Infection, Genetics and Evolution 12 (2012) 1381–1391

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Genotypic linkages of gene segments of rotaviruses circulating in pediatricpatients with acute gastroenteritis in Thailand

Natthawan Chaimongkol a, Pattara Khamrin a, Rungnapa Malasao a, Aksara Thongprachum b,Hiroshi Ushijima c, Niwat Maneekarn a,⇑a Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailandb Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japanc Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan

a r t i c l e i n f o

Article history:Received 6 March 2012Received in revised form 11 April 2012Accepted 14 April 2012Available online 4 May 2012

Keywords:RotavirusEpidemiologyGenotypic linkageThailand

1567-1348/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.meegid.2012.04.015

⇑ Corresponding author. Address: Department oMedicine, Chiang Mai University, Chiang Mai 50200, Tfax: +66 53 217144.

E-mail address: nmaneeka@med.cmu.ac.th (N. Ma

a b s t r a c t

Rotavirus is a major cause of morbidity and mortality of infants and young children with diarrheathroughout the world. In Thailand, extensive studies of rotavirus infections have been reported continu-ally and rotavirus diarrhea remains a common illness. To monitor the epidemiological situation of rota-virus in Chiang Mai, Thailand, surveillance of rotavirus circulating in pediatric patients was conducted. Atotal of 160 fecal specimens collected from children hospitalized with diarrhea were tested for rotavirus-es groups A, B, and C by RT-PCR and their genotypes were identified by multiplex PCR and nucleotidesequencing. Group A rotavirus was detected at 29.4% but none of group B and C was found in this study.Molecular characterizations of G- and P-genotypes revealed three different G–P combinations, G1P[8]was the most predominant genotype with the prevalence of 72.3% followed by G2P[4] at 19.2%, andG3P[8] at 8.5%. Phylogenetic analyses of VP7 and VP4 genes of the representative strains detected inthe present study, G1, G2, G3, and P[4] and P[8], respectively, revealed that G1 belonged to G1–Ic andG1–II, G2 belonged to G2–II, and G3 belonged to G3–III–S4 lineages while P[4] and P[8] were identifiedas P[4]–V and P[8]–III lineages. Analyses of VP6, NSP4, and NSP5 genes demonstrated that these repre-sentative strains belonged to genotypes I1 and I2, E1 and E2, and H1 and H2, respectively. Analyzingthe association of G- and P-genotypes with I, E, H genotypes revealed unique patterns of genotypic link-age. The G1P[8] and G3P[8] were intimately linked with I1, E1, H1 genotypes and displayed the geneticfeatures of G1–P[8]–I1–E1–H1 and G3–P[8]–I1–E1–H1, respectively, while G2P[4] was closely linked toI2, E2, H2 genotypes and showed the genetic pattern of G2–P[4]–I2–E2–H2. This study provides epidemi-ological information and insight into the genetic background of rotaviruses circulating in pediatricpatients in Chiang Mai, Thailand.

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1. Introduction

Rotavirus is the most common and remains the major cause ofsevere diarrhea in infants and young children worldwide. It has beenestimated recently that 453,000 children with the age under 5 yearsdie from rotavirus diarrhea each year and more than half of deathsoccur in developing countries in Africa and Asia (Tate et al., 2012).

Rotavirus is a non-enveloped virus containing 11 segments ofdouble-stranded RNA genome which encode six structural proteins(VPs), and six non-structural proteins (NSPs). The two outer-layerproteins VP7 and VP4 form the basis of the dual classification sys-tem of group A rotavirus into G and P genotypes (Estes and Kapikian,

ll rights reserved.

f Microbiology, Faculty ofhailand. Tel.: +66 53 945332;

neekarn).

