (n = 32) that were periodically predominant in 2006, 2007–2009, and 2009– 2011, respectively (Fig.2). 3.4. Abrupt increase in prevalence of new lineage of minor genotype GII.13 Interestingly, the minor genotype GII.13 accounted for only 1.7% (4/230) of norovirus positive samples between November 2005 and December 2008 (hereafter referred to as period 1 in this study) but increased substantially to 26.2% (33/126) of genotyped specimens between January 2009 and January 2011 (period 2), being dominant in 2009 and 2010 (Fig. 1B and Table 1). A phylogenetic tree for the capsid gene of 35 of the 37 GII.13 norovirus-positive specimens was drawn in reference to all the GII.13 strains for which the entire capsid sequences were available in GenBank. The 35 Nepalese norovirus VP1 sequences were sepa- rated into two lineages: NPL2008 and NPL2009 to which two per- iod 1 strains and 33 period 2 strains belonged, respectively; the clustering of two period 1 strains and 33 period 2 strains into their respective lineages was with 100% bootstrap supports (Fig. 3). The strains in the lineage NLP2009 were further grouped into two sub- lineages, six Nepalese samples collected between March and April 2009 (sublineage NPL2009.1), and the remaining 27 samples col- lected between July 2009 and December 2010 (sublineage NPL2009.2) (Fig. 3). Four Germany GII.13 strains, Oranienburg1190/2012/DE, Ber- lin1162/2012/DE, Luckenwalde1378/2012/DE, and Berlin1195/ 2012/DE clustered together with 27 Nepalese period 2 strains in sublineage NPL2009.2 with a 100% bootstrap support (Fig. 3). While VA173/2010/USA clustered together with period 2 strains with a 100% bootstrap support, it did not belong to either sub-line- age NPL2009.1 or NPL2009.2 (Fig. 3). The aa divergence of the VP1 sequence of Nepalese strains within lineage NPL2008 was 0.2%; that within lineage NPL2009 ranged from 0% to 1.5%, and the aa divergence between 33 Nepa- lese period 2 strains and four German strains within lineage NPL2009 ranged from 0% to 1.2%; however, the divergence between NPL2008 and NPL2009 ranged from 4.6% to 5.4%, which was significantly larger than that within each lineage. 3.5. Amino acid variations at the putative HBGA-binding sites of Nepalese GII.13 The copying method using the crystal structures of GII.4 VA387 (2obs) and GII.4 TCH005 (3sej) as templates indicated that the putative HBGA-binding residues in the P monomer of GII.13 con- sisted of stretches of aa from 348 to 352 (loop 1/site 1), from 377 to 378 (loop 2/site 1), from 441 to 444 (loop 3/site 1), and from 394 to 399 (site 2) (Fig. 4). Interestingly, there were two hitherto undescribed substitutions E377G and H378D in loop 2/site 1 of two Nepalese NLP2008 strains, and another two substitutions H378N and V394Q that provided two new amino acids capable of forming hydrogen bonds, in the putative HBGA-binding sites NLP2009_loop 2/site 1: aa 377 to 378 NLP2009_loop 1/site 1: aa 348 to 352 NLP2009_loop 3/site 1: aa 441 to 444 NLP2009_site 2: aa 394 to 399 VA387_loop 2/site 1: aa 373 to 374 VA387_loop 1/site 1: aa 343 to 347 VA387_loop 3/site 1: aa 440 to 443 VA387_site 2: aa 390 to 395 (A) (B) Fig. 4. Prediction of the residues involved in the formation of the HBGA-binding sites of GII.13 by the copying method based on the known template of GII.4/VA378 (pdb: 2obs). The prediction of residues in the HBGA-binding sites 1 and 2 based on template of GII.4/VA387 was consistent with the predictions based on the other known templates of GII.4/TCH005 (data not shown). (A) Superposition of P monomers of GII.4/VA387 (red backbone) and GII.13 (strain 09N3688, lineage NPL2009) (green backbone) and the location of known regions involved in the formation of the HBGA-binding sites of GII.4 (black backbone). (B) Superposition of P monomers of GII.13 (strain 09N3688, lineage NPL2009) (green backbone) and GII.4/VA387 (white backbone) and the location of predicted regions involved in the formation of the HBGA-binding sites of GII.13 (black backbone). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 32 T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 in loop2/site 1 and site 2, respectively, of Nepalese NPL2009 strains (Fig. 5). Similar substitutions H378N and V394Q were also found in the putative HBGA-binding sites of four Germany GII.13 strains with sublineage NPL2009 (Fig. 5). 