Prevalence and phylogenetic analysis of rotavirus genotypes in Thailand between 2007 and 2009

Infection, Genetics and Evolution 10 (2010) 537–545

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Prevalence and phylogenetic analysis of rotavirus genotypes in Thailand between 2007 and 2009 Kamonwan Khananurak a, Viboonsak Vutithanachot b, Nipat Simakachorn c, Apiradee Theamboonlers a, Voranush Chongsrisawat a, Yong Poovorawan a,* a b c

Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Chumphae Hospital, Khon Kaen, Thailand Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima, Thailand

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 November 2009 Received in revised form 3 February 2010 Accepted 5 February 2010 Available online 13 February 2010

Rotaviruses are the most common cause of severe diarrhea among infants and young children worldwide, especially in developing countries. In Thailand, rotavirus has presented a major public health problem causing severe diarrhea in infants and young children. It was responsible for about one-third of diarrheal diseases in hospitalized patients. In this study, we have analyzed the distribution and performed molecular characterization of rotaviruses circulating in infants and young children with diarrhea admitted to the city and rural hospitals in Thailand between July 2007 and May 2009. Group A human rotavirus was detected in 158 (28.4%) of 557 fecal specimens by RT-PCR. The peak incidence of infection was found in the winter months between December and March. The G1P[8] strain was identified as the most prevalent (49.4%) followed by G9P[8] (22.2%), G2P[4] (20.2%) and G3P[8] (0.6%). The uncommon strains G12P[8], G12P[6] and G3P[9] were also detected. Phylogenetic analysis of selected G and P genotypes isolated in this study was performed to compare with the reference strains from different countries. Emergence of G12 in the northern part of Thailand was observed and phylogenetic analysis demonstrated close relation between Thai isolates and strains from India. The present study reveals the recurring changing genotypes of rotavirus circulating in Thailand. The genetic association between isolates from Thailand and other countries ought to be considered with regard to local and global dissemination of rotavirus as it is crucial for prevention especially, with respect to vaccine implementation. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Rotavirus Prevalence Phylogenetic analysis Genotype

1. Introduction Rotaviruses are the most common cause of severe diarrhea among infants and young children worldwide. Each year, rotavirus-associated gastroenteritis results in an estimated 527,000 fatalities (range: 475,000–580,000) of children below 5 years of age, with the fatality rate highest in developing countries (CDC, 2008; WHO, 2004). Rotaviruses are classified as belonging to the genus Rotavirus, family Reoviridae. They are non-enveloped, icosahedral viruses displaying a wheel-shaped appearance under electron microscopy. Rotavirus particles are triple-layered and enclose a genome of 11 double-stranded RNA segments. Each double-stranded RNA segment encodes a specific protein essential for the viral replication cycle. Rotaviruses are classified into groups A–G by antigenicity of VP6. Groups A–C mostly identified in humans and

* Corresponding author. Tel.: +66 2 256 4909; fax: +66 2 256 4929. E-mail address: [email protected] (Y. Poovorawan). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.02.003

animals. Of these, group A rotaviruses are a major cause of human rotavirus-associated gastroenteritis (Estes and Kapikian, 2001). The rotavirus outer capsid consists of two neutralization antigens, VP7 and VP4, which were classified into G (glycoprotein) and P (protease-sensitive) types, respectively. Based on molecular analysis of the VP7 and VP4 genes, a total of 23 G genotypes and 31 P genotypes have been identified as previously described (Abe et al., 2009; Schumann et al., 2009; Solberg et al., 2009; Trojnar et al., 2009; Ursu et al., 2009). More than 120 different G/P combinations have been described in the literature as recently reviewed by Matthijnssens et al. (2009). The globally most common strains are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. Other G types such as G5, G6, G8, G10 and G12 in various combinations with P[4], P[6], P[8], P[9] and P[14] types have been detected in some regions of the world (Matthijnssens et al., 2008). For example, G5P[8], G8P[6] and G9P[6] were detected as the major strains in Brazil, Malawi and India, respectively, but they do not appear to be of global impact (CDC, 2008; Gentsch et al., 2005; Santos and Hoshino, 2005). In addition, the emergence of G12 was also reported in several countries (Rahman et al., 2007).