2007). To date, 27G-genotypes and 35P-genotypes have beenreported (Matthijnssens et al., 2011). Globally, human infectionwith group A rotavirus mainly cause by G1, G2, G3, G4, and G9 incombination with P[4], P[6], and P[8] to form various G–P combina-tions (Santos and Hoshino, 2005). In addition, under the novel clas-sification system proposed by Rotavirus Classification WorkingGroup (RCWG) to define the VP6, NSP4, and NSP5 genes as I, E,and H genotypes, respectively, and at least 16I, 14E, and 11H geno-types have been identified (Matthijnssens et al., 2008, 2011).

In Thailand, numerous studies of epidemiological surveillanceof rotavirus infection and distribution of rotavirus genotypes dur-ing 1977–1997 have been reviewed (Maneekarn and Ushijima,2000). Most studies reported the prevalence of rotavirus infectionin Thailand within the range of 27–34% and G1 was the most pre-dominant genotype during that period, followed by G2, G4, and G3.The studies in Chiang Mai city during 2000–2001 reported theG9P[8] with exceptionally high prevalence of 91.6% followed by

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G3P[8] (4.7%), G2P[4] (2.8%), and G3P[3] (0.9%) genotypes(Khamrin et al., 2006). In addition, during 2002–2004 the preva-lence of G9P[8] declined to 40.8% followed by G1P[8] (33.7%),G2P[4] (23.5%), and G3P[9] (2%) (Khamrin et al., 2007). In 2005,G1P[8] was re-emerged with high prevalent rate of 62.8% followedby G2P[4] (27.9%), G9P[8] (4.7%), G3P[8] and G3P[10] each of 2.3%(Khamrin et al., 2010). Analyzing the G- and P-genotypes of thesestrains in association with their I, E, and H genotypes revealedunique patterns of genotypic linkages. The G1P[8], G3P[8], andG9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1,and H1, respectively, while G2P[4] was exclusively linked to I2,E2, and H2 (Khamrin et al., 2010).

In an attempt to provide more information on epidemiology,genotypic distribution, and evolutionary characteristics of rotavi-rus in Chiang Mai city, Thailand, the followed-up epidemiologicalsurveillance was carried out in children hospitalized with acutegastroenteritis in 2007. The G- and P-genotypes of rotavirusesand the patterns of their genotypic linkages with I, E, and Hgenotypes were described.

Table 1Distribution of G–P genotype combinations in group A rotaviruses detected frompediatric patients with acute gastroenteritis.

G-genotypes P-genotypes Total (%)

P[4] P[8]

G1 34 72.3G2 9 19.2G3 4 8.5

Total 19.1 80.9 100

2. Materials and methods

2.1. Specimen collection

One hundred and sixty stool samples were collected frompediatric patients at the age of younger than 5 years, who wereadmitted to the McCormick Hospital, Chiang Mai, Thailand with aclinical diagnosis of acute gastroenteritis throughout the year2007. The specimens were stored at �20 �C until a batch-testingwas performed.

2.2. RNA extraction and RT reaction

The viral RNA genome was extracted from 10% stool suspensionusing QIAamp viral RNA Mini Kit (QIAGEN, Hamburg, Germany)and reverse transcription (RT) reaction was then performedaccording to the manufacturer’s instruction (Fermentas, Vilnius,Lithuania). Briefly, 10 lL of viral genome were added to 1 lL of50% dimethyl sulfoxide (DMSO) before heating at 95 �C for 5 min.Then, reverse transcription (RT) reaction using random hexamerprimers (Takara, Shiga, Japan) was carried out at 42 �C for 1 h, fol-lowed by 70 �C for 10 min and then immediately chilled on ice.

2.3. Detection of group A, B, and C rotaviruses by RT-multiplex PCR

The presence of group A, B, and C rotaviruses were detected byRT-multiplex PCR using the protocol described previously (Yanet al., 2004). The presence of each virus was assigned based onthe detection of the expected fragment length of PCR amplicons,395 bp (group A rotavirus), 814 bp (group B rotavirus), and351 bp (group C rotavirus).