3.6. The polymerase genotypes observed in combination with GII.13 When partial RdRp sequences of Nepalese GII.13 strains were examined, they were shown to combine with various polymerase genotypes, indicating that some of them were ORF1/ORF2 recom- binants. More specifically, the two period 1 strains possessed GII.Pm/GII.13; two and four period 2 strains with sub-lineage NPL2009.1 possessed GII.P21/GII.13 and GII.P13/GII.13, respec- tively; the remaining 27 period 2 strains with sub-lineage NPL2009.2 possessed GII.P16/GII.13. Similarity plot analysis for the continuous sequences of the 30 end of the RdRp and the ORF2 of those strains predicted that the recombination sites occurred at the 30 end of the ORF1 and in the vicinity of the first nucleotide of the ORF2 (Fig. 6). A phylogenetic tree for the 30 end of the RdRp region (about 700 nucleotides) of the 35 Nepalese strains with the capsid genotype GII.13 was constructed in reference to all GII.13 recombinant strains for which the sequence information was available in Gen- Bank (Fig. 7). In this phylogenetic tree, two Nepalese strains belonging to sub-lineage NPL2009.1, i.e., 09N3180 and 09N3206 (Fig. 3) possessed the genotype combination of GII.P21/GII.13 and clustered together with three strains isolated in Korea in 2010 (Fig. 7). On the other hand, the remaining four Nepalese strains belonging to sub-lineage NPL2009.1, i.e., 09N3289, 09N3145, 09N3223 and 09N3120, possessed the genotype combination of GII.P13/GII.13 and clustered together with strains isolated in India between 2006 and 2009 (Fig. 7). The rest of Nepalese strains belonging to sublineage NPL2009.2, 09N3758 and other 26 strains possessed the genotype combination of GII.P16/GII.13, and clus- tered together with Indian, Spanish, Italian, and German GII.P16/ GII.13 strains with a 100% bootstrap support. It should be noted that three Kolkata GII.13 recombinants originally reported as GII.1/GII.13, GII.3/GII.13, and GII.5/GII.13 (Nataraju et al., 2011b) are re-classified as GII.Pm/GII.13, GII.P13/GII.13, and GII.P16/ GII.13, respectively, and Korean GII.13 recombinant, GII.b/GII.13 (Han et al., 2010) as GII.P21/GII.13 in accordance with the current nomenclature system. 4. Discussion This study is the first long-term investigation on the molecular epidemiology of norovirus infections among diarrheal children less than 5 years of age in Nepal, and provided baseline information about the detection rate, seasonality of noroviruses, prevalent genotypes, and their change over time. Although Nepal is a small and land-locked country, a consider- able diversity of genotypes was found during our study period. Thus, there were five, 16, and one genotypes among nine GI, 22 GII, and one GIV genotypes, respectively, which were previously reported to be associated with human infection (Kroneman et al., 22 90 21 3 23 7 25 6 28 3 28 5 28 6 28 7 29 1 29 4 29 5 29 7 30 2 30 6 30 9 32 7 32 8 32 9 33 1 34 0 34 1 34 2 34 3 34 4 34 5 34 8 34 9 35 0 35 1 35 2 35 4 36 7 37 7 37 8 37 9 38 0 38 1 39 2 39 4 39 5 39 6 39 7 39 8 39 9 40 1 40 9 41 1 41 2 41 3 41 4 41 5 41 9 42 0 42 2 44 1 44 2 44 3 44 4 44 5 44 9 47 0 47 1 47 2 47 3 47 4 47 6 48 3 Prototype Goulburn Valley G5175 B/1983/AUS G A I L N I S Y R V N G N L T N D F S M N T N V S A A K N A K I I E H V Y P V V N E N T P Q A S L A L N A P V V Q G Q Q V S - - - - A Y Fayetteville/1998/US . . . . T V . . . . . . . . . . . . . . . V . . . T . . . . . . V . . . H . . . . . . . . . . . . . . . . . . . . . L . A . - - - - . . Kashiwa47 . . . . T V F L Q . . . . . . . . . . . . V . . . T . . . . . . . . . . H . . . D . . . . . . . . . . . . . . . . . L . A . - - - - . . Maizuru/000324/2000/JP/2468 . . . . T V . . . . . . . . . . . . . . . V . . . T . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . L . A A P A P S . . Pune/PC25/2006/India . . . . T V . . . . S . . . . . . . . . . V . . . T . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . L . A . - - - - . . Hy-718/KOR . . . . T V . . . . . . . . . . . . . . . V . . . T . . . . . . . . . . H . . . . . . . . . . . T R S H G T L . . . L . A . - - - - . . 