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Due to mortality rate and the large burden of rotavirus disease, development of preventive vaccines has become necessary. Two new rotavirus vaccines, Rotarix (a monovalent G1P[8] human attenuated vaccine) and RotaTeq (a pentavalent human bovine reassorted vaccine), have recently been licensed in many countries (Santosham et al., 2007). Both vaccines have proven highly efficient in reducing the hospitalization of the most common rotavirus infections, although different vaccines may vary in efficacy against various strains (Patel et al., 2008; Ward and Bernstein, 2009). Therefore, vaccine efficiency against strains that do not share any G/P combination with the vaccine strains has been questioned. Thus, surveillance of rotavirus G and P types in different areas of the world is important in order to monitor new G/ P combinations and arrive at a better understanding of the effect of strain variation on vaccine efficacy. In Thailand since 1974, various studies have demonstrated the correlation between the peak of rotavirus infection and the coldest months in winter (December, January and February) (Jiraphongsa et al., 2005; Maneekarn and Ushijima, 2000). A previous study performed on rotavirus infection in Thailand from 1982 to 1997, indicated the fluctuation of predominant rotavirus serotypes. However, on average all previous studies (since 1982–1997) revealed that G1 was the most frequently detected serotype in Thailand (Maneekarn and Ushijima, 2000). A rotavirus surveillance study in different regions of Thailand during 2001–2003 demonstrated that G9 became the most predominant genotype in 2001 and co-predominant with G2 in 2003 (Jiraphongsa et al., 2005). Research performed in Bangkok, Thailand during 2002–2004, reported the reemergence of G2 as the most predominant genotype followed by G9 and G1, respectively (Theamboonlers et al., 2005). A recent study conducted from 2004 to 2006 revealed that the prevalence of G9 was decreasing while G1 has been reemerging in high frequency (Khamrin et al., 2007b). In addition to the changing distribution of common human rotavirus strains since 1982, the uncommon human rotavirus strains such as G3P[3], G3P[9], G3P[10], G3P[19] and G12P[9] have been detected in some regions of Thailand (Khamrin et al., 2006, 2007a, 2009; Pongsuwanna et al., 2002; Theamboonlers et al., 2008). In Thailand, rotavirus represents the major cause of diarrhea in infants and children, and is responsible for about one-third of diarrheal diseases in hospitalized patients (Jiraphongsa et al., 2005; Maneekarn and Ushijima, 2000). Previous projects have investigated the change of G genotype circulation and the emergence of uncommon rotavirus strains in Thailand. In this study, we have described the distribution and molecular characterization of rotaviruses circulating in infants and children with diarrhea admitted in four hospitals of Thailand between July 2007 and May 2009.

Fig. 1. Locations of four areas in this study.

2.2. Viral RNA extraction Fecal specimens were suspended in 10% phosphate buffered saline (PBS), centrifuged at 3000 rpm for 10 min and the supernatant was collected for viral RNA extraction. RNA was extracted from 150 ml of each diluted specimen applying the guanidium-isothiocyanate method (Cha et al., 1991) as described elsewhere, and the extracted RNA was subjected to 1-step RT-PCR for the amplification of the VP7 and VP4 genes in separate reactions.

2. Materials and methods 2.3. Detection of group A rotaviruses by RT-PCR 2.1. Study population and fecal specimens Five hundred and fifty-seven stool specimens were collected from infants and children below 10 years of age with acute gastroenteritis or diarrhea admitted in four hospitals representative for four provinces of Thailand (Bangkok (BK), Khon Kaen (KK), Nakhon Ratchasima (NR) and Tak (TK)) between July 2007 and May 2009. Most of the stool samples were collected from Khon Kaen (269/557), followed by Bangkok (236/557), Nakhon Ratchasima (40/557) and Tak (12/557). The locations of each area in this study were plotted on the map of Thailand (Fig. 1). The stool specimens were collected as anonymous and subsequently stored at 70 8C until further examination. The study protocol was approved by the Ethics Committee, Ministry of Public Health and Faculty of Medicine, Chulalongkorn University, Bangkok.