2.4. G- and P-genotyping of group A rotavirus by multiplex PCR

To characterize G- and P-genotypes of group A rotavirus, thecDNA of rotavirus derived from positive samples was used as atemplate in the multiplex PCR reaction using consensus and type-specific primers as described previously by Gouvea et al. (1990)Gentsch et al. (1992). In the first round amplification of VP7 andVP4 genes, a 1062 bp-VP7 gene fragment was generated by usinga primer pair Beg9 (forward) and End9 (reverse) while a 877bp-partial VP4 gene fragment was generated by using a consensusprimers Con2 (reverse) and Con3 (forward). In the second roundamplification, the G genotyping was performed using a pool offorward primer (BT1, CT2, ET3, DT4, AT8, and FT9) in combination

with a reverse primer (End9) for amplification of the VP7 genes ofG1–G4, G8, and G9, respectively. The P genotype was identifiedby using a forward primer Con3 and a pool of reverse primers(1T-1, 2T-1, 3T-1, 4T-1, 5T-1, and ND2) for amplification of theVP4 genes of P[8], P[4], P[6], P[9], P[10] and P[11], respectively.All PCR products were evaluated by electrophoresis in 1.5% agarosegel containing 0.5 lg/mL ethidium bromide and visualized underUV light. The virus of which their genotypes could not be deter-mined by multiplex PCR was subjected to be identified by nucleo-tide sequencing.

2.5. Sequence and phylogenetic analyses of VP4, VP6, VP7, NSP4, andNSP5 genes

The representative strains of rotaviruses detected in 2007 wererandomly selected, based on different genotypes distributed in eachmonths throughout the year, for nucleotide sequencing and phylo-genetic analysis. The VP7, VP4, and VP6 genes were amplified usingconsensus primers Beg9/End9 (Gouvea et al., 1990), Con3/Con2(Gentsch et al.,1992), and VP6-F/VP6-R (Shen et al., 1994), respec-tively. In addition, the amplifications of NSP4 and NSP5 genes werecarried out using primers NSP4-1a and NSP4-2b (Kudo et al., 2001),and primers GEN-NSP5F and GEN–NSP5R (Matthijnssens et al.,2006), respectively. The PCR products of the VP4, VP6, VP7, NSP4,and NSP5 genes were then purified using QIAquick Gel ExtractionKit (QIAGEN, Germany) and sequenced using BigDye� Terminatorv3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The nucleo-tide sequences of the representative strains obtained from thisstudy were compared with those of the reference strains availablein the NCBI GenBank database using BLAST server (www.ncbi.nlm.nih.gov/blast) and analyzed using CLUSTAL X program for multiplesequence alignment and MEGA 4.0 (Tamura et al., 2007) for phylo-genetic tree construction.

2.6. Nucleotide sequence accession numbers

The nucleotide sequences of group A rotaviruses described in thepresent study have been deposited in the GenBank database. Theaccession numbers are as follows: VP7 (JQ043266–JQ043278);VP4 (JQ043279–JQ043291); VP6 (JQ043292–JQ043299); NSP4(JQ043300–JQ043312); NSP5 (JQ043313–JQ043323).

3. Results

3.1. Distribution of G- and P-genotypes of group A rotavirus

The G- and P-genotypes of 47 strains of group A rotavirusesidentified by RT- multiplex PCR and nucleotide sequencing are pre-sented in Table 1. Three different G–P combinations, G1P[8],G2P[4], and G3P[8] were detected in this study. All of G1 and G3rotavirus strains were found to associate exclusively with P[8],while G2 strain was found to associate with P[4]. Among the rota-virus strains detected, G1P[8] was the most predominant strainwith the prevalence of 72.3% (34 of 47), followed by G2P[4], and

(a)

Fig. 1. Phylogenetic analysis of VP7 nucleotide sequences of (a) G1, (b) G2, and (c) G3 of rotavirus representative strains circulating in Chiang Mai in 2007. The tree wasconstructed based on the neighbor–joining method using the MEGA 4.0 program. Bootstrap values >80% are shown at the branch nodes. The lineages and sub-lineages areindicated at the right side. The strains from the present study are indicated in bold with a solid circle. Name of each rotavirus strain is described with accession number andcountry or origin of animals.