08N2045 A S F M T . . . . L . - S . S . . . . I . V T E G T . . . . . . . G D . H H I . . . . . . K S . . . . . . . . . . . L H A . - - - - T F 08N2250 A S F M T . . . . L . - S . S . . . . I . V T E G T . . . . . . . G D . H H I . . . . . . K S . . . . . . . . . . . L H A . - - - - T F 09N3120 A . . . . V . . . . S . . . S S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3145 A . . . . V . . . . S . . . . S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3180 A . . . . V . . . . S . . . . S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3206 A . . . . V . . . . S . . . . S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3223 A . . . . V . . . . S . . . . S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3289 A . . . T V . . . . S . . . . S . . . I . V S E R T . . . . . . . . N . H . . Q . . . . . R . A . . . . . . . . . . L . A . - - - - . . 09N3546 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3564 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3609 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3617 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3645 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3683 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3688 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3698 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3721 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3751 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3758 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . 09N3779 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3796 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3798 A . . . . V . . . . S . . . . S . . . I . E R E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 09N3816 A . . . . V . . . . S . . . . S . . . I . V S E A T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N3852 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N3917 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N3922 A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4254 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4358 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4439 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4441 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4487 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4488 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4489 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4555 A . . . . V . . . . R . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . 10N4598 A . . . . V . . . . S . . . . S E L R I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . Oranienburg1190/2012/DE A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . Berlin1162/2012/DE A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . Luckenwalde1378/2012/DE A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . Berlin1195/2012/DE A . . . . V . . . . S . . . . S . . . I . V S E G T . . . . . V . . N . H . . Q . . . . . . . A . . . . . . . . . . L R A . - - - - . . VA173/2010/USA (JN899242) A . . . . V . . . . S . . V . S . . . I . V R E N T . . . . . V . . D . R . . Q . . . . . . . A . . . . . . . . . . L . A . - - - - . . Subdomain NPL2008 strains NPL2009 strains Outlier strains P1.1 P2 P1.2 Position of residues in VP1 Fig. 5. Amino acid variations in the P1 and P2 sub-domains of GII.13 strains over time. The scheme started with the sequence of Goulburn Valley G5175 B/1983/AUS as the prototype strain and ended with those of the most recent strains detected in USA and Germany that clustered into lineage NPL2009. Of note was the deletion of one amino acid at position 295 of strains in the NPL2008 lineage and the insertion of 4 continuous amino acids at positions from 470 to 473 in Maizuru/000324/2000/JP; hence, the length of VP1 was not consistent within GII.13 strains. T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 33 2011, 2013). The distribution of many norovirus genotypes associ- ated with human gastroenteritis in other countries was also previ- ously reported (Okame et al., 2006; Park et al., 2010; Kirby et al., 2011; Nataraju et al., 2011a). The presence of a variety of norovirus genotypes in Nepal and the periodic shift of prevalent GII.4 vari- ants, i.e., GII.4 Yerseke_2006a, GII.4 Osaka_2007, and GII.4 New_- Orleans_2009 (Fig. 2) that was in line with the pattern of the emergence of GII.4 variants in the rest of the world (Hoa Tran et al., 2013) confirmed that the distribution of norovirus strains has no geographical restriction. Nevertheless, a lower detection rate of GII.4 variant Den Haag_2006b in Nepal revealed in this study differed from observations made in the majority of the rest in the world (Hoa Tran et al., 2013). The most interesting observation capturing our attention in this study was the increase in prevalence of GII.13 since 2009 (period 2). Although the first GII.13 norovirus was detected in Australia in 1983, it was not until the end of the 1990s and in the 2000s that the circulation of GII.13 was globally recognized in outbreaks (Iritani et al., 2010; Okada et al., 2006; Kim et al., 2005; Bruggink and Marshall, 2009) as well as in sporadic gastroenteritis cases albeit at a low frequency (Chhabra et al., 2009; Han et al., 2010; Trang et al., 2012; Kaplan et al., 2011; Kirby et al., 2011). Here, to avoid confusion in the literature, a caveat may be necessary; in a few previously published studies (Kaplan et al., 2011; Okada et al., 2006; Kim et al., 2005), what is now classified as GII.13 strains including Kashiwa47/JP (Shirato et al., 2008) were described as GII.14. On the other hand, strain OIF (Operation Iraqi Freedom) 031998 (AY675554), which was described as GII.13 (Huang et al., 2005), is GII.21 according to the current nomencla- ture system (Kroneman et al., 2011, 2013). Interestingly, there were recent reports on the emergence and predominance of GII.P13/GII.13 and GII.P16/GII.13 in India (Nataraju et al., 2011a,b) and GII.P16/GII.13 in German and Italy (Mäde et al., 2013; Medici et al., 2014a,b). Thus, taken together, this study sug- gests that genotype GII.13 which was previously regarded as a minor genotype has a potential to become an epidemiologically important genotype. Variants of the GII.13 strains, however, have never been defined unlike GII.4 noroviruses in which variants are defined and well characterized in many molecular epidemiological studies (Bull et al., 2006; Kamel et al., 2009; Mans et al., 2010). Phylogenetic analysis of VP1 sequences showed that the 33 Nepalese period 2 strains belonged to a lineage completely different from the lineage containing two period 1 strains. Following the precedent of the definition of a new GII.4 variant (i.e., approximately 5% in the amino acid sequence of VP1 of new GII.4 variants was observed from those of previously circulating GII.4 variants (Bull et al., 2006), the lineages NPL2008 and NPL2009 of GII.13 noroviruses were determined to contain distinct variants. Thus, the period 1 strains (NPL2008) were not a precursor of the period 2 strains (NPL2009), and the predominance of GII.13 in Nepal since 2009 was due to the replacement of strains independent from each other rather than the acquisition of mutations by previously circulating strains. The clustering of the period 2 strains with European GII.13 strains (Figs. 3 and 7) and Indian GII.13 strains (Fig. 7) that were recently reported to be emergent (Nataraju et al., 2011a; Mäde et al., 2013; Medici et al., 2014a,b) indicates that very similar, if not identical, strains of the GII.13 variants (within lineage NPL2009) circulated not in a restricted geographic location but there was an ongoing spread of these new GII.13 variants in much wider regions of the world. Fig. 6. Similarity plots for four Nepalese recombinant strains 08N2045 (representing strains belonging to lineage NPL2008), 09N3180 (sublineage NPL2009.1), 09N3223 (sublineage NPL2009.1), and 09N3751 (sublineage NPL2009.2) which were determined to be GII.Pm/GII.13, GII.P21/GII.13, GII.P13/GII.13, and GII.P16/GII.13 recombinants, respectively. The vertical axis represents the nucleotide sequence similarity, and the horizontal axis represents the position of the query sequence which was in total about 2300 nucleotides in length. The sequence spanned from about 600–700 nucleotides before the first nucleotide of the ORF2 to the end of the capsid gene. Breakpoints were located about 2–28 nucleotides before the first nucleotide of the ORF2. In each graph, the red line represents the nucleotide sequence similarity between the query and the prototype GII.13 strain, Goulburn Valley G5175 B/1983/AUS. The green line represents the nucleotide similarity between the queries and the reference strains which were most genetically closely related with the queries in their polymerase gene sequence. The other reference strains include Hu/Pune/PC24/2006/India (EU921353) (GII.Pm/ GII.12) (A), Hu/Saga/5424/03/JP (AB242256) (GII.P21/GII.3) (B), Hu/Briancon870/2004/France (EF529741) (GII.P13/GII.17) (C), and Hu/NLV/GII/Neustrelitz260/2000/DE (AY772730) (GII.P16/GII.16) (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 34 T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 However, the abrupt increase in prevalence of new variants belonging to the lineage NPL2009 also raises a question on the molecular basis by which the minor genotype GII.13 to emerge in the human population, because the mechanism by which an increase of a minor genotype to become prevalent should be differ- ent from the mechanism by which GII.4 variants maintain the glo- bal predominance (Siebenga et al., 2007; Bull et al., 2006). In the latter case, the mechanism was hypothesized to be the continuous emergence of new antigenic variants that escape the neutralization by blocking antibodies. This is unlikely to explain the increase of a minor genotype. The observations on the mutations in the putative HBGA-binding sites and the RdRp sequences of GII.13 strains in this study may provide clues to postulate working hypothesis. The first hypothesis is related to the appearance of two new amino acids highly capable of forming hydrogen bonds in the puta- tive HBGA-binding sites of Nepalese and German strains belonging to lineage NPL2009 that might allow the mutants to gain a broader or stronger HBGA-binding specificity. Specifically, amino acid resi- due 378 in the P domain of GII.13 strains corresponded to residue 374, 374, 385, or 375 described for GII.4, GII.9, GII.10, or GII.12 strains, respectively, where there is aspartic acid which forms two stable hydrogen bonds between its side chains and the secre- tor fucose ring of HBGAs (Cao et al., 2007; Shanker et al., 2011; Hansman et al., 2011) or the Lewis fucose ring of the non-secretor HBGA, i.e., the Lewis X tetra-saccharide (Chen et al., 2011). Substi- tutions H378N (period 2 strains) may ensure the binding capacity to HBGAs since asparagine also has the capacity to form two hydrogen bonds between their side chains and the secretor or the Lewis fucose ring. Amino acid at residue 394 in HBGA-binding site 2 of GII.13 strains corresponded to Q390 of GII.4 strains that was involved in water-mediated hydrogen bonds between the P domain of GII.4 and the b-galactose ring of carbohydrates A and B of HBGAs (Cao et al., 2007; Shanker et al., 2011). Therefore, the V394Q mutation, an amino acid lacking the capacity for forming a hydrogen bond to the one capable of forming two hydrogen bonds, is assumed to enhance the binding capacity of the period 2 strains with the b-galactose ring of HBGA A and B types. The speculation is supported by the similar observations made for GII.4: there was a strong correlation between the richness in hydrogen-bond forming capacity of residues in HBGA-binding site 2 and the enhancement in the HBGA-binding affinity (Cao et al., 2007; Shanker et al., 2011). The second hypothesis is related to the acquisition of various polymerase genotypes of strains in the lineage NPL2009 that might provide new variant an opportunity for acquiring an RdRp that has a higher replication capacity and resulting in a better fitness for the mutants in the population (Bull et al., 2007). We observed that GII.P16/GII.13 and GII.P13/GII.13 (period 2 strains belonging to lineage NPL2009) were not only dominant in Nepal but also fre- quently detected, even causing large-scale outbreaks in other countries in the world. Thus, it is assumed that the emerging Nepa- lese GII.13 strains might possess RdRps with higher replication capacity that led to recent emergence of GII.13, similar to the mechanism driving