The dsRNA gene segments 9 and 4 of group A rotaviruses encoding proteins VP7 and VP4, respectively, were detected by 1step RT-PCR using the Superscript III platinum One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA) in separate tubes. The reaction mixture consisted of 1 ml of extracted RNA, 10 ml of 2 reaction Mix buffer (Invitrogen, Carlsbad, CA), 2 U of superscript reverse transcriptase III platinum Taq polymerase (Invitrogen, Carlsbad, CA), 0.5 mM of each primer, and adjusted to a final volume of 25 ml with nuclease-free water. A reaction mixture without template was used as negative control. The VP7 gene was amplified with Beg9 and End9 primers (Gouvea et al., 1990) to obtain a full-length genome segment 9. The VP4 gene was amplified with Con2 and Con3 primers to produce an 876-bp fragment covering the VP8* fragment of genome segment 4

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(Gentsch et al., 1992). The RT step and PCR amplification of each gene were performed simultaneously in an Eppendorf Mastercycler personal (Eppendorf, Hamburg, Germany) under the following conditions: reverse transcription at 50 8C for 30 min, followed by initial denaturation at 95 8C for 2 min, 40 cycles at 94 8C for 1 min (denaturation), 55 8C (for Beg9 and End9) or 48 8C (for Con2 and Con3) for 1 min (primer annealing), 72 8C for 1 min 30 s (extension), and concluded by a final extension at 72 8C for 10 min. The PCR products were subjected to electrophoresis in a 2% agarose gel and stained with ethidium bromide for 20 min. The expected 1062 and 876 bp bands for the VP7 and VP4 products, respectively, were visualized on a Gel Doc 1000 UV trans-illuminator (BIO-RAD, Hercules, CA). 2.4. Nucleotide sequencing The G and P genotypes of group A rotaviruses were determined by direct sequencing of the PCR products. The PCR products were purified from agarose gel using HiYield Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience, Taipei, Taiwan) according to the manufacturer’s instructions. Direct sequencing was performed by First BASE Laboratories Sdn Bhd (Selangor Darul Ehsan, Malaysia). 2.5. Genetic characterization and phylogenetic analysis The nucleotide sequences of the VP7 and VP4 gene were analyzed using the BLAST program available in GenBank (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were prepared and multiple aligned using Clustal X version 1.8.3 (Thompson et al., 1997). The nucleotide regions used for analysis were VP7 (40–985) and VP4 (40–840). Phylogenetic trees of alignment were constructed using the neighbor-joining method by bootstrapping with 1000 replicates and phylogenetic distances were measured by Kimura’s two-parameter model implemented in the MEGA4 version 4.0 program (Tamura et al., 2007). 3. Results 3.1. Distribution of group A rotavirus From a total of 557 stool specimens collected from infants and children hospitalized with diarrhea between July 2007 and May 2009, Group A rotaviruses were detected by RT-PCR in 158 (28.4%) specimens, of which 99 (30.4%) of 326 originated from males and 59 (25.5%) of 231 from females. Age distribution of rotavirus infected patients in this study ranged from 1 month to 8 years, with the most rotavirus-positive patients (36.1%; 57/158) between 12 months and 17 months of age (Table 1).

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Table 1 Distribution of rotavirus diarrhea among infants and children hospitalized with diarrhea in Thailand between July 2007 and May 2009. Age (months)

Rotavirus screening by RT-PCR Positive

0–2 3–5 6–11 12–17 18–23 24–35 36–47 48–59 60–71 72–83 84–95 96–119 120–144 Missing data Total