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G3P[8], which accounted for 19.2% (9 of 47), and 8.5% (4 of 47),respectively.

3.2. Phylogenetic analysis of VP7 gene

The VP7 nucleotide sequences of the representative strains ofG1, G2, and G3 genotypes detected in this study were analyzed phy-logenetically together with those of reference strains reportedworldwide (Fig. 1). Within G1 strains, the VP7 nucleotide sequencesof six representative G1 strains (CMH042/07, CMH050/07,CMH056/07, CMH060/07, CMH090/07, and CMH110/07) showed93.2–99.7% identity to each other. Phylogenetic analysis (Fig. 1a)

revealed that all of these G1 strains were found to cluster intotwo different lineages. Two strains (CMH042/07 and CMH056/07)belonged to sub-lineage c of lineage I which clustered exclusivelywith Germany strain GER15-08 with 99% nucleotide sequence iden-tity. However, these two strains were less closely related to thestrains isolated previously in the same region of Chiang Mai cityin 2004, which clustered in distinct branches of lineage Ic, than tothe G1 strains isolated from other parts of Thailand during the per-iod of 2007–2009. In addition, the other four strains of G1(CMH050/07, CMH060/07, CMH090/07, and CMH110/07) fell intolineage II and showed genetically closely related with other Thaistrains reported previously during 2005–2009.

(b)

Fig. 1 (continued)

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The VP7 nucleotide sequences of five strains of G2 showed98.3–99.9% similarity among themselves and all strains belongedto lineage II (Fig. 1b). Most of these strains (CMH028/07,CMH030/07, CMH043/07, and CMH049/07) showed their geneticbackgrounds closely related to the G2 strains previously identifiedfrom Thailand, Brazil, and Bangladesh (97–99%), except for onestrain, CMH070/07. The CMH070/07, however, showed nucleotidesequence identity more than 99% with the strains ISO-8 from Indiaand Dhaka2-04/2004 from Bangladesh and formed a separatecluster.

Two strains of G3 (CMH014/07 and CMH055/07) showed VP7sequence of 99.4% identity to each other and both of them be-longed to lineage III, sub-lineage S4, which clustered together withseveral G3–III-S4 strains from China, Vietnam, Malaysia, Sri Lanka,Ireland, Russia, and Thailand (Fig. 1c).

3.3. Phylogenetic analysis of VP4 gene

For VP4 gene, 13 representative strains of P[4] and P[8] wereanalyzed in this study. Strains of P[4] (CMH028/07, CMH030/07,

(c)

Fig. 1 (continued)

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CMH043/07, CMH049/07, and CMH070/07) shared 98.9–100% se-quence identity to each other. Phylogenetic tree of P[4] (Fig. 2a)demonstrated that all five strains of P[4] belonged to lineage V. Itis interesting to note that most strains (4 of 5) were more closelyrelated (98–99%) with strains detected from different countries,Russia and India, than those from Thailand while only one strainfound to be clustered with Thai strains located in a separatebranch.

The strains of P[8] (CMH014/07, CMH042/07, CMH050/07,CMH055/07, CMH056/07, CMH060/07, CMH090/07, andCMH110/07) showed 96.1–99.7% identity to each other and allstrains belonged to lineage III (Fig. 2b). It was found that P[8]strains detected in this study formed their own cluster and com-bined with specific G-genotypes. Four P[8] strains CMH050/07,CMH060/07, CMH090/07, and CMH110/07 were combined withG1–II, 2 P[8] strains CMH042/07 and CMH056/07 were combined

(a)

Fig. 2. Phylogenetic analysis of VP4 nucleotide sequences of (a) P[4], and (b) P[8] of representative strains circulating in Chiang Mai in 2007. The tree was constructed basedon the neighbor–joining method using the MEGA 4.0 program. Bootstrap values >80% are shown at the branch nodes. The lineages and sub-lineages are indicated at the rightside. The strains from the present study are indicated in bold with a solid circle. Name of each rotavirus strain is described with accession number and country or origin ofanimals.

