Infection, Genetics and Evolution 23 (2014) 65–73
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Molecular epidemiology of norovirus associated with gastroenteritis and emergence of norovirus GII.4 variant 2012 in Japanese pediatric patients Aksara Thongprachum a,b, Wisoot Chan-it a, Pattara Khamrin c, Patchreenart Saparpakorn d, Shoko Okitsu a,b, Sayaka Takanashi a, Masashi Mizuguchi a, Satoshi Hayakawa b, Niwat Maneekarn c, Hiroshi Ushijima a,b,⇑ a
Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand d Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand b c
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 17 January 2014 Accepted 19 January 2014 Available online 4 February 2014 Keywords: Norovirus Epidemiology Gastroenteritis Japanese Variant
a b s t r a c t In late 2012, an outbreak of acute gastroenteritis due to norovirus variant Sydney_2012 occurred and have been reported from many counties. In this study, we described surveillance study of the incidence of norovirus infections among Japanese pediatric patients in association with gastroenteritis and investigated the antigenic change of the new variant Sydney_2012 circulated in Japanese populations. A total of 2381 fecal specimens collected from children with acute gastroenteritis in Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from 2009 to 2013 were examined for norovirus and further analyzed molecularly. A high proportion (39.3%) of norovirus positive samples and several genotypes were detected. Norovirus GII.4 dominated over other genotypes (71.4%). The Den_Haag_2006b (43.2%) was detected as the predominant variant and co-circulated with New_Orleans_2009 (17.8%) until March 2012. Subsequently, they were displaced by Sydney_2012. The Sydney_2012 variant has been responsible for the majority of norovirus infections in 2012–2013 (85.7%). Although Sydney_2012 variant has a common ancestor with New_Orleans_2009 variant, analysis of P2 sub-domain showed a high level of diversity in comparison with other variants in four amino acid changes at the antigenic sites. The change in particular residue 393 of new variant may affect HBGA recognition. Analysis of noroviruses circulating in the past 4 years revealed a change of predominant variant of norovirus GII.4 in each epidemic season. The change of amino acid in putative epitopes may have led the virus escape from the existing herd immunity and explain the increase of new variant outbreaks. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Norovirus is a major etiologic agent responsible for epidemic gastroenteritis worldwide. It is a single-stranded, positive sense RNA virus that belongs to the Caliciviridae family. The norovirus genome is 7.5 kb long and contains three open reading frames; ORF1 encodes the non-structural proteins, ORF2 encodes the VP1 major capsid protein, and ORF3 encodes the VP2 minor capsid protein (Prasad et al., 1999).
⇑ Corresponding author at: Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3509; fax: +81 3 5841 3629. E-mail address: [email protected] (H. Ushijima). http://dx.doi.org/10.1016/j.meegid.2014.01.030 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Based on genetic difference in the capsid gene, noroviruses are genetically highly diverse and divided into five genogroups (GI-V). They are further subdivided into several different genotypes, at least nine genotypes of GI, 21 genotypes of GII, 2 genotypes of GIII, and only one genotype of GIV and GV have been identified (Zheng et al., 2010; Kroneman et al., 2013). Genotype II.4 (GII.4) norovirus has been shown to spread rapidly and is the most commonly detected worldwide, particularly in association with outbreaks. Recent molecular surveillances indicate that GII.4 norovirus has evolved a series of genetic variants over the last 20 years, some of which persist and replace the previous circulating variants (Siebenga et al., 2010). Norovirus epidemic patterns in human populations include epidemic outbreaks of disease every 2–3 years, punctuated by the emergence of an antigenically distinct GII.4 strain that appears to escape human herd immunity to the previous circulating strains (Debbink et al., 2012; Lindesmith et al.,
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2013). The continuous evolution of the P2 sub-domain of the surface exposed major capsid protein (VP1) has been proposed as a key mechanism by which new antigenic variants are generated. Norovirus recognizes histo-blood group antigen (HBGAs), which play an important role in host susceptibility to norovirus infection (Tan and Jiang, 2011). HBGAs are a diverse family of carbohydrates expressed on red blood cells, mucosal epithelia, saliva, milk and other body fluids, which are highly polymorphic and are related to the ABO, secretor and Lewis families. They have been hypothesized to be receptors or coreceptors that allow norovirus to attach and enter permissive cells (Debbink et al., 2012). The HBGA binding interfaces are located at the top of the P dimer, corresponding to the outermost surface of the capsid. The carbohydrate binding pockets involve several scattered amino acid residues in the P domain that form extensive hydrogen bond network with individual saccharides, and thus stabilizing the binding of HBGAs to the capsid protein (Zhang et al., 2013). The increased detections of norovirus GII.4 variants have been observed globally. Since the mid-90s, norovirus GII.4 variants have been shown to be associated with at least six pandemics of acute gastroenteritis and continue to cause millions of infections across the globe annually. The norovirus GII.4 variants reported previously include the US 1995/96 in 1996 (Noel et al., 1999; White et al., 2002), Farmington_Hills_2002 in 2002 (Lopman et al., 2004; Widdowson et al., 2004), Hunter_2004 in 2004 (Bull et al., 2006), Minerva_2006b and Den_Haag_2006b virus in 2006–2008 (Eden et al., 2010), New_Orleans_2009 in 2009–2012 (Yen et al., 2011), and most recently Sydney_2012 (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). The periodic increase of norovirus outbreaks have been shown to be related to the emergence of new variant strains, probably owing to evasion of population immunity (Zheng et al., 2010). The emergence of such variants has been shown to be associated with substantial increases in cases worldwide (Pang et al., 2010). Since late 2012, surveillance systems in many countries showed the increase of norovirus activity levels compared to the previous seasons, and associated with the emergence of a new variant GII.4 named Sydney_2012 (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). Therefore, rapid antigenic variations are important evolutionary forces that shape epidemiological success and persistence of the GII.4 viruses in the population. The objectives of this study were: (1) to describe a surveillance of norovirus infections that are associated with gastroenteritis in Japanese pediatric patients; and (2) to investigate P2 sub-domain of the new variant Sydney_2012 which may affect antigenic property.
2. Materials and methods 2.1. Patients and samples A total of 2381 fecal specimens were collected from non-hospitalized children with acute gastroenteritis in pediatric clinics in six prefectures from northern to southern Japan including Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from July 2009 to June 2013. Among these, 515, 536, 592, and 738 samples were obtained during the period of 2009–2010, 2010–2011, 2011–2012, and 2012–2013, respectively. In this study, the annual observation period for norovirus gastroenteritis began in July and ended in June of the following year. The spectrum of clinical signs exhibited by patients with acute gastroenteritis included acute watery diarrhea, vomiting, abdominal cramps, and fever of various severity and in various combinations. The age of the patients ranged from neonate to 15 years (0–178 months; mean age 24.4 months). For subsequent data analysis, seven different age-groups were established:
60 months (156 until use.
samples), 6–11 months (673 samples), 12– samples), 24–35 months (334 samples), 36– samples), 48–59 months (63 samples), and samples). The specimens were stored at 20 °C
2.2. Viral RNA extraction The fecal specimens were prepared as 10% fecal suspensions in distilled water. Viral genome was extracted from the supernatant of 10% suspension using the QIAamp Viral RNA MiniKit, according to the manufacturer’s instructions (QIAgen, Hilden, Germany). The extracted viral RNAs were stored at 80 °C until use. 2.3. Detection of norovirus The reverse transcription (RT) was performed with RNA template using SuperScriptIII reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The presence of norovirus was detected by RT-PCR using the protocol described previously (Yan et al., 2003). The specimens were tested for the presence of norovirus GI and GII by amplification of partial gene. For norovirus GI, a combination of forward primer G1-SKF (nt 5342–5361) 50 -CTGCCCGAATTYGTAAATGA-30 and reverse primer G1SKR (nt 5653–5671) 50 -CC AACCCARCCATTRTACA-30 were used. For norovirus GII, a forward primer COG2F (nt 5003–5028; 50 -CARGARBCNATGTTYAGRTGGATGAG-30 ) was used in combination with the reverse primer G2SKR (nt 5367–5389; 50 -CCRCCNGCATRHCCRTTRTACAT-30 ). Additionally for norovirus GII, semi-nested PCR was performed using G2SKF (nt 5389–5367; 50 -CNTGGGAGGGCGATCGCAA-30 ) and G2SKR primers for the samples that were negative by the first set of primer for norovirus GII. 2.4. Amplification of polymerase and capsid genes of norovirus The partial polymerase and full-length capsid region of a new variant was amplified using two pairs of primers, GV21 (50 -GTB GGNGGYCARATGGGNATG-30 )/G2SKR (50 -CCRCCNGCATRHCCRT TRTACAT-30 ) and COG2F (50 -CARGARBCNATGTTYAGRTGGATGAG30 )/TX30SXN (50 -GACTAGTTCTAGATCGCGAGCGGCCGCCC(T)30-30 ). Long-range PCR was amplified with High Fidelity DNA polymerase KOD-Plus (Toyobo, Osaka, Japan). The PCR was carried out with 2 ll of cDNA using TX30SXN primer, in the reagent mixture containing 2.5 ll of 10 buffer for KOD-Plus, 1 ll of 2 mM dNTPs, 1 ll of 25 mM MgSO4, 0.75 ll of each specific primer (20 lM) and 0.5 ll of KOD Plus enzyme. MilliQ water was added to make up a total volume of 25 ll. The PCR was performed at 94 °C for 2 min, followed by 35 cycles of 98 °C 20 s, 50 °C or 55 °C 30 s, 68 °C 2 min, and a final extension at 68 °C for 10 min, and then held at 4 °C. 2.5. Sequence and Phylogenetic Analyses The genotypes of the norovirus were characterized by direct nucleotide sequencing of partial capsid gene and then performing phylogenetic analysis as described previously by Kageyama et al. (2004). The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced by using the BigDye Terminator Cycle Sequencing kit (Perkin Elmer-Applied Biosystems, Inc., Foster City, CA) on an automated DNA sequencer (ABI 3100; Perkin Elmer-Applied Biosystems, Inc., Foster City, CA). The nucleotide sequences were analysed in comparison with those of norovirus strains deposited in GenBank database. Phylogenetic relationship of norovirus was examined by aligning sequences with the ClustalX program. A phylogenetic tree was constructed
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according to the maximum likelihood (ML) method using MEGA version 5 (Tamura et al., 2011).
930), GII.7 (1.4%; 13/930), GII.6 (1.3%; 12/930), GII.13 (1.1%; 10/ 930), GII.12 (0.8%; 7/930), and GII.17 (0.1%; 1/930) (Table 1). The norovirus GI was detected at 0.3% (8/2381), comprising GI.1 (0.04%), GI.4 (0.04%), and GI.6 (0.25%). Out of total samples, 28.1% of the patients were infected by norovirus GII alone, while 11.0% were co-circulated with other enteric viruses. Double-infection and triple-infection were found at 10.5% (250/2381) and 0.5% (11/2381), respectively. Mix-infection between norovirus GII and group A rotavirus was the most predominant (4.5%; 106/2381), followed by human parechovirus (2.3%), adenovirus (1.9%), enterovirus (1.4%), sapovirus (0.3%), astrovirus (0.1%), and norovirus GI (0.1%).
2.6. Nucleotide sequence accession numbers The partial nucleotide sequences of the capsid gene of norovirus identified in this study were deposited in GenBank under accession numbers KF658275–KF658339. Accession number for partial polymerase and full-length capsid sequences of norovirus GII.4 Sydney_2012 variant used in this study is KF145149. 2.7. Three-dimensional structure
3.2. Identification of epidemic norovirus GII.4 variants
The three-dimensional structures of norovirus genomes of variants were constructed by homology modeling technique using Geno3D web server (Combet et al., 2002), the structures were checked the quality of geometry by PROCHECK (Laskowski et al., 1993). For the model structures, all sequences were based on the structure of a norovirus GII.4 2004 norovirus using PDB code 3sej (Shanker et al., 2011).
Phylogenetic tree of nucleotide sequences of the partial capsid region of norovirus GII isolates detected in the present study was constructed in comparison with those of the reference strains available in GenBank database (Fig. 1). The norovirus GII.4 consisted of three major variants strains detected in pediatric patients in this study (Fig. 2). The Den_Haag_2006b strain that appeared during the study period comprised 287 of 664 norovirus GII.4 strains (43.2%). The sequences of these norovirus strains exhibited 98.0–100% nucleotide identity and 98.7–100% amino acid identity with Hu/GII.4/DenHaag89/2006/NL (GenBank accession number EF126965). The second variant, New_Orleans_2009 strain, was detected in 17.8% (118/664) and displayed 96.0–100% nucleotide sequence identity and 97.5–100% amino acid identity with the capsid region of Hu/GII.4/New Orleans1805/2009/USA (GU445325). Interestingly, the new Sydney_2012 variant was detected at 36.4% (242/ 664) and clustered within the same cluster of the Sydney/ NSW0514/2012/AU (JX459908) and Hong Kong/CUHK3655/2012/ CHN (JX629456) reference strains and showed 98.7–100% nucleotide sequence identity and 100% amino acid sequence identity with reference strains. However, a number of additional GII.4 variants have also been identified, including Hunter 2004, Yerseke_2006a, Apeldoorn_2007, and Hokkaido1_2008. These variants were less common in this study.
3. Results 3.1. Detection and genotypic distribution of norovirus A high proportion (39.3%; 936/2381) of norovirus were detected in the pediatric patients during 2009–2013, comprising 0.3% of GI (6/2381), 39.0% of GII (928/2381), and 0.1% mixed GI/GII (2/2381). Most of norovirus belonged to norovirus GII genogroup (39.1%; 930/2381). As shown in Table 1, the detection rate of norovirus GII were 40.8% (210/515) in 2009–2010, 40.1% (215/536) in 2010–2011, 31.8% (188/592) in 2011–2012, and 43.0% (317/738) in 2012–2013. Based on the analysis of partial sequence of capsid gene of norovirus GII, nine distinct genotypes were identified. Of these, GII.4 was the most predominant genotype (71.4%; 664/930), followed by GII.3 (13.1%; 122/930), GII.2 (6.5%; 60/930), GII.14 (4.4%; 41/
Table 1 Genotype distributions of noroviruses detected in Japanese pediatric patients with diarrhea from 2004 to 2013. No. of genogroup positive (%)
No. of genotype cases
Reference
Year
No. of samples tested
No. of norovirus positive (%)
GI
GII
GII.1
GII.2
GII.3
GII.4
GII.6
GII.7
GII.12
GII.13
GII.14
GII.17
2004–2005
752
139 (18.5)
2 (0.3)
137 (18.2)
1
0
22
108
6
0
0
0
0
0
2005–2006
603
51 (8.5)
0 (0)
51 (8.5)
1
1
27
19
2
1
0
0
0
0
2006–2007
628
97 (15.4)
2
6
84
0
3
0
0
0
0
477
125 (26.2)
95 (15.1) 118 (24.7)
0
2007–2008
2 (0.3) 7 (1.5)
0
0
6
92
6
0
0
0
14
0
2008–2009
477
129 (27.0)
0 (0)
129 (27.0)
1
4
7
92
18
1
3
0
3
0
2009–2010
515
210 (40.8)
0 (0)
0
24
30
135
1
6
5
1
7
1
2010–2011
536
215 (40.1)
0
17
60
118
2
1
1
2
14
0
2011–2012
592
188 (31.8)
1 (0.2) 0 (0)
0
7
30
132
3
0
0
5
11
0
2012–2013
738
323 (43.8)
210 (40.8) 215 (40.1) 188 (31.8) 317 (43.0)
0
12
2
279
6
6
1
2
9
0
7 (0.9)
Phan et al. (2006) Phan et al. (2007) Dey et al. (2012) Chan-it et al. (2011) Chan-it et al. (2011) This study This study This study This study
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Fig. 1. Phylogenetic analysis of the partial capsid sequence of norovirus GII.4 strains. The phylogenetic tree constructed using the maximum likelihood method. Representative strains detected in this study are presented in boldface.
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Fig. 2. Monthly distribution of norovirus GII.4 norovirus infection in Japan pediatric patients during the epidemic seasons of 2009/2010–2012/2013. (A) norovirus genotypes. (B) Norovirus GII.4 variants.