Total

Negative

1 13 33 57 19 17 10 3 3 1 1 0 0 0

15 36 105 86 24 35 23 13 6 5 5 8 13 25

16 49 138 143 43 52 33 16 9 6 6 8 13 25

158

399

557

The occurrence of rotavirus diarrhea varied according to seasonal temperature. The peaks of infection were detected in the cold months between December and March (41.3%). Based on our results, rotavirus infection was the most prevalent during the winter months in Thailand (Fig. 2). Furthermore, the prevalence of rotavirus infections was investigated in urban compared to rural areas. In Bangkok, the capital of Thailand, hospitalized patients due to rotavirus diarrhea amounted to 22.0% increasing to 33.0% in rural areas (data not shown). 3.2. Distribution of G and P genotypes A total of 158 rotavirus-positive specimens were characterized for G and P genotypes by BLAST. The distribution of group A rotavirus genotypes during the study period is shown in Supplementary Table S1. The majority of G genotypes detected were G1 (49.4%) followed by G9 (22.2%), G2 (20.3%), G12 (7.0%) and G3 (1.3%). As for P genotypes, P[8] was the most prevalent (78.5%), followed by P[4] (20.3%), P[6] (0.6%) and P[9] (0.6%). With respect to the distribution of common G/P combination strains, G1P[8] (49.4%) was the most predominant, followed by G9P[8] (22.2%) and G2P[4] (20.2%), while G3P[8] (0.6%) was rarely detected. Interestingly, as for the uncommon rotavirus strains of Thailand, G12P[8] (6.3%) was detected as the fourth most prevalent strain in this study, followed by G3P[9] (0.6%) and G12P[6] (0.6%). The distribution of group A rotavirus genotypes in four areas of Thailand is shown in Table 2 and the locations of these areas were plotted on the map of Thailand as shown in Fig. 1.

Fig. 2. Seasonal pattern of rotavirus diarrhea among infants and children hospitalized with diarrhea in Thailand between July 2007 and May 2009.

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540

Table 2 Distribution of group A rotavirus G/P combination strains among infants and children with diarrhea in four areas of Thailand between July 2007 and May 2009. Area

Bangkok Khon Kaen Nakhon Ratchasima Tak

No. of G/P combination strains (%)

Total

G1P[8]

G2P[4]

G3P[8]

G3P[9]

G9P[8]

G12P[6]

G12P[8]

20(38.5) 53(65.4) 3(20.0) 2(20.0)

19(36.5) 12(14.8) 1(6.7) 0

0 1(1.2) 0 0

0 1(1.2) 0 0

12(23.1) 13(16.0) 10(66.7) 0

0 0 1(6.7) 0

1(1.9) 1(1.2) 0 8(80.0)

52 81 15 10

Fig. 3. Phylogenetic analysis of nucleotide sequence of VP7 gene of (a) G1, (b) G2, (c) G12, and VP4 gene of (d) P[8] rotavirus strains from Thailand between July 2007 and May 2009. Closed rhombi (^) indicate the G- and P-type rotavirus strains in this study. Reference sequences were obtained from the GenBank database. Percent bootstrap support was indicated by the values at each node, and the values less than 70 were omitted. The bar indicated the variation scale.

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Fig. 3. (Continued )

541

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VP7 gene. Phylogenetic trees for G1, G2, G3, G9 and G12 were constructed applying the neighbor-joining method. Reference strains of those genotypes were randomly selected from the predominant strains circulating in different regions worldwide. The VP7 nucleotide sequences of rotavirus were submitted to GenBank database and assigned accession numbers GQ996833– GQ996935. As shown in Fig. 3, The G1 rotaviruses segregated into seven major lineages (I–VII) as reported by Arista et al. (2006) (Fig. 3a). In this study, phylogenetic analysis of 49 G1 rotavirus strains showed that they clustered into two genetic lineages (I and II). Most clustered in lineage I with high nucleotide similarity (>96%) to sub-lineage Ic, which includes many strains from Asian countries. In addition, four strains of G1 rotavirus clustered into lineage II with 97.0–97.1% nucleotide similarity to strains PA11/89 and PA33/89 from Italy. The G2 rotaviruses segregated into four major lineages (I–IV) (Khamrin et al., 2007b). All G2 rotavirus strains clustered into lineage II (Fig. 3b). On the phylogenetic tree, 10 strains clustered with strain KO-2 from Japan with high nucleotide similarity (99.5– 99.8%). One strain clustered with 99.9–100% similarity with the G2 rotaviruses detected previously in Thailand in 2004. In addition, we found that three G2 rotavirus strains, isolated in 2008 formed the same cluster with strain MMC84 from Bangladesh with a nucleotide similarity of 97.8–98.8%. In this study, a total of 29 G9 rotavirus strains clustered in lineage III (Khamrin et al., 2007b) (Supplementary Fig. S1). All G9 rotavirus strains were closely related to strain CU33G9, isolated in Thailand between 2002 and 2004, with 98.8–99.3% nucleotide similarity. The phylogenetic tree of G12 rotavirus strains segregated into four major lineages as reported by Sharma et al. (2008) (Fig. 3c). In this study, all strains clustered in lineage III. Nine strains (eight from Tak and one from Bangkok) clustered with strain ISO26 and 14B2 from India with high nucleotide similarity (99.0–99.6%). In addition, two strains formed the same cluster with strain MS04107 from Thailand with a nucleotide similarity of 99.6–99.8%. Throughout the study period, the G3 rotaviruses proved to be the least prevalent. All of these were isolated from diarrheal children hospitalized in Khon Kaen. The phylogenetic tree of G3 shows that two G3 strains clustered in lineage I (Dey et al., 2009; Trinh et al., 2007) (Supplementary Fig. S2). Upon genetic distance analysis, we found that strain CU444-KK/09 was closely related to strains E885 from China with 97.9% nucleotide similarity, while CU365-KK/08 showed the highest nucleotide similarity (96.8%) to the G3 rotavirus strains previously detected in Chiang Mai, Thailand (CMH 134-04 and CMH 120-04). 3.4. Nucleotide sequence and phylogenetic analysis of VP4 genes