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with G1–Ic while the other 2 P[8] CMH014/07 and CMH055/07were combined with G3–III–S4.

3.4. Phylogenetic analysis of VP6 gene

Among 13 representative strains of rotavirus detected in thisstudy, eight strains could be successfully obtained theirfull-length VP6 nucleotide sequences. Phylogenetic tree showedthat these strains fell into two distinct I genotypes, I1 and I2(Fig. 3). Within I1 genotype, two strains (CMH060/07 andCMH110/07) showed the highest relatedness with the strain Hos-okawa from Japan (98%), while the other two strains (CMH014/07and CMH042/07) were closely related with Thai strains. Addition-ally, the other four strains of I2 genotype (CMH028/07, CMH030/07, CMH049/07, and CMH070/07) showed high degree of VP6sequence identity (99%) to each other and also showed more than97% identity to rotavirus strains isolated previously in Chiang Maicity.

3.5. Phylogenetic analysis of NSP4 and NSP5 genes

Phylogenetic analysis of NSP4 nucleotide sequences of 13 repre-sentative strains revealed two E genotypes, E1 and E2. Most of rota-virus strains (CMH014/07, CMH042/07, CMH050/07, CMH055/07,CMH056/07, CMH060/07, CMH090/07, and CMH110/07) belongedto E1 genotype and the others (CMH028/07, CMH030/07, CMH043/07, CMH049/07, and CMH070/07) were classified as E2 genotype(Fig. 4). Likewise, 11 strains of NSP5 sequences identified in thisstudy, eight strains (CMH014/07, CMH042/07, CMH050/07,CMH055/07, CMH056/07, CMH060/07, CMH090/07, and CMH110/07) belonged to H1 genotype and the rests (CMH030/07, CMH043/07, and CMH049/07) belonged to H2 genotype (Fig. 5).

3.6. Genotypic linkages of VP7, VP4, VP6, NSP4, and NSP5 genes ofgroup A rotavirus

According to a novel classification system of group A rotavirus,multiple genes, VP7 (G), VP4 (P), VP6 (I), NSP4 (E), and NSP5 (H),

(b)

Fig. 2 (continued)

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were analyzed for their genotypic linkages. In the present study,three different G–P combinations were identified; G1P[8], G2P[4],and G3P[8], and contained three conserved genotypic linkages pat-terns (Table 2). The strains of G1P[8] and G3P[8] carried the I1, E1,H1 genotypes which displayed their genotype patterns of G1–P[8]–I1–E1–H1 and G3–P[8]–I1–E1–H1, respectively. In contrast, theG2P[4] strains were intimately linked to the I2, E2, H2 genotypesand showed the genotype pattern of G2–P[4]–I2–E2–H2.

4. Discussion

In the present study, the overall picture of epidemiological sit-uation of rotavirus circulating in Chiang Mai, Thailand in 2007 is

presented. Rotavirus was detected at 29.4% which was relativelylower than those reported previously in Chiang Mai at 34% in2000–2001, 37.3% in 2002–2004, but with similar rate of 2005 at29.3%. However, the data is still in the range of the prevalence ofrotavirus infection in children hospitalized with acute gastroenter-itis in Chiang Mai at the range of 27–37.3% (Maneekarn and Ushij-ima, 2000; Khamrin et al., 2006, 2007).

A changing distribution of rotavirus has been reported fromseveral countries, although variations from country to countrywere observed. The present study, investigated genotypic distribu-tion of rotaviruses in Chiang Mai, Thailand in 2007 showing the cir-culation of G1, G2, G3, P[4], and P[8] in this area. Rotavirus G1 wasfound to combine only with P[8] and the G1P[8] continues to be

Fig. 3. Phylogenetic analysis of VP6 nucleotide sequences of rotavirus representative strains circulating in Chiang Mai in 2007. The tree was constructed based on theneighbor–joining method using the MEGA 4.0 program. Bootstrap values >80% are shown at the branch nodes. The lineages and sub-lineages are indicated at the right side.The strains from the present study are indicated in bold with a solid circle. Name of each rotavirus strain is described with accession number and country or origin of animals.

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the most common genotype, followed by G2P[4], and G3P[8].These results indicate that G1 remains to be an important strainthat affects pediatric population in Chiang Mai, Thailand since2004 (Khamrin et al., 2007). Sequence analysis of VP7 gene re-vealed that most of G1 detected in this study were classified inthe lineage II which is more closely related to G1 reported fromother regions of Thailand (2007–2009) than those reported previ-ously in Chiang Mai (2004), which fell mostly into sub-lineage Ic.In addition, some of G1 that clustered in sub-lineage Ic showedthe closest genetic relationship with the strain from Germany.

G2 has also been considered to be the common genotype amongrotavirus strains reported worldwide but the prevalent rate in

Chiang Mai, Thailand in 2007 is much lower than those of G1 de-tected in the same epidemic season. However, recent studies con-ducted in other regions of Thailand (Bangkok, Khon Kaen, Tak,Nakhon Rachasima) demonstrated that the prevalent rate of G2strain increased and became co-predominant with G1 during2008 and 2009 (Khananurak et al., 2010). Phylogenetic analysisof G2 detected in this study demonstrated that their VP7 gene seg-ments were closely related to the viruses isolated in Asian coun-tries, including Thailand, Bangladesh, India, and Taiwan andgrouped in the same cluster of lineage II. The finding implies thatG2 rotavirus has been prevailing and spreading in a wide area inAsia. In addition, G2 is seen exclusively in combination with P[4]

Fig. 4. Phylogenetic analysis of NSP4 nucleotide sequences of rotavirus representative strains circulating in Chiang Mai in 2007. The tree was constructed based on theneighbor–joining method using the MEGA 4.0 program. Bootstrap values >80% are shown at the branch nodes. The lineages and sub-lineages are indicated at the right side.The strains from the present study are indicated in bold with a solid circle. Name of each rotavirus strain is described with accession number and country or origin of animals.

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genotype and the representative P[4] strains were identified inlineage V. Of note, only one P[4] strain (CMH070/07) was phyloge-netically closely related to the strains from Thailand, suggestingthat most P[4] rotavirus strains found in this surveillance mightnot have originated from the strains circulating in the area. It is,therefore, interesting to perform the follow-up study to monitoringthe trend and evolution of G2P[4] in Chiang Mai in the next epi-demic seasons.

G3 and G4 in Thailand have been reported as a lesser frequentgenotypes compared to G1 and G2 (Maneekarn and Ushijima,2000; Khamrin et al., 2006, 2007). This situation has been con-firmed in the present study that G3 was detected as the third pre-dominant genotype with a far lesser prevalence than G1 and G2 in

Chiang Mai, Thailand. Phylogenetic analysis revealed that all G3detected in the present study belonged to lineage III, sub-lineageS4 and showed a close genetic relationship with strains from Thai-land, China, Vietnam, Japan, Russia, Sri Lanka, Malasia, and Ireland.Also, all of G3 strains were found to combine only with P[8]. Epide-miological studies of G4 genotype in Songkhla and Tak provinces,Thailand revealed a high prevalence of G4 only in 2001–2002 epi-demic season but was rarely detected in the year 2000, 2003–2004,and finally disappeared from 2005 to 2009 (Pongsuwannna et al.,2010; Khananurak et al., 2010). In Chiang Mai, G4 was first de-tected with low prevalence of 3% during 1995–1996 (Zhou et al.,2001), and disappeared completely since 1997, and remains unde-tectable in this study.