the evolution of GII.4 variants since 2001 (Bull et al., 2010). In conclusion, this study showed an increase in prevalence of new variants of GII.13 noroviruses in Nepal. Taken together with recent reports describing the emergence of GII.13 strains in India and in European countries, this study predicts that genotype GII.13 which was previously regarded as a minor genotype has a N10N4489 10N4555 10N4487 10N4441 10N4439 10N4358 10N4254 10N4488 IDH1873/2009/IND 09N3816 ESP/2010/GII.P16 GII.13/SanSebastian356037 10N4598 Berlin1195/2012/DE PR6717/2010/ITA PR1395/2012/ITA Luckenwalde1378/2012/DE Berlin1162/2012/DE Oranienburg1190/2012/DE 09N3564 09N3688 09N3798 09N3609 10N3922 09N3796 10N3852 09N3645 09N3683 09N3617 09N3546 09N3698 09N3721 09N3751 09N3758 09N3779 10N3917 IDH2651/2009/IND IDH2770/2010/IND GII.P16/GII.13 Seoul/0950/2010/KOR Seoul/0954/2010/KOR Seoul1009/KOR/2010 09N3180 09N3206 GII.P21/GII.13 Hu/GII/Hy-718/KOR Hiroshima/32-754/2003/JP Pont de Roide 671/2004/France Hokkaido/203A/2004/JP Hokkaido/77/2001/JP Hokkaido/146A/2003/JP GII.P12/GII.13 08N2045 08N2250 IDH1501/2009/IND GII.Pm/GII.13 GII.Pg/GII.13 Goulburn Valley G5175 B/1983/AUS Pune/PC25/2006/India IDH883/2008/IND IDH1390/2009/IND V1668/06/IND 09N3223 09N3289 09N3120 09N3145 GII.P13/GII.13 92 100 91 100 98 100 100 100 100 77 97 73 76 85 87 100 0.05 Fig. 7. A phylogenetic tree for the 30 end of the RdRp (about 700 nucleotides) of 35 Nepalese norovirus strains with the capsid genotype GII.13 and all GII.13 recombination strains for which continuous, partial RdRp and N/S sequences were available in GenBank. The tree was constructed with the MEGA 6 software package, by using the maximum likelihood method, the genetic distances were computed according to the Kimura 2-parameter model, the rate variation model allowed for some sites to be evolutionarily invariable, bootstrap values were obtained after 1000 replicate trials. The bootstrap values lower than 70% were omitted. Of note was that by using the current nomenclature, three Kolkata GII.13 recombinants, GII.1/GII.13, GII.3/GII.13, and GII.5/GII.13 (Nataraju et al., 2011b) were re-classified as GII.Pm/GII.13, GII.P13/GII.13, and GII.P16/GII.13, respectively, the Korean GII.13 recombinant, GII.b/GII.13 (Han et al., 2010) was re-classified as GII.P21/GII.13. T.N. Hoa-Tran et al. / Infection, Genetics and Evolution 30 (2015) 27–36 35 potential to become an epidemiologically important genotype. Thus, experimental studies are warranted to elucidate the roles of the two substitutions in the HBGA-binding sites and the various polymerase genotypes of strains belonging to the lineage NPL2009. Acknowledgements Thi Nguyen Hoa-Tran was a Ph.D. student supported by a schol- arship from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Nigel A. Cunliffe and Miren Iturriza-Gomara involved in this study under the Agreement on Academic Partner- ship between the University of Liverpool and Nagasaki University. We thank Winifred Dove for her excellent technical support in strain characterization. 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http://refhub.elsevier.com/S1567-1348(14)00449-3/h0230 http://refhub.elsevier.com/S1567-1348(14)00449-3/h0235 http://refhub.elsevier.com/S1567-1348(14)00449-3/h0235 http://refhub.elsevier.com/S1567-1348(14)00449-3/h0240 http://refhub.elsevier.com/S1567-1348(14)00449-3/h0240 http://refhub.elsevier.com/S1567-1348(14)00449-3/h0240 Molecular epidemiology of noroviruses detected in Nepalese children with acute diarrhea between 2005 and 2011: Increase and predominance of minor genotype GII.13 1 Introduction 2 Materials and methods 2.1 Study setting 2.2 Detection and identification of norovirus genogroups, genotypes and genetic lineages/variants 2.3 Sequence and phylogenetic analysis 2.4 Homology modeling 2.5 Accession numbers 3 Results 3.1 Detection of noroviruses 3.2 The capsid genotypes of noroviruses in Nepal 3.3 Periodic shift of GII.4 variants in Nepal 3.4 Abrupt increase in prevalence of new lineage of minor genotype GII.13 3.5 Amino acid variations at the putative HBGA-binding sites of Nepalese GII.13 3.6 The polymerase genotypes observed in combination with GII.13 4 Discussion Acknowledgements References