Fig. 3. (Continued ).

3.3. Nucleotide sequence and phylogenetic analysis of VP7 genes A total of 103 rotavirus strains with high quality sequences of at least 900-bp length were subjected to phylogenetic analysis for the

On the basis of 800-bp nucleotide sequences of 103 rotavirus VP4 genes, phylogenetic trees for P[8], P[4], P[6] and P[9] were constructed using the neighbor-joining method. Reference strains of those genotypes were randomly selected from the predominant strains circulating in different regions worldwide. The VP4 nucleotide sequences of rotavirus were submitted to GenBank database and assigned accession numbers GQ996730–GQ996832. P[8] segregated into four major lineages (I–IV) as reported by Arista et al. (2006) (Fig. 3d). All of those clustered into lineage II, which includes many strains from Asian countries with 96.8–99.2% nucleotide similarity except for CU581-BK/09. A single strain clustered into lineage IV with high nucleotide similarity (98.4%) with MW670 and OP530 from Malawi. The phylogenetic tree of eleven P[4] rotaviruses was constructed into five major lineages (I–V) (Espı´nola et al., 2008) (Supplementary Fig. S3). Of all P[4] rotaviruses in this study,

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543

Table 3 Distribution of G genotypes among infants and children hospitalized with diarrhea in Thailand during 1998–1999, 2002–2006 and 2007–2009. Year

Area

Typing method

No. of isolates tested

No. of G genotype (%) G1

G2

G3

G4

G9

G12

0

Unclassified

0

0

0

7 (16.7)

1998–1999

Bangkok

Sequence

42

35 (83.3)

2002–2004

Bangkok

Sequence

36

2 (5.6)

25 (69.4)

0

0

9 (25.0)

0

0

2004–2005

Bangkok, Buriram

Sequence

51

36 (70.6)

4 (7.8)

0

0

11 (21.6)

0

0

2005–2006

Bangkok, Buriram

Sequence

75

72 (96.0)

0

2 (2.6)

0

0

1 (1.4)

2007–2008

Bangkok, Khon Kaen, Tak, Nakhon Ratchasima Bangkok, Khon Kaen, Tak, Nakhon Ratchasima

Sequence

83

38 (45.8)

8 (9.6)

1 (1.2)

0

1 (1.2)

0

Noppornpanth et al. (2001) Theamboonlers et al. (2005) Theamboonlers et al. (2008) Theamboonlers et al. (2008) This study

Sequence

70

37 (52.9)

22 (31.4)

1 (1.4)

0

10 (14.3)