Fig. 5. Phylogenetic analysis of NSP5 nucleotide sequences of rotavirus representative strains circulating in Chiang Mai in 2007. The tree was constructed based on theneighbor–joining method using the MEGA 4.0 program. Bootstrap values >80% are shown at the branch nodes. The lineages and sub-lineages are indicated at the right side.The strains from the present study are indicated in bold with a solid circle. Name of each rotavirus strain is described with accession number and country or origin of animals.

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The present study also investigated the genetic characteristicsof VP6, NSP4, and NSP5 genes of group A rotaviruses. Several stud-ies have demonstrated the evidence for the genetic linkagebetween NSP4 and VP6 genes of human rotavirus strains(Iturriza-Gomara et al., 2003;; Araujo et al., 2007; Tavares Tdeet al., 2008; Khamrin et al., 2010). The linkage between NSP4 andVP6 genes has also been demonstrated in the present study thatNSP4 genotype E1 is consistently linked to VP6 genotype I1, andNSP5 genotype H1, while NSP4 genotype E2 is consistently linkedto VP6 genotype I2, and NSP5 genotype H2. Moreover, sequences ofVP6, NSP4 and NSP5 genes of human rotavirus detected in thisstudy were found to cluster in a distinct branch separated frombranches of animal rotaviruses but clustered with those of human

rotaviruses particularly with strains that have been emerged previ-ously in human in Chiang Mai in 2005. These observations confirmthe species-specific nature of rotaviruses.

Analysis of possible genotypic linkages of VP7, VP4, VP6, NSP4,and NSP5 genes of rotaviruses detected in the present studyrevealed three possible genotypic linkages includings G1–P[8]–I1–E1–H1, G2–P[4] I2–E2–H2, and G3–P[8]–I1–E1–H1. The dataare totally agree with those reported previously in 2005 in ChiangMai, Thailand (Khamrin et al., 2010). This possible genotypic link-age patterns observed in our studies of rotaviruses in Thailand mayneed further more extensive study in other countries to see if thesepatterns of genetic linkages are also true in other countries andcould be generalized for rotaviruses in other geographical area.

Table 2Genotypic linkage of VP7, VP4, VP6, NSP4, and NSP5 gene segments of group Arotaviruses circulating in Chiang Mai during January to December, 2007.

Samples/strains Genotypes

VP7 VP4 VP6 NSP4 NSP5

G1P[8]CMH042/07 G1 P[8] I1 E1 H1CMH050/07 G1 P[8] I1a E1 H1CMH056/07 G1 P[8] I1a E1 H1CMH060/07 G1 P[8] I1 E1 H1CMH090/07 G1 P[8] I1a E1 H1CMH110/07 G1 P[8] I1 E1 H1

G2P[4]CMH028/07 G2 P[4] I2 E2 –b

CMH030/07 G2 P[4] I2 E2 H2CMH043/07 G2 P[4] I2a E2 H2CMH049/07 G2 P[4] I2 E2 H2CMH070/07 G2 P[4] I2 E2 –b

G3P[8]CMH014/07 G3 P[8] I1 E1 H1CMH055/07 G3 P[8] I1a E1 H1

a Rotavirus strains that full-length VP6 nucleotide sequences could not beobtained but 351–480 bp were amplified by VP6 genogroup-specific primers (Shenet al., 1994), and the nucleotide sequences of these strains were identified as Igenotype by comparing with the reference strains available in the NCBI GenBankdatabase.

b Nucleotide sequences were not obtained.

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Taken together, the present study describes the overall pictureof rotavirus infection and provides data insight into the diversityand evolution of rotaviruses by investigating not only the geno-types but also elucidating the lineages and genotypic linkages ofVP7, VP4, VP6, NSP4, and NSP5 genes of rotavirus strains circulatingin Chiang Mai, Thailand.

Acknowledgements

This research was supported by the Grants from the Faculty ofMedicine, Chiang Mai University (Medical research fund) andGraduate School of Chiang Mai University, Chiang Mai, Thailand.

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