3.3. Seasonal pattern and age distribution of norovirus infection The seasonal pattern of norovirus infection is shown in Fig. 2. Norovirus was detected all year round with a highest peak in December. From this study, it was clearly observed that many noroviruses genotypes were common in winter season in Japan (Fig. 2A). The highest prevalence of norovirus was found in December (23.6%; 221/936), followed by January (19.6%; 183/936), November (12.3%; 115/936), and February (9.8%; 92/936), respectively. Focusing on norovirus GII.4 variants, most of norovirus GII.4 occurred between November and January, with a highest peak in December (25.9%). The Den_Haag_2006b variant was detected as the most predominant variant until March 2012 in co-circulation with New_Orleans_2009 variant. Subsequently, they were replaced by variant Sydney_2012 which was described in June 2012 (Fig. 2B). However, the Sydney_2012 was first detected in a Japanese patient in Tokyo since November 2011 before spreading throughout Japan from mid-2012. Of the patients with acute gastroenteritis, norovirus infection was detected in all age groups. The mean age of norovirus infected patients was 22.4 months. The highest prevalence of norovirus was found in infants and children aged 12–23 months (42.4%, n = 397),
followed by 6–11 months (25.6%, n = 240), 24–36 months (14.9%, n = 139), less than 6 months (6.3%, n = 59), 36–47 months (4.5%, n = 42), more than 60 months (3.5%, n = 33), and the lowest in children aged 48–59 months (1.1%, n = 26). 3.4. Analysis of polymerase and capsid of norovirus GII.4 Sydney_2012 To determine antigenic differences between GII.4 Sydney_2012 and the recent circulating ancestral strains Den_Haag_2006b and New_Orleans_2009 strains. We analysed VP1 and P2 sub-domain especially in blockade epitopes of Sydney_2012 and compared with other variant strains. The analysis of partial VP1 capsid of GII.4 variant 2012 representative strains revealed a high diversity of 6 positions of amino acid substitutions compared to Den_Haag_2006b and New_Orleans_2009 variants; positions 294(A2006b ? P2009 ? T2012), 359(T2006b ? S2009 ? A2012), 368(S2006b ? A2009 ? E2012), 377(T2006b ? T2009 ? A2012), 393(S2006b ? S2009 ? G2012), and 413(V2006b ? I2009 ? T2012), as shown in Fig. 2A and B. In the analysis of P2 sub-domain, the Japanese norovirus GII.4 variant 2012 strains showed P98.9% nucleotide and P99.2% amino acid sequence identities with the Sydney/NSW0514/2012 (JX459908). Moreover, analysis of polymerase region of the Sydney_2012 variant was found to be similar to Apeldoorm_2007 but distinct from
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other variants and accounted for three amino acid substitutions in polymerase region.
3.5. Effect of HBGA binding site In order to investigate the HBGA binding site, the superimposition between the crystal structure of Hunter_2004 strain (pdb code 3sej) and other variants were studied as shown in Fig. 4A. The structure of Hunter_2004 strain revealed the hydrogen bond interaction between Lewisb hexasaccharide and some amino acid residues in both dimers (i.e. Chain A: Gln390, Asp391, Gly392, Ser393, Thr394, Thr395, Ser442, and Gly443, Chain C: Ser343, Thr344, Arg345, and Asp374), as shown in Fig. 4B. For the strains of Sydney_2012 (JP41), the amino acid residue position 393 was glycine which was different from other variant strains. From the crystal structure, the hydroxyl group of Ser393 formed the hydrogen bond interaction to Lewis fucose (LeFuc) while Gly393 in Sydney_2012 (JP41) strain could not form the hydrogen bond interaction to the LeFuc (Fig. 4A).
4. Discussion Viral gastroenteritis is still a health burden and one of the most frequently encountered problems in developed and developing countries. Global outbreaks of acute gastroenteritis by norovirus have frequently reported. Our group also previously reported the epidemic occurrence of norovirus in a Japanese population (Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). The current study extended these findings by investigating different epidemic patterns. A high prevalence (39.3%) of norovirus infection was found in these Japanese pediatric patients with acute gastroenteritis in Japan during July 2009 to June 2013; 99.4% of all norovirus cases were infected by norovirus GII. In contrast, the prevalences of rotavirus, human parechovirus, enterovirus, adenovirus, sapovirus, astrovirus, and Aichi virus were 20.1%, 6.6%, 6.1%, 5.6%, 4.8%, 2.3%, and 0.1%, respectively (unpublished data). The prevalence of norovirus is higher than that found in the preceding studies conducted in the same geographical area during 2004–2009 which showed the prevalence rates of norovirus ranged from 8.5% to 27.0% (Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). Taken together, results in the present and preceding studies confirmed that the detection rate increased year by year and norovirus is the most predominant agent causing acute gastroenteritis in Japan (Kageyama et al., 2004; Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). This study documents 11.0% mixed infection between norovirus and other enteric viruses, in which 4.5% mixed infection between norovirus and group A rotavirus was most predominant. Differences in rotavirus and norovirus seasonality may relate to co-infection rate in this study. Moreover, mixed infection was not detected more frequently in any particular age group. Regarding norovirus seasonality, the seasonal distribution of norovirus infection exhibits a peak in winter seasons, a highest peak of incidence in December (23.6%). It is clearly observed that norovirus infection, especially norovirus GII.4 is commonly detected in cold months in Japan; 65.3% of confirmed norovirus infection cases occurred between December and February. Our data are in good agreement with the other surveillance on pediatric cases which demonstrated the main peak of norovirus during the winter months in Japan (Iritani et al., 2010; Dey et al., 2010). The temperature and humidity had highly consistent effects on driving norovirus epidemic patterns (Lopman et al., 2009). A high number of norovirus cases detected in every winter season in this study might be associated with cold and dry temperature.
Several studies reported that norovirus infection occurred frequently in young children under 2 years-old (Sai et al., 2013; Yoneda et al., 2014). In agreement with this, our results showed a marked trend toward higher rates of infection in children under-2-years of age (74.4%, 696/936), particularly children between 12 and 23 months are the most frequently affected group. The findings show that norovirus infections usually occur in early childhood. It is possible that the children younger than 2 years old might lack of maternal antibody protection. Based on antigenic analyses and more recent extensive sequence analyses, the circulation of norovirus in nature have been shown to be highly variable. In this study, genetic analysis among norovirus GII demonstrated that GII.4 genotype is the most predominant genotype accounting for 71.4%, followed by GII.3, GII.2, GII.14, GII.7, GII.6, GII.13, GII.12, and GII.17. The highest prevalence of GII.4 is consistent with those obtained from earlier studies conducted in the same settings as well as other studies conducted elsewhere in Japan (Phan et al., 2006; Dey et al., 2011; Chan-it et al., 2011; Hoa Tran et al., 2013; Yoneda et al., 2014). Norovirus GII.4 strains evolved rapidly and spread globally with the emerging variants. On the other hand, the increase of norovirus GII.3 has been identified and this strain is the second leading genotype in Japan after dropping during 2006–2009. A sharp decrease in the number of patients infected by GII.3 was observed when the new emerging variant was spread in 2012–2013 (14.3% in 2009–2010; 27.9% in 2010–2011; 16.0% in 2012–2013; 0.6% in 2012–2013). However, norovirus GI was low detectable in this surveillance study. Focusing on norovirus GII.4 variants, analysis of noroviruses circulating in the past 4 years in Japanese pediatric patients revealed a change of the predominant variant of norovirus GII.4 in each epidemic season over time. Among norovirus GII.4 identified in this study, three separate norovirus GII.4 variants were mainly identified as the etiologic agents of norovirus-associated acute gastroenteritis, including Den_Haag_2006b (43.2%), New_Orleans_2009 (17.8%), and Sydney_2012 (36.4%) variants. The Sydney_2012 new variant was shown to emerge since late 2011 and this emergence has been shown to be associated with the increase of norovirus infections in Japanese pediatric patients from May 2012. Interestingly, the previous dominant Den_Haag_2006b and New_Orleans_2009 variants were displaced by new emergence variant Sydney_2012 (Fig. 2B). The Sydney_2012 variant has been shown to be responsible for the majority of norovirus infection in 2012–2013, accounting for 85.7% of all norovirus GII.4 detected in 2012–2013. Even though many studies reported the detection of Sydney_2012 variant from late 2012 (Bennett et al., 2013; Fonager et al., 2013; van Beek et al., 2013), the present study described the detection of Sydney_2012 variant among pediatric patients in Tokyo since November 2011 before spreading throughout Japan from mid-2012. This study clearly indicated that norovirus variant Sydney_2012 was the predominant group causing acute gastroenteritis among Japanese pediatric populations since mid-2012. Interestingly, the Sydney_2012 strains detected in the current study were clustered in the same lineage and showed a high nucleotide sequence (P98.7%) and amino acid sequence (100%) similar to norovirus GII found previously Australia, the United States, Europe, Canada, and China (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). Then, the Sydney_2012 had emerged as the etiologic agent in acute gastroenteritis across the United States, Europe and Asia, including Japan in the same period. Although the Sydney_2012 variant has common ancestor with the dominant New_Orleans_2009 variants, analysis of partial VP1 capsid of GII.4 variant 2012 representative strains revealed a high diversity with 6 positions of amino acid substitutions, positions 294, 359, 368, 377, 393, and 413, compared to Den_Haag_2006b
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A
Strain CHDC5191 Camberwell Dresden Farmington_Hills Guangzhou Hunter Yerseke Den_Haag Apeldoorm Hokkaido1 New_Orleans Sydney JP41
Accession NO. FJ537134 AF145896 AY741811 AY502023 DQ369797 DQ078814 EF126963 EF126965 AB445395 AB541261 GU445325 JX459908 KF145149
Variant 1974 1994 1997 2002 2003 2004 2006a 2006b 2007 2008 2009 2012 2012
294 G V A A P A A A T A P T T
296 S S S T T T T S S S S S S
Epitope A 297 298 368 H D T H D T H D T H N N R I A Q N S Q E S R N S R N A R N S R N A R N E R N E
372 N N N N D S S E D E D D D
Epitope D 393 394 395 D T D N N G T S S A S T T S T T S T T D T A S T T S T T G T T G T T
Epitope E 407 412 413 N S G N T G N T G S T G D T V D D S D D S S N V S N N S D V S N I S N T S N T
B
Fig. 3. Norovirus GII.4 variation of representative strains. (A) Amino acid variation over time in blockade-epitope regions. Twelve representative sequences of norovirus GII.4 variants and the representative of variant 2012 detected in this study were selected from each major phylogenetic cluster. Each color represents amino acid changes that occurred within the subcluster. (B) The three-dimensional structural model of norovirus GII.4 variants showing locations of amino acid changes in blockade-epitope regions of the dominant strains; (a) Den_Haag_2006b, (b) New_Orleans_2009, (c) JP41 [Sydney_2012] variants.
and New_Orleans_2009 variants which previously emerged as predominant variants that cause outbreaks (Fig. 3A and B). The P2 sub-domain of VP1 contains potential neutralizing antibody epitopes and interacts with HBGAs, which is a diverse family of carbohydrates and serve as putative receptors for norovirus attachment (Shanker et al., 2011). The analysis of P2 sub-domain of norovirus GII.4 variant Sydney_2012 detected in this study compared with the reference strains revealed four amino acid changes in epitope A (positions 294 and 368), epitope D (position 393), and epitope E (position 413), which has mapped as the blockade epitope (Lindesmith et al., 2012; Debbink et al., 2013). Although it is unclear how many and which amino acid in the blockade epitopes are needed to mediate an escape mutant phenotype, previous studies demonstrates that using MAbs mapping the blockade epitope showed a significant reduces of reactivity and blockade of Sydney_2012 (Debbink et al., 2013). So, evolving of these epitopes may result in a difference of antigenic determinants between strains which drives GII.4 escape from herd immunity. Residue 393–395 in the epitope D consistently identified as a putative epitope that is commonly altered norovirus binding affinity and specificity to HBGAs as well as antibody binding and blockade among epidemic GII.4 strains (Lindesmith et al., 2012). In this study, we predicted the binding structure of HBGA binding interface of the Lewis binding norovirus. As a member of norovirus GII.4, Sydney_2012 share highly conserved location, amino acid composition and overall structures of the binding pockets with Den_Haag_2006b and New_Orleans_2009 variants. The Lewis fucose of Lewisb with its exocyclic carboxyl groups, interacts extensively and making a total of four hydrogen bonds involving residue Asp391, Gly392, and Ser393 (Shanker et al., 2011). In the current study, from the superimposition between the crystal structures of Sydney_2012 with other variants, it was found that amino acid residue 393 of Sydney_2012 variant differed from the other
variants changing from serine to glycine (Fig. 4). Amino acid glycine at residue 393 in Sydney_2012 strain could not form the hydrogen bond interaction to the Lewisb. The P2 structure of Sydney_2012 variants clearly shows that chain A of the Sydney_2012 variant is positioned slightly away from chain C compared to that in Den_Haag_2006b and New_Orleans_2009 strains. Therefore, the change of the amino acid residue at the position 393 in Sydney_2012 may change the HBGA recognition and represent the selective force that drives antigenic variation within around the receptor binding pocket. The current study showed the norovirus infection among the Japanese pediatric patient with gastroenteritis. The limitation concerns the generalizability of the findings to the entire Japanese populations. Although data were collected from six prefectures from northern to southern part of Japan, they may not represent the national situation of norovirus outbreak activity. This study indicates a high prevalence (39.3%) of norovirus infection in Japanese pediatric patients with diarrhea in 2009– 2013. Analysis of noroviruses circulating in the past 4 years revealed a change of the predominant variant of norovirus GII.4 in each epidemic season over time. The new variant termed ‘‘Sydney_2012’’ was shown to emerge in Japanese populations and this emergence has been shown to be associated with the increase of norovirus infections in pediatric patients throughout Japan from Mid-2012. Interestingly, the Sydney_2012 appears to replace the previous predominant strains, Den_Haag_2006b and New_ Orleans_2009, and shows a high level of diversity across P2 subdomain comparison with those of previous dominant variant strains. These observations suggest that the two existing variants, Den_Haag_2006b and New_Orleans_2009, might be currently competing for persistence in human populations by evolving a novel variant 2012 strain. Our observation serves as a further piece of evidence for this possibility. It has been postulated that
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Fig. 4. HBGA binding of norovirus GII. 4 variant representative strains. (A) Hydrogen-bond interaction (red dash line) in HBGA binding site between Lewisb hexasaccharide and Hunter_2004 strain (pdb code 3sej) (Grey). Structures of Den_Haag_2006 (Blue), New Orleans_2009 (Green), Sydney_2012 (Pink) and JP41 (Orange) are also shown. ⁄SeFuc (secretor fucose), LeFuc (Lewis fucose), Gal (galactose), GlcNAc (N-acetylglucosamine), and Glc (glucose). (B) Amino acid residues interacting with Secretor Lewis HBGA (Lewisb). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
diversification of the P2 sub-domain through accumulated mutations in antigenic blockade epitopes and HBGAs binding pocket in Sydney_2012 may affect HBGA binding and has been linked to antigenic escape from host immune responses directed to previous infection that permits the emergence of widespread a new variant Sydney_2012. Acknowledgements We are grateful to Dr. Shuichi Nishimura, Dr. Hideaki Kikuta, Dr. Atsuko Yamamoto, Dr. Kumiko Sugita, Dr. Masaaki Kobayashi, and Dr. Tsuneyoshi Baba for specimen collections. This study was supported by Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Grant Number; 23406036 and 24390266). References Bennett, S., MacLean, A., Miller, R.S., Aitken, C., Gunson, R.N., 2013. Increased norovirus activity in Scotland in 2012 is associated with emergence of a new norovirus GII.4 variant. Euro. Surveill. 18, 20349. Bull, R.A., Tu, E.T., Mciver, C.J., Rawlinson, W.D., White, P.A., 2006. Emergence of a new norovirus genotype II.4 variant associated with global outbreaks of gastroenteritis emergence of a new norovirus genotype II.4 variant associated with global outbreaks of gastroenteritis. J. Clin. Microbiol. 44, 327–333. Chan-It, W., Thongprachum, A., Okitsu, S., Nishimura, S., Kikuta, H., Baba, T., Yamamoto, A., Sugita, K., Hashira, S., Tajima, T., Mizuguchi, M., Ushijima, H., 2011. Detection and genetic characterization of norovirus infections in children with acute gastroenteritis in Japan, 2007–2009. Clin. lab. 57, 213–220. Combet, C., Jambon, M., Deléage, G., Geourjon, C., 2002. Geno3D: automatic comparative molecular modeling of protein. Bioinformatics 18, 213–214. Debbink, K., Donaldson, E.F., Lindesmith, L.C., Baric, R.S., 2012. Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity. J. Virol. 86, 1214–1226.
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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Molecular epidemiology of norovirus associated with gastroenteritis and emergence of norovirus GII.4 variant 2012 in Japanese pediatric patients Aksara Thongprachum a,b, Wisoot Chan-it a, Pattara Khamrin c, Patchreenart Saparpakorn d, Shoko Okitsu a,b, Sayaka Takanashi a, Masashi Mizuguchi a, Satoshi Hayakawa b, Niwat Maneekarn c, Hiroshi Ushijima a,b,⇑ a
Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand d Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand b c
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 17 January 2014 Accepted 19 January 2014 Available online 4 February 2014 Keywords: Norovirus Epidemiology Gastroenteritis Japanese Variant
a b s t r a c t In late 2012, an outbreak of acute gastroenteritis due to norovirus variant Sydney_2012 occurred and have been reported from many counties. In this study, we described surveillance study of the incidence of norovirus infections among Japanese pediatric patients in association with gastroenteritis and investigated the antigenic change of the new variant Sydney_2012 circulated in Japanese populations. A total of 2381 fecal specimens collected from children with acute gastroenteritis in Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from 2009 to 2013 were examined for norovirus and further analyzed molecularly. A high proportion (39.3%) of norovirus positive samples and several genotypes were detected. Norovirus GII.4 dominated over other genotypes (71.4%). The Den_Haag_2006b (43.2%) was detected as the predominant variant and co-circulated with New_Orleans_2009 (17.8%) until March 2012. Subsequently, they were displaced by Sydney_2012. The Sydney_2012 variant has been responsible for the majority of norovirus infections in 2012–2013 (85.7%). Although Sydney_2012 variant has a common ancestor with New_Orleans_2009 variant, analysis of P2 sub-domain showed a high level of diversity in comparison with other variants in four amino acid changes at the antigenic sites. The change in particular residue 393 of new variant may affect HBGA recognition. Analysis of noroviruses circulating in the past 4 years revealed a change of predominant variant of norovirus GII.4 in each epidemic season. The change of amino acid in putative epitopes may have led the virus escape from the existing herd immunity and explain the increase of new variant outbreaks. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Norovirus is a major etiologic agent responsible for epidemic gastroenteritis worldwide. It is a single-stranded, positive sense RNA virus that belongs to the Caliciviridae family. The norovirus genome is 7.5 kb long and contains three open reading frames; ORF1 encodes the non-structural proteins, ORF2 encodes the VP1 major capsid protein, and ORF3 encodes the VP2 minor capsid protein (Prasad et al., 1999).
⇑ Corresponding author at: Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3509; fax: +81 3 5841 3629. E-mail address: [email protected] (H. Ushijima). http://dx.doi.org/10.1016/j.meegid.2014.01.030 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Based on genetic difference in the capsid gene, noroviruses are genetically highly diverse and divided into five genogroups (GI-V). They are further subdivided into several different genotypes, at least nine genotypes of GI, 21 genotypes of GII, 2 genotypes of GIII, and only one genotype of GIV and GV have been identified (Zheng et al., 2010; Kroneman et al., 2013). Genotype II.4 (GII.4) norovirus has been shown to spread rapidly and is the most commonly detected worldwide, particularly in association with outbreaks. Recent molecular surveillances indicate that GII.4 norovirus has evolved a series of genetic variants over the last 20 years, some of which persist and replace the previous circulating variants (Siebenga et al., 2010). Norovirus epidemic patterns in human populations include epidemic outbreaks of disease every 2–3 years, punctuated by the emergence of an antigenically distinct GII.4 strain that appears to escape human herd immunity to the previous circulating strains (Debbink et al., 2012; Lindesmith et al.,
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2013). The continuous evolution of the P2 sub-domain of the surface exposed major capsid protein (VP1) has been proposed as a key mechanism by which new antigenic variants are generated. Norovirus recognizes histo-blood group antigen (HBGAs), which play an important role in host susceptibility to norovirus infection (Tan and Jiang, 2011). HBGAs are a diverse family of carbohydrates expressed on red blood cells, mucosal epithelia, saliva, milk and other body fluids, which are highly polymorphic and are related to the ABO, secretor and Lewis families. They have been hypothesized to be receptors or coreceptors that allow norovirus to attach and enter permissive cells (Debbink et al., 2012). The HBGA binding interfaces are located at the top of the P dimer, corresponding to the outermost surface of the capsid. The carbohydrate binding pockets involve several scattered amino acid residues in the P domain that form extensive hydrogen bond network with individual saccharides, and thus stabilizing the binding of HBGAs to the capsid protein (Zhang et al., 2013). The increased detections of norovirus GII.4 variants have been observed globally. Since the mid-90s, norovirus GII.4 variants have been shown to be associated with at least six pandemics of acute gastroenteritis and continue to cause millions of infections across the globe annually. The norovirus GII.4 variants reported previously include the US 1995/96 in 1996 (Noel et al., 1999; White et al., 2002), Farmington_Hills_2002 in 2002 (Lopman et al., 2004; Widdowson et al., 2004), Hunter_2004 in 2004 (Bull et al., 2006), Minerva_2006b and Den_Haag_2006b virus in 2006–2008 (Eden et al., 2010), New_Orleans_2009 in 2009–2012 (Yen et al., 2011), and most recently Sydney_2012 (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). The periodic increase of norovirus outbreaks have been shown to be related to the emergence of new variant strains, probably owing to evasion of population immunity (Zheng et al., 2010). The emergence of such variants has been shown to be associated with substantial increases in cases worldwide (Pang et al., 2010). Since late 2012, surveillance systems in many countries showed the increase of norovirus activity levels compared to the previous seasons, and associated with the emergence of a new variant GII.4 named Sydney_2012 (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). Therefore, rapid antigenic variations are important evolutionary forces that shape epidemiological success and persistence of the GII.4 viruses in the population. The objectives of this study were: (1) to describe a surveillance of norovirus infections that are associated with gastroenteritis in Japanese pediatric patients; and (2) to investigate P2 sub-domain of the new variant Sydney_2012 which may affect antigenic property.
2. Materials and methods 2.1. Patients and samples A total of 2381 fecal specimens were collected from non-hospitalized children with acute gastroenteritis in pediatric clinics in six prefectures from northern to southern Japan including Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from July 2009 to June 2013. Among these, 515, 536, 592, and 738 samples were obtained during the period of 2009–2010, 2010–2011, 2011–2012, and 2012–2013, respectively. In this study, the annual observation period for norovirus gastroenteritis began in July and ended in June of the following year. The spectrum of clinical signs exhibited by patients with acute gastroenteritis included acute watery diarrhea, vomiting, abdominal cramps, and fever of various severity and in various combinations. The age of the patients ranged from neonate to 15 years (0–178 months; mean age 24.4 months). For subsequent data analysis, seven different age-groups were established:
60 months (156 until use.
samples), 6–11 months (673 samples), 12– samples), 24–35 months (334 samples), 36– samples), 48–59 months (63 samples), and samples). The specimens were stored at 20 °C
2.2. Viral RNA extraction The fecal specimens were prepared as 10% fecal suspensions in distilled water. Viral genome was extracted from the supernatant of 10% suspension using the QIAamp Viral RNA MiniKit, according to the manufacturer’s instructions (QIAgen, Hilden, Germany). The extracted viral RNAs were stored at 80 °C until use. 2.3. Detection of norovirus The reverse transcription (RT) was performed with RNA template using SuperScriptIII reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The presence of norovirus was detected by RT-PCR using the protocol described previously (Yan et al., 2003). The specimens were tested for the presence of norovirus GI and GII by amplification of partial gene. For norovirus GI, a combination of forward primer G1-SKF (nt 5342–5361) 50 -CTGCCCGAATTYGTAAATGA-30 and reverse primer G1SKR (nt 5653–5671) 50 -CC AACCCARCCATTRTACA-30 were used. For norovirus GII, a forward primer COG2F (nt 5003–5028; 50 -CARGARBCNATGTTYAGRTGGATGAG-30 ) was used in combination with the reverse primer G2SKR (nt 5367–5389; 50 -CCRCCNGCATRHCCRTTRTACAT-30 ). Additionally for norovirus GII, semi-nested PCR was performed using G2SKF (nt 5389–5367; 50 -CNTGGGAGGGCGATCGCAA-30 ) and G2SKR primers for the samples that were negative by the first set of primer for norovirus GII. 2.4. Amplification of polymerase and capsid genes of norovirus The partial polymerase and full-length capsid region of a new variant was amplified using two pairs of primers, GV21 (50 -GTB GGNGGYCARATGGGNATG-30 )/G2SKR (50 -CCRCCNGCATRHCCRT TRTACAT-30 ) and COG2F (50 -CARGARBCNATGTTYAGRTGGATGAG30 )/TX30SXN (50 -GACTAGTTCTAGATCGCGAGCGGCCGCCC(T)30-30 ). Long-range PCR was amplified with High Fidelity DNA polymerase KOD-Plus (Toyobo, Osaka, Japan). The PCR was carried out with 2 ll of cDNA using TX30SXN primer, in the reagent mixture containing 2.5 ll of 10 buffer for KOD-Plus, 1 ll of 2 mM dNTPs, 1 ll of 25 mM MgSO4, 0.75 ll of each specific primer (20 lM) and 0.5 ll of KOD Plus enzyme. MilliQ water was added to make up a total volume of 25 ll. The PCR was performed at 94 °C for 2 min, followed by 35 cycles of 98 °C 20 s, 50 °C or 55 °C 30 s, 68 °C 2 min, and a final extension at 68 °C for 10 min, and then held at 4 °C. 2.5. Sequence and Phylogenetic Analyses The genotypes of the norovirus were characterized by direct nucleotide sequencing of partial capsid gene and then performing phylogenetic analysis as described previously by Kageyama et al. (2004). The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced by using the BigDye Terminator Cycle Sequencing kit (Perkin Elmer-Applied Biosystems, Inc., Foster City, CA) on an automated DNA sequencer (ABI 3100; Perkin Elmer-Applied Biosystems, Inc., Foster City, CA). The nucleotide sequences were analysed in comparison with those of norovirus strains deposited in GenBank database. Phylogenetic relationship of norovirus was examined by aligning sequences with the ClustalX program. A phylogenetic tree was constructed
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according to the maximum likelihood (ML) method using MEGA version 5 (Tamura et al., 2011).
930), GII.7 (1.4%; 13/930), GII.6 (1.3%; 12/930), GII.13 (1.1%; 10/ 930), GII.12 (0.8%; 7/930), and GII.17 (0.1%; 1/930) (Table 1). The norovirus GI was detected at 0.3% (8/2381), comprising GI.1 (0.04%), GI.4 (0.04%), and GI.6 (0.25%). Out of total samples, 28.1% of the patients were infected by norovirus GII alone, while 11.0% were co-circulated with other enteric viruses. Double-infection and triple-infection were found at 10.5% (250/2381) and 0.5% (11/2381), respectively. Mix-infection between norovirus GII and group A rotavirus was the most predominant (4.5%; 106/2381), followed by human parechovirus (2.3%), adenovirus (1.9%), enterovirus (1.4%), sapovirus (0.3%), astrovirus (0.1%), and norovirus GI (0.1%).
2.6. Nucleotide sequence accession numbers The partial nucleotide sequences of the capsid gene of norovirus identified in this study were deposited in GenBank under accession numbers KF658275–KF658339. Accession number for partial polymerase and full-length capsid sequences of norovirus GII.4 Sydney_2012 variant used in this study is KF145149. 2.7. Three-dimensional structure
3.2. Identification of epidemic norovirus GII.4 variants
The three-dimensional structures of norovirus genomes of variants were constructed by homology modeling technique using Geno3D web server (Combet et al., 2002), the structures were checked the quality of geometry by PROCHECK (Laskowski et al., 1993). For the model structures, all sequences were based on the structure of a norovirus GII.4 2004 norovirus using PDB code 3sej (Shanker et al., 2011).
Phylogenetic tree of nucleotide sequences of the partial capsid region of norovirus GII isolates detected in the present study was constructed in comparison with those of the reference strains available in GenBank database (Fig. 1). The norovirus GII.4 consisted of three major variants strains detected in pediatric patients in this study (Fig. 2). The Den_Haag_2006b strain that appeared during the study period comprised 287 of 664 norovirus GII.4 strains (43.2%). The sequences of these norovirus strains exhibited 98.0–100% nucleotide identity and 98.7–100% amino acid identity with Hu/GII.4/DenHaag89/2006/NL (GenBank accession number EF126965). The second variant, New_Orleans_2009 strain, was detected in 17.8% (118/664) and displayed 96.0–100% nucleotide sequence identity and 97.5–100% amino acid identity with the capsid region of Hu/GII.4/New Orleans1805/2009/USA (GU445325). Interestingly, the new Sydney_2012 variant was detected at 36.4% (242/ 664) and clustered within the same cluster of the Sydney/ NSW0514/2012/AU (JX459908) and Hong Kong/CUHK3655/2012/ CHN (JX629456) reference strains and showed 98.7–100% nucleotide sequence identity and 100% amino acid sequence identity with reference strains. However, a number of additional GII.4 variants have also been identified, including Hunter 2004, Yerseke_2006a, Apeldoorn_2007, and Hokkaido1_2008. These variants were less common in this study.
3. Results 3.1. Detection and genotypic distribution of norovirus A high proportion (39.3%; 936/2381) of norovirus were detected in the pediatric patients during 2009–2013, comprising 0.3% of GI (6/2381), 39.0% of GII (928/2381), and 0.1% mixed GI/GII (2/2381). Most of norovirus belonged to norovirus GII genogroup (39.1%; 930/2381). As shown in Table 1, the detection rate of norovirus GII were 40.8% (210/515) in 2009–2010, 40.1% (215/536) in 2010–2011, 31.8% (188/592) in 2011–2012, and 43.0% (317/738) in 2012–2013. Based on the analysis of partial sequence of capsid gene of norovirus GII, nine distinct genotypes were identified. Of these, GII.4 was the most predominant genotype (71.4%; 664/930), followed by GII.3 (13.1%; 122/930), GII.2 (6.5%; 60/930), GII.14 (4.4%; 41/
Table 1 Genotype distributions of noroviruses detected in Japanese pediatric patients with diarrhea from 2004 to 2013. No. of genogroup positive (%)
No. of genotype cases
Reference
Year
No. of samples tested
No. of norovirus positive (%)
GI
GII
GII.1
GII.2
GII.3
GII.4
GII.6
GII.7
GII.12
GII.13
GII.14
GII.17
2004–2005
752
139 (18.5)
2 (0.3)
137 (18.2)
1
0
22
108
6
0
0
0
0
0
2005–2006
603
51 (8.5)
0 (0)
51 (8.5)
1
1
27
19
2
1
0
0
0
0
2006–2007
628
97 (15.4)
2
6
84
0
3
0
0
0
0
477
125 (26.2)
95 (15.1) 118 (24.7)
0
2007–2008
2 (0.3) 7 (1.5)
0
0
6
92
6
0
0
0
14
0
2008–2009
477
129 (27.0)
0 (0)
129 (27.0)
1
4
7
92
18
1
3
0
3
0
2009–2010
515
210 (40.8)
0 (0)
0
24
30
135
1
6
5
1
7
1
2010–2011
536
215 (40.1)
0
17
60
118
2
1
1
2
14
0
2011–2012
592
188 (31.8)
1 (0.2) 0 (0)
0
7
30
132
3
0
0
5
11
0
2012–2013
738
323 (43.8)
210 (40.8) 215 (40.1) 188 (31.8) 317 (43.0)
0
12
2
279
6
6
1
2
9
0
7 (0.9)
Phan et al. (2006) Phan et al. (2007) Dey et al. (2012) Chan-it et al. (2011) Chan-it et al. (2011) This study This study This study This study
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Fig. 1. Phylogenetic analysis of the partial capsid sequence of norovirus GII.4 strains. The phylogenetic tree constructed using the maximum likelihood method. Representative strains detected in this study are presented in boldface.
A. Thongprachum et al. / Infection, Genetics and Evolution 23 (2014) 65–73
69
Fig. 2. Monthly distribution of norovirus GII.4 norovirus infection in Japan pediatric patients during the epidemic seasons of 2009/2010–2012/2013. (A) norovirus genotypes. (B) Norovirus GII.4 variants.
3.3. Seasonal pattern and age distribution of norovirus infection The seasonal pattern of norovirus infection is shown in Fig. 2. Norovirus was detected all year round with a highest peak in December. From this study, it was clearly observed that many noroviruses genotypes were common in winter season in Japan (Fig. 2A). The highest prevalence of norovirus was found in December (23.6%; 221/936), followed by January (19.6%; 183/936), November (12.3%; 115/936), and February (9.8%; 92/936), respectively. Focusing on norovirus GII.4 variants, most of norovirus GII.4 occurred between November and January, with a highest peak in December (25.9%). The Den_Haag_2006b variant was detected as the most predominant variant until March 2012 in co-circulation with New_Orleans_2009 variant. Subsequently, they were replaced by variant Sydney_2012 which was described in June 2012 (Fig. 2B). However, the Sydney_2012 was first detected in a Japanese patient in Tokyo since November 2011 before spreading throughout Japan from mid-2012. Of the patients with acute gastroenteritis, norovirus infection was detected in all age groups. The mean age of norovirus infected patients was 22.4 months. The highest prevalence of norovirus was found in infants and children aged 12–23 months (42.4%, n = 397),
followed by 6–11 months (25.6%, n = 240), 24–36 months (14.9%, n = 139), less than 6 months (6.3%, n = 59), 36–47 months (4.5%, n = 42), more than 60 months (3.5%, n = 33), and the lowest in children aged 48–59 months (1.1%, n = 26). 3.4. Analysis of polymerase and capsid of norovirus GII.4 Sydney_2012 To determine antigenic differences between GII.4 Sydney_2012 and the recent circulating ancestral strains Den_Haag_2006b and New_Orleans_2009 strains. We analysed VP1 and P2 sub-domain especially in blockade epitopes of Sydney_2012 and compared with other variant strains. The analysis of partial VP1 capsid of GII.4 variant 2012 representative strains revealed a high diversity of 6 positions of amino acid substitutions compared to Den_Haag_2006b and New_Orleans_2009 variants; positions 294(A2006b ? P2009 ? T2012), 359(T2006b ? S2009 ? A2012), 368(S2006b ? A2009 ? E2012), 377(T2006b ? T2009 ? A2012), 393(S2006b ? S2009 ? G2012), and 413(V2006b ? I2009 ? T2012), as shown in Fig. 2A and B. In the analysis of P2 sub-domain, the Japanese norovirus GII.4 variant 2012 strains showed P98.9% nucleotide and P99.2% amino acid sequence identities with the Sydney/NSW0514/2012 (JX459908). Moreover, analysis of polymerase region of the Sydney_2012 variant was found to be similar to Apeldoorm_2007 but distinct from
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other variants and accounted for three amino acid substitutions in polymerase region.
3.5. Effect of HBGA binding site In order to investigate the HBGA binding site, the superimposition between the crystal structure of Hunter_2004 strain (pdb code 3sej) and other variants were studied as shown in Fig. 4A. The structure of Hunter_2004 strain revealed the hydrogen bond interaction between Lewisb hexasaccharide and some amino acid residues in both dimers (i.e. Chain A: Gln390, Asp391, Gly392, Ser393, Thr394, Thr395, Ser442, and Gly443, Chain C: Ser343, Thr344, Arg345, and Asp374), as shown in Fig. 4B. For the strains of Sydney_2012 (JP41), the amino acid residue position 393 was glycine which was different from other variant strains. From the crystal structure, the hydroxyl group of Ser393 formed the hydrogen bond interaction to Lewis fucose (LeFuc) while Gly393 in Sydney_2012 (JP41) strain could not form the hydrogen bond interaction to the LeFuc (Fig. 4A).
4. Discussion Viral gastroenteritis is still a health burden and one of the most frequently encountered problems in developed and developing countries. Global outbreaks of acute gastroenteritis by norovirus have frequently reported. Our group also previously reported the epidemic occurrence of norovirus in a Japanese population (Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). The current study extended these findings by investigating different epidemic patterns. A high prevalence (39.3%) of norovirus infection was found in these Japanese pediatric patients with acute gastroenteritis in Japan during July 2009 to June 2013; 99.4% of all norovirus cases were infected by norovirus GII. In contrast, the prevalences of rotavirus, human parechovirus, enterovirus, adenovirus, sapovirus, astrovirus, and Aichi virus were 20.1%, 6.6%, 6.1%, 5.6%, 4.8%, 2.3%, and 0.1%, respectively (unpublished data). The prevalence of norovirus is higher than that found in the preceding studies conducted in the same geographical area during 2004–2009 which showed the prevalence rates of norovirus ranged from 8.5% to 27.0% (Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). Taken together, results in the present and preceding studies confirmed that the detection rate increased year by year and norovirus is the most predominant agent causing acute gastroenteritis in Japan (Kageyama et al., 2004; Phan et al., 2006; Chan-it et al., 2011; Dey et al., 2011). This study documents 11.0% mixed infection between norovirus and other enteric viruses, in which 4.5% mixed infection between norovirus and group A rotavirus was most predominant. Differences in rotavirus and norovirus seasonality may relate to co-infection rate in this study. Moreover, mixed infection was not detected more frequently in any particular age group. Regarding norovirus seasonality, the seasonal distribution of norovirus infection exhibits a peak in winter seasons, a highest peak of incidence in December (23.6%). It is clearly observed that norovirus infection, especially norovirus GII.4 is commonly detected in cold months in Japan; 65.3% of confirmed norovirus infection cases occurred between December and February. Our data are in good agreement with the other surveillance on pediatric cases which demonstrated the main peak of norovirus during the winter months in Japan (Iritani et al., 2010; Dey et al., 2010). The temperature and humidity had highly consistent effects on driving norovirus epidemic patterns (Lopman et al., 2009). A high number of norovirus cases detected in every winter season in this study might be associated with cold and dry temperature.
Several studies reported that norovirus infection occurred frequently in young children under 2 years-old (Sai et al., 2013; Yoneda et al., 2014). In agreement with this, our results showed a marked trend toward higher rates of infection in children under-2-years of age (74.4%, 696/936), particularly children between 12 and 23 months are the most frequently affected group. The findings show that norovirus infections usually occur in early childhood. It is possible that the children younger than 2 years old might lack of maternal antibody protection. Based on antigenic analyses and more recent extensive sequence analyses, the circulation of norovirus in nature have been shown to be highly variable. In this study, genetic analysis among norovirus GII demonstrated that GII.4 genotype is the most predominant genotype accounting for 71.4%, followed by GII.3, GII.2, GII.14, GII.7, GII.6, GII.13, GII.12, and GII.17. The highest prevalence of GII.4 is consistent with those obtained from earlier studies conducted in the same settings as well as other studies conducted elsewhere in Japan (Phan et al., 2006; Dey et al., 2011; Chan-it et al., 2011; Hoa Tran et al., 2013; Yoneda et al., 2014). Norovirus GII.4 strains evolved rapidly and spread globally with the emerging variants. On the other hand, the increase of norovirus GII.3 has been identified and this strain is the second leading genotype in Japan after dropping during 2006–2009. A sharp decrease in the number of patients infected by GII.3 was observed when the new emerging variant was spread in 2012–2013 (14.3% in 2009–2010; 27.9% in 2010–2011; 16.0% in 2012–2013; 0.6% in 2012–2013). However, norovirus GI was low detectable in this surveillance study. Focusing on norovirus GII.4 variants, analysis of noroviruses circulating in the past 4 years in Japanese pediatric patients revealed a change of the predominant variant of norovirus GII.4 in each epidemic season over time. Among norovirus GII.4 identified in this study, three separate norovirus GII.4 variants were mainly identified as the etiologic agents of norovirus-associated acute gastroenteritis, including Den_Haag_2006b (43.2%), New_Orleans_2009 (17.8%), and Sydney_2012 (36.4%) variants. The Sydney_2012 new variant was shown to emerge since late 2011 and this emergence has been shown to be associated with the increase of norovirus infections in Japanese pediatric patients from May 2012. Interestingly, the previous dominant Den_Haag_2006b and New_Orleans_2009 variants were displaced by new emergence variant Sydney_2012 (Fig. 2B). The Sydney_2012 variant has been shown to be responsible for the majority of norovirus infection in 2012–2013, accounting for 85.7% of all norovirus GII.4 detected in 2012–2013. Even though many studies reported the detection of Sydney_2012 variant from late 2012 (Bennett et al., 2013; Fonager et al., 2013; van Beek et al., 2013), the present study described the detection of Sydney_2012 variant among pediatric patients in Tokyo since November 2011 before spreading throughout Japan from mid-2012. This study clearly indicated that norovirus variant Sydney_2012 was the predominant group causing acute gastroenteritis among Japanese pediatric populations since mid-2012. Interestingly, the Sydney_2012 strains detected in the current study were clustered in the same lineage and showed a high nucleotide sequence (P98.7%) and amino acid sequence (100%) similar to norovirus GII found previously Australia, the United States, Europe, Canada, and China (Bennett et al., 2013; Fonager et al., 2013; Mai et al., 2013; van Beek et al., 2013). Then, the Sydney_2012 had emerged as the etiologic agent in acute gastroenteritis across the United States, Europe and Asia, including Japan in the same period. Although the Sydney_2012 variant has common ancestor with the dominant New_Orleans_2009 variants, analysis of partial VP1 capsid of GII.4 variant 2012 representative strains revealed a high diversity with 6 positions of amino acid substitutions, positions 294, 359, 368, 377, 393, and 413, compared to Den_Haag_2006b
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A
Strain CHDC5191 Camberwell Dresden Farmington_Hills Guangzhou Hunter Yerseke Den_Haag Apeldoorm Hokkaido1 New_Orleans Sydney JP41
Accession NO. FJ537134 AF145896 AY741811 AY502023 DQ369797 DQ078814 EF126963 EF126965 AB445395 AB541261 GU445325 JX459908 KF145149
Variant 1974 1994 1997 2002 2003 2004 2006a 2006b 2007 2008 2009 2012 2012
294 G V A A P A A A T A P T T
296 S S S T T T T S S S S S S
Epitope A 297 298 368 H D T H D T H D T H N N R I A Q N S Q E S R N S R N A R N S R N A R N E R N E
372 N N N N D S S E D E D D D
Epitope D 393 394 395 D T D N N G T S S A S T T S T T S T T D T A S T T S T T G T T G T T
Epitope E 407 412 413 N S G N T G N T G S T G D T V D D S D D S S N V S N N S D V S N I S N T S N T
B
Fig. 3. Norovirus GII.4 variation of representative strains. (A) Amino acid variation over time in blockade-epitope regions. Twelve representative sequences of norovirus GII.4 variants and the representative of variant 2012 detected in this study were selected from each major phylogenetic cluster. Each color represents amino acid changes that occurred within the subcluster. (B) The three-dimensional structural model of norovirus GII.4 variants showing locations of amino acid changes in blockade-epitope regions of the dominant strains; (a) Den_Haag_2006b, (b) New_Orleans_2009, (c) JP41 [Sydney_2012] variants.
and New_Orleans_2009 variants which previously emerged as predominant variants that cause outbreaks (Fig. 3A and B). The P2 sub-domain of VP1 contains potential neutralizing antibody epitopes and interacts with HBGAs, which is a diverse family of carbohydrates and serve as putative receptors for norovirus attachment (Shanker et al., 2011). The analysis of P2 sub-domain of norovirus GII.4 variant Sydney_2012 detected in this study compared with the reference strains revealed four amino acid changes in epitope A (positions 294 and 368), epitope D (position 393), and epitope E (position 413), which has mapped as the blockade epitope (Lindesmith et al., 2012; Debbink et al., 2013). Although it is unclear how many and which amino acid in the blockade epitopes are needed to mediate an escape mutant phenotype, previous studies demonstrates that using MAbs mapping the blockade epitope showed a significant reduces of reactivity and blockade of Sydney_2012 (Debbink et al., 2013). So, evolving of these epitopes may result in a difference of antigenic determinants between strains which drives GII.4 escape from herd immunity. Residue 393–395 in the epitope D consistently identified as a putative epitope that is commonly altered norovirus binding affinity and specificity to HBGAs as well as antibody binding and blockade among epidemic GII.4 strains (Lindesmith et al., 2012). In this study, we predicted the binding structure of HBGA binding interface of the Lewis binding norovirus. As a member of norovirus GII.4, Sydney_2012 share highly conserved location, amino acid composition and overall structures of the binding pockets with Den_Haag_2006b and New_Orleans_2009 variants. The Lewis fucose of Lewisb with its exocyclic carboxyl groups, interacts extensively and making a total of four hydrogen bonds involving residue Asp391, Gly392, and Ser393 (Shanker et al., 2011). In the current study, from the superimposition between the crystal structures of Sydney_2012 with other variants, it was found that amino acid residue 393 of Sydney_2012 variant differed from the other
variants changing from serine to glycine (Fig. 4). Amino acid glycine at residue 393 in Sydney_2012 strain could not form the hydrogen bond interaction to the Lewisb. The P2 structure of Sydney_2012 variants clearly shows that chain A of the Sydney_2012 variant is positioned slightly away from chain C compared to that in Den_Haag_2006b and New_Orleans_2009 strains. Therefore, the change of the amino acid residue at the position 393 in Sydney_2012 may change the HBGA recognition and represent the selective force that drives antigenic variation within around the receptor binding pocket. The current study showed the norovirus infection among the Japanese pediatric patient with gastroenteritis. The limitation concerns the generalizability of the findings to the entire Japanese populations. Although data were collected from six prefectures from northern to southern part of Japan, they may not represent the national situation of norovirus outbreak activity. This study indicates a high prevalence (39.3%) of norovirus infection in Japanese pediatric patients with diarrhea in 2009– 2013. Analysis of noroviruses circulating in the past 4 years revealed a change of the predominant variant of norovirus GII.4 in each epidemic season over time. The new variant termed ‘‘Sydney_2012’’ was shown to emerge in Japanese populations and this emergence has been shown to be associated with the increase of norovirus infections in pediatric patients throughout Japan from Mid-2012. Interestingly, the Sydney_2012 appears to replace the previous predominant strains, Den_Haag_2006b and New_ Orleans_2009, and shows a high level of diversity across P2 subdomain comparison with those of previous dominant variant strains. These observations suggest that the two existing variants, Den_Haag_2006b and New_Orleans_2009, might be currently competing for persistence in human populations by evolving a novel variant 2012 strain. Our observation serves as a further piece of evidence for this possibility. It has been postulated that
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A
B Strain
Variant
Chain A
Chain C
390
391
392
393
442
443
343
344
345
374
Hunter
2004
Q
D
G
S
S
G
S
T
R
D
Den_Haag
2006b
Q
D
G
S
S
G
S
T
R
D
New_Orleans
2009
Q
D
G
S
S
G
S
T
R
D
Sydney
2012
Q
D
G
G
S
G
S
T
R
D
JP41
2012
Q
D
G
G
S
G
S
T
R
D
Fig. 4. HBGA binding of norovirus GII. 4 variant representative strains. (A) Hydrogen-bond interaction (red dash line) in HBGA binding site between Lewisb hexasaccharide and Hunter_2004 strain (pdb code 3sej) (Grey). Structures of Den_Haag_2006 (Blue), New Orleans_2009 (Green), Sydney_2012 (Pink) and JP41 (Orange) are also shown. ⁄SeFuc (secretor fucose), LeFuc (Lewis fucose), Gal (galactose), GlcNAc (N-acetylglucosamine), and Glc (glucose). (B) Amino acid residues interacting with Secretor Lewis HBGA (Lewisb). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
diversification of the P2 sub-domain through accumulated mutations in antigenic blockade epitopes and HBGAs binding pocket in Sydney_2012 may affect HBGA binding and has been linked to antigenic escape from host immune responses directed to previous infection that permits the emergence of widespread a new variant Sydney_2012. Acknowledgements We are grateful to Dr. Shuichi Nishimura, Dr. Hideaki Kikuta, Dr. Atsuko Yamamoto, Dr. Kumiko Sugita, Dr. Masaaki Kobayashi, and Dr. Tsuneyoshi Baba for specimen collections. This study was supported by Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Grant Number; 23406036 and 24390266). References Bennett, S., MacLean, A., Miller, R.S., Aitken, C., Gunson, R.N., 2013. Increased norovirus activity in Scotland in 2012 is associated with emergence of a new norovirus GII.4 variant. Euro. Surveill. 18, 20349. Bull, R.A., Tu, E.T., Mciver, C.J., Rawlinson, W.D., White, P.A., 2006. Emergence of a new norovirus genotype II.4 variant associated with global outbreaks of gastroenteritis emergence of a new norovirus genotype II.4 variant associated with global outbreaks of gastroenteritis. J. Clin. Microbiol. 44, 327–333. Chan-It, W., Thongprachum, A., Okitsu, S., Nishimura, S., Kikuta, H., Baba, T., Yamamoto, A., Sugita, K., Hashira, S., Tajima, T., Mizuguchi, M., Ushijima, H., 2011. Detection and genetic characterization of norovirus infections in children with acute gastroenteritis in Japan, 2007–2009. Clin. lab. 57, 213–220. Combet, C., Jambon, M., Deléage, G., Geourjon, C., 2002. Geno3D: automatic comparative molecular modeling of protein. Bioinformatics 18, 213–214. Debbink, K., Donaldson, E.F., Lindesmith, L.C., Baric, R.S., 2012. Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity. J. Virol. 86, 1214–1226.
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