0

This study

2008–2009

0

References

CU128-BK/08 is the only strain that clustered with the strains detected previously in Thailand. Seven strains isolated in Bangkok, the capital city of Thailand, formed the same cluster with Korea strains (KMR029) with 99.7–99.9% nucleotide similarity. In the same lineage, three P[4] rotaviruses clustered with strain MMC6 from Bangladesh and H676 from Russia with 99.2–100.0% nucleotide similarity. In this study, uncommon P[6] and P[9] rotaviruses were detected. The P[6] rotaviruses, CU331-NR/08, clustered into lineage I (La´szlo´ et al., 2009) with the highest nucleotide similarity(99.4%) with strain Dhaka13-06 form Bangladesh (Supplementary Fig. S4). Phylogenetic analysis of the rare P[9] rotavirus (CU365-KK/08) showed this strain clustered with strain P[9] rotaviruses previously detected in Chiang Mai, Thailand (CMH 13404 and CMH 120-04) (Supplementary Fig. S5) with 98.0–98.2% nucleotide similarity. 3.5. Distribution of group A rotavirus strains The distribution of G genotypes throughout the period of this study is shown in Table 3. During 2007–2008, G9 was detected in large amounts (42.2%) as a co-predominant genotype with G1 (45.8%), followed by G2 (9.6%) and G3 (1.2%). During 2008–2009, G1 was the most predominant genotype (52.9%), followed by G2 (31.4%) and G3 (1.4%). Interestingly, G12 was detected at a low frequency of 1.4% during 2007–2008 with its percentage increasing to 14.3% during 2008–2009. 4. Discussion Rotaviruses have been described as a major cause of severe diarrhea among infants and young children in Thailand for the last 30 years (Maneekarn and Ushijima, 2000). Two live oral vaccines have been introduced to protect against rotavirus diarrhea as commercially available vaccines. Rotarix and RotaTeq have been licensed in Thailand in 2006 and 2008, respectively (Thai Drug Control Division, 2008). Due to the high cost of rotavirus vaccine, most vaccine introduction has been limited to the high-income population, especially in Bangkok. Based on previous studies performed between 1998 and 2005, prior to rotavirus vaccine introduction, the prevalence of hospitalized patients due to rotavirus diarrhea in Thailand was 33.3–50.6% (average 42.6%) (Jiraphongsa et al., 2005; Theamboonlers et al., 2005, 2008; Nelson et al., 2008; Intusoma et al., 2008; Khamrin et al., 2007b; Sungkapalee et al., 2006). In this study, we described the epidemiology of rotavirus infection among infants and young children with diarrhea admitted in four hospitals in Thailand between July 2007 and May 2009. A total of 557 stool

0 35 (42.2)

0

specimens were tested for rotavirus using RT-PCR and 28.4% were positive. The majority of infected patients were between 12 and 17 months of age. Approximately 30.1% of children below the age of 5 years showed evidence of rotavirus infection. The peak of rotavirus infections was found between January and March which are the coldest months of the year in Thailand. This finding supported the seasonal pattern of rotaviruses in Thailand as described in the previous surveillance report (Jiraphongsa et al., 2005). Furthermore, the prevalence of rotavirus infections was investigated in urban compared to rural areas. The difference of rotavirus prevalence between urban and rural areas may result from poor sanitation and poor access to vaccine introductions in those areas. The VP7 and VP4 genes were analyzed to identify G and P genotypes of rotaviruses. In this study, most genotype identifications revealed the globally common rotavirus genotypes (G1P[8], G2P[4] and G9P[8]). A changing pattern of rotavirus genotypes was observed. Based on previous research, G1 had been reported with high prevalence as a predominant strain between 1998 and 1999 (Noppornpanth et al., 2001). Although predominance of G2 was detected between 2002 and 2004 (Theamboonlers et al., 2005), G1 has still remained an important strain (Theamboonlers et al., 2008). In this study, the co-predominance of G1 and other G genotypes was described. We found co-predominance of G1 and G9 between 2007 and 2008. G9 strains had decreased and disappeared in 2009 while G2 strains increased and became copredominant with G1 between 2008 and 2009 (Table 3). In addition, the circulation of G2P[4] rotaviruses was observed. Interestingly, we detected G2P[4] with high frequency in Bangkok and with low frequency in a rural area (Khon Kean) as shown in Table 2. The high prevalence of G2P[4] in areas with access to the vaccine such as Bangkok supported the data from previous research that the vaccine may be less efficient against G2P[4] rotaviruses (Ruiz-Palacios et al., 2006; Ward and Bernstein, 2009). However, since G2 has predominantly been detected from 2002 to 2004 as shown in Table 3, temporary circulation of G2P[4] in the population should be considered. In this study, G3P[9], G12P[6] and G12P[8], the uncommon rotavirus strains, were also detected. Most uncommon strains were isolated from hospitalized patients in rural areas. In Thailand, the G3P[9] rotaviruses were first detected in Chiang Mai, Thailand in 2004. Those strains were documented to be closely related to feline rotavirus strains (Khamrin et al., 2007a). The first detection of G12 rotaviruses was reported in the Philippines in 1987, and since then, the emergence of G12 has been reported in many countries around the world as recently reviewed by Matthijnssens et al. (2008). In addition, G12 was detected with high prevalence in India and has become an important strain in that country since 2005 (Sharma et al., 2008). In Thailand, the first

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detection of G12 was reported in 1998 and found in combination with P[9] (Pongsuwanna et al., 2002). In this study, we described the emergence of G12 in hospitals located in four areas of Thailand. Interestingly, most G12 strains were detected in Tak, the province located 426 km from Bangkok. The western edge of the province has a long border with Myanmar. Hence, it is possible that G12 was imported from South Asia to Thailand by immigrants. Based on phylogenetic analysis, G12 rotaviruses clustered into lineage III which had the highest nucleotide similarity with Indian G12 rotavirus strains. The predominant members of lineage III were G12 rotaviruses isolated from India, Bangladesh and Nepal. In conclusion, the present study has shown the difference of rotavirus distribution among hospitalized children with diarrhea in urban and rural areas of Thailand between July 2007 and May 2009. The results demonstrated the co-predominance of G1P[8] and G2P[4] in urban areas as opposed to rural areas where G2P[4] was hardly detected. In addition, emergence of G12 strains in Thailand was also reported. It remains to be seen if the relative frequency of other G and P genotypes which are not covered by current vaccines, will increase in prevalence once the rotavirus vaccines have been routinely administered to infants in Thailand. Detection of potential vaccine breakthrough strains may indicate that additional specificities should be considered to be included in the next generation of rotavirus vaccines. Thus, surveillance of rotavirus epidemiology is important in order to monitor new rotavirus strains and arrive at a better understanding of the effect of strain variation on vaccine efficacy. Acknowledgments We are grateful to the Center of Excellence in Clinical Virology Fund, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society. Commission on Higher Education, Ministry of Education, Chulalongkorn University, CU Centernary Academic Development Project. Also, we would like to express our gratitude to the entire staff of the Center of Excellence in Viral Hepatitis, Chulalongkorn University and the staff from the Chumphae Hospital, Maharat Nakhon Ratchasima Hospital and Umphang Hospital for the specimen collection. We also would like to thank Ms. Petra Hirsch for reviewing the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2010.02.003. References Abe, M., Ito, N., Morikawa, S., Takasu, M., Murase, T., Kawashima, T., Kawai, Y., Kohara, J., Sugiyama, M., 2009. Molecular epidemiology of rotaviruses among healthy calves in Japan: isolation of a novel bovine rotavirus bearing new P and G genotypes. Virus Res. 144 (1–2), 250–257. Arista, S., Giammanco, G.M., De Grazia, S., Ramirez, S., Lo Biundo, C., Colomba, C., Cascio, A., Martella, V., 2006. Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population. J. Virol. 80 (21), 10724– 10733. Centers for Disease Control Prevention (CDC), 2008. Rotavirus surveillanceworldwide, 2001–2008. MMWR Morb. Mortal. Wkly. Rep. 57 (46), 1255– 1257. Cha, T.A., Kolberg, J., Irvine, B., Stempien, M., Beall, E., Yano, M., Choo, Q.L., Houghton, M., Kuo, G., Han, J.H., et al., 1991. Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma. J. Clin. Microbiol. 29 (11), 2528–2534. Dey, S.K., Thongprachum, A., Islam, A.R., Phan, G.T., Rahman, M., Mizuguchi, M., Okitsu, S., Ushijima, H., 2009. Molecular analysis of G3 rotavirus among infants and children in Dhaka City, Bangladesh after 1993. Infect. Genet. Evol. 9 (5), 983–986. Estes, M.K., Kapikian, A.Z., 2001. Rotaviruses. In: Knipe, D.M., Howley, P.M., Griffin, D.E. (Eds.), Virology. 4th ed. Lippincott Williams and Wilkins, Philadelphia, pp. 1787–1833.

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