Osong Public Health and Research Perspectives

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Osong Public Health Res Perspect > 3(1); 2012 > Article

Oh, Hong, Ahn, and Min: Phylogenetic Analysis of the Rotavirus Genotypes Originated from Children < 5 Years of Age in 16 Cities in South Korea, between 2000 and 2004

Abstract

ObjectivesThe purpose of this study was to examine the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 to 2004.

MethodsWe selected 38 Group A rotavirus isolates among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in 8 hospitals representative of five provinces of South Korea between 2000 and 2004. Rotavirus P- and G-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed.

ResultsOne G1P[4] consisted G1-Id-P[4]-V; one G1P[6] consisted G1-Id-P[6]-Ia; nine G1P[8] consisted G1-Ib-P[8]-Ia (n=3), G1-Ic-P[8]-Ia (n=1), and G1-Id-P[8]-Ia (n=5); 13 G2P[4] consisted G2-V-P[4]-V; two G3P[4] consisted G3-IIId-P[4]-V; five G3P[8] consisted G3-IIId-P[8]-Ia; four G4P[6] consisted G4-Ie-P[6]-Ia; two G4P[8] consisted G4-Ie-P[8]-II; one G9P[6] consisted G9-III-P[6]-Ia.

ConclusionsA considerable amount of rotavirus genotypic diversity was detected in South Korea from 2000 to 2004. These findings are important to develop the effective vaccines and to undertake epidemiologic studies.

Keywords: human rotavirus vaccine; phylogenetic analysis; rotavirus; South Korea; viral gastroenteritis

1. IntroductionGroup A rotavirus, the most common etiologic agent of severe diarrhea in children, causes about 600,000 deaths per year [1]. Rotavirus, which is a genus belonging to the Reoviridae family, has a genome of 11 segments of double-stranded RNA surrounded by a triple-layered capsid consisting of a core, inner capsid, and outer capsid. The outer capsid is composed of two structural proteins, VP4 and VP7, which define virus G(VP4) or P(VP7) serotype specificity [2]. Although at least 15 G genotypes and 26 P genotypes are known [3-8], the most prevalent P-G combinations in humans are G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. In Korea, rotavirus is still the most common viral agent of acute diarrhea in young children. Although G1P[8] was the most prevalent strain until 1997 regardless of geographic area or season [6,9], the predominant G type strain has shifted to other genotypes including G4, G2 or G9 [10-13]. In the present study, we examined the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 – 2004 periods. As a result, we confirmed that total nine P-G genotypic isolates were identified.

2. Methods

2.1. Sample collectionA total of 38 rotavirus isolates were selected among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in eight hospitals representative for five provinces of South Korea between 2000 and 2004. Human rotaviruses were detected in 354 of 652 (54.3%) fecal samples by enzyme-linked immunosorbent assay (ELISA). G and P genotypes were detected by multiplex polymerase chain reaction (PCR) in 316 (89.3%) and 327 (92.4%) of these sample, respectively. The location of these areas was plotted on the map of South Korea is shown Figure 1.

2.2. Nucleotide sequencingHuman rotavirus (HRV) double-stranded RNA (dsRNA) was extracted using a QIAamp Viral RNA kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. The dsRNA samples

Figure 1.

Location of the five provinces used in this study. Numbers in parentheses indicate the number of case.

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were subjected to seminested multiplex Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) using conserved and type specific primers (VP7-G1, G2, G3, G4, and G9, and VP4-P[4], P[6], and P[8])[14-18]. The PCR amplicons were purified using a commercial spin column method (Qiagen GmbH, Hilden, Germany) and sequenced automatically using the ABI PRISM 3100 automated DNA sequencer (Applied Biosystems, Inc, Foster city, California, USA).

2.3. Phylogenetic analysis of nucleotide sequencesThe VP4 and VP7 sequences obtained were aligned and compared with others VP4 and VP7 sequences of rotaviruses available in the Genebank database (http://www.ncbi.nlm.nih.gov/genbank/). Phylogenetic trees of alignment were constructed using the neighbor-joining method by bootstrapping with 1000 replicates and phylogenetic distances were measured by the Tajima-Nei model [19] implements in the Molecular Evolutionary Genetics Analysis (MEGA) (http://www.megasoftware.net/) analytical package (version 5.05, Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University, University Park, Pennsylvania state, USA).

2.4. Nucleotide sequence accession numbersThe VP4 nucleotide sequences of the human rotavirus in this study were assigned accession numbers EF015885, EF077317-EF077322, EF077324-EF077342, EF077344, EF077345, and EF0773347-EF077356. The VP7 nucleotide sequences were deposited in NCBI Gen-Bank under accession numbers HQ425255-HQ425292. The referenced sequences in the GenBank database are shown in Table 1.

3. ResultsMost of the fecal samples were collected from Gyunggi province (306/652), followed by Gyeongsang province (130/652), Chungcheong province (88/652), Gangwon province (85/652), Jeolla province (43/652). These are shown in Figure 1. Among 652 stool samples, 354 (54.3%) children below 5 years of age were reactive in ELISA. G and P genotypes in parallel were detected by multiplex PCR in 314(88.7%) of these samples.During the 2000–2004 period, nine P-G combinations (N=314) are prevalent in South Korea. Overall, G2P[4](53.5%) was the most dominant, followed by G1P[8](16.6%), G1P[6](13.7%), G4P[6](7.3%), G4P[8](5.7%), G3P[8](1.3%), G3P[4](1.0%), G1P[4] (0.6%) and G9P[6] (0.3%), which are shown Table 2. G2P[4] [Gyunggi (47.0%), Gangwon (58.1%), Chungcheong (46.9%), Jeolla (36.8%), and Gyeongsang (70.4%)] was the dominant combination of genotypes. However, subdominant combinations were different in five provinces. G1P[6] and

Table 1.
Reference sequence of this study
Strains VP4 VP7 Strains VP4 VP7 Strains VP4 VP7

MMC71 EU979382 EU839912 AK26 JF304929 JF304931 D205 JF304918 JF304920
mcs/13-07 EU753965 EU753963 Wa M96825 M21843 NIV929893 DQ887060 DQ886957
GER31-08 GU979199 GU979198 GER198-08 GU393007 GU393006 GER173-08 GU392999 GU392998
GER125-08 GU392991 GU392990 GER96-08 GU392987 GU392986 GER84-08 GU392981 GU392980
GER67 GU392979 GU392978 GER15-08 GU392975 GU392974 CMH146/05 GU288635 288626
GER20-08 GU392977 GU392976 CMH032/05 GU288631 GU288623 CMH008/05 GU288627 GU288621
CMH054/05 GU288633 GU288624 GR846/86 AF161829 AF161822 NB123/86 AF161828 AF161821
NB187/86 AF161827 AF161820 GR442/86 AF161824 AF161817 Kagawa/88-349 AB039937 AB039033
Kagawa/88-104 AB039935 AB039030 Kagawa/90-544 AB039939 AB039026 Kagawa/90-554 AB039941 AB039025
Hochi/Tokyo-1980 AB039943 AB039035 Odelia/Tokyo-1984 AB039942 AB039034 YO AB008279 D86284
CAU160 EU679396 EU679390 CAU164 EU679398 EU679392 KR/Seoul-661 HM131007 HM130944
KR/Seoul-697 HM131012 HM130949 OM67 HQ127436 AJ491179 F45 U30716 AB180970
AU19 AB017917 AB018697 ST3 EF672612 EF672616 TE56 AF183869 AF183856
Py9856 EU045216 DQ015681 DS-1 AB118025 AB118023 KR/Seoul-638 HM131021 HM130965
MW333 AJ278256 AJ278257 rj5323/02 DQ857926 DQ857953 MMC6 EU839950 EU839923
Py99449 EU045222 DQ015687 BP1227/02 AJ621505 BP271/00 AJ621502
VN846/2003 EF179117 MW670 AJ302146 VN-281 DQ508167
Kor-64 U26378 PA5/90 DQ377573 CH631 AF183857
VN846/2003 EF545000 KR/Seoul-708 HM130955 KR/Seoul-710 HM130956
64SB/96 AY261341 MO D86280 T108 AF450293
CC425 AJ311738 CHW17 D86276 A131 L35055
CMH222 AY707792 Arg928 AF373918 VA75 M86833
MW4086 FJ386453 KUMS00-74 DQ478420
G1P[8] prevailed in Gyunggi province (20.1%, 17.2%), G1P[8] prevailed in Gangwon province (25.8%), G1P[8], G4P[6] and G4P[8] prevailed in Chungcheong province (18.4%, 14.3% and 14.3%), and G1P[6] prevailed in Gyeongsang province (13.6%). Also, G1P[8] and G3P[8] prevailed in Jeolla province.To examine the VP7 and VP4 nucleotide sequences for 38 isolates among 314 identified isolates, phylogenetic trees for G type (G1, G2, G3, G4, and G9) and P type [P(4), P(6), P(8)) were constructed by applying the neighbor-joining method. Sequences of VP7 and VP4 were determined from 38 representative rotaviruses, comprising the different genotypes and intra genotypic lineages detected by partial sequencing. Sequences of the representative isolates were submitted to GenBank (Table 3) and included in the phylogenetic analysis (Figures 2 and 3). In this study, the 11 G1 rotavirus isolates showed that they are a part of the lineage I and are clustered into five
Table 2.
Distribution of group A rotavirus P-G combination strains among infants and children below 5 years of age with diarrhea in five provinces of South Korea between 2000 and 2004
Provinces Number of P-G combination strains (%)

G1P[4] G1P[6] G1P[8] G2P[4] G3P[4] G3P[8] G4P[6] G4P[8] G9P[6] Total

Gyunggi 2(1.5) 27(20.1) 23(17.2) 63(47.0) 3(2.2) 0(0.0) 11(8.2)  5(3.7) 0(0.0) 134(42.7)
Gangwon 0(0.0) 1(3.2)   8(25.8) 18(58.1) 0(0.0) 0(0.0)   1(3.2)  2(6.5) 1(3.2)   31(9.9)
Chungcheong 0(0.0) 3(6.1)   9(18.4) 23(46.9) 0(0.0) 0(0.0)   7(14.3)  7(14.3) 0(0.0)   49(15.6)
Jeolla 0(0.0) 1(5.3)   6(31.6) 7(36.8) 0(0.0) 4(21.1)   1(5.3)  0(0.0) 0(0.0)   19(6.1)
Gyeongsang 0(0.0) 11(13.6)   6(7.4) 57(70.4) 0(0.0) 0(0.0)   3(3.7)  4(4.9) 0(0.0)   81(25.8)
Total 2(0.6) 43(13.7) 52(16.6) 168(53.5) 3(1.0) 4(1.3) 23(7.3) 18(5.7) 1(0.3) 314
minor lineages(Ia-Ie) in the phylogenetic analysis. The G1 rotaviruses segregated into seven major lineages (I–VII ) as reported by Arista and colleagues [20]. Most of these isolates are clustered in sub-lineage Id (n=7), followed by sublineage Ib (n=3) and sublineage Ic (n=1). Sublineage Id isolates showed 97.8%~99.2% nucleotide sequence similarity to strain GER15-08, and sublineage Ib isolates showed 99.3%~99.6%nucleotide similarity with strains VN-281.KMR267 isolates in sublineage Ic showed 99.5% nucleotide similarity to Py9856. Among eleven G1 rotavirus isolates, the three G1-Ib isolate was associated with P[8]-IIIa. One G1-Ic was associated with P[8]-IIIa and seven G1-Id isolates were associated with P[4]-V, P[6]-Ia, and P[8]-IIIa.The G2 rotaviruses segregated into five major lineages (I–V) [21]. The thirteen G2 rotavirus isolates clustered under lineage V. They showed 99.2~99.5% nucleotide similarity to strain GER84-08 from Germany. All G2-V
Table 3.
The G and P genotypes of the 38 representative rotavirus strains of this study are given. The VP7 and VP4 sequences were submitted to GenBank and the accordant accession numbers are provided in brackets. atient age, gender, city of sample collection, and the year of sample collection are indicated
Isolates G genotype (Acc.no.) P genotypes (Acc.no.) Patient age (month) Patient sex City of collection Year of collection

KMR004 G1-Id (HQ425255) P[8]-IIIa (EF077330) 11 M Seoul 2002
KMR010 G3-IIId (HQ424279) P[4]-V (EF077353) 11 F Seoul 2003
KMR012 G3-IIId (HQ425280) P[8]-IIIa (EF077336) 11 M Seoul 2003
KMR021 G2-V (HQ422566) P[4]-V (EF077344) 47 F Seoul 2002
KMR023 G1-Id (HQ425256) P[8]-IIIa (EF077350) 47 M Seoul 2002
KMR024 G2-V (HQ425267) P[4]-V (EF077345) 8 M Seoul 2002
KMR028 G4-Ie (HQ425287) P[6]-Ia (EF077334) 47 F Seoul 2003
KMR025 G4-Ie (HQ425286) P[6]-Ia (EF077348) 11 F Seoul 2002
KMR029 G2-V (HQ425268) P[4]-V (EF077354) 2 F Seoul 2003
KMR037 G2-V (HQ425269) P[4]-V (EF015885) 4 M Seoul 2000
KMR044 G1-Id (HQ425257) P[4]-V (EF077332) 6 M Seoul 2003
KMR053 G4-Ie (HQ425288) P[6]-Ia (EF077341) 23 F Incheon 2004
KMR057 G2-V (HQ425270) P[4]-V (EF077317) 31 F Incheon 2000
KMR058 G1-Ib (HQ425258) P[8]-IIIa (EF077342) 1 F Incheon 2004
KMR060 G3-IIId (HQ425281) P[4]-V (EF077347) 11 F Incheon 2004
KMR101 G1-Id (HQ425259) P[8]-IIIa (EF077338) 23 M Seoul 2003
KMR106 G2-V (HQ425271) P[4]-V (EF077356) 4 M Seoul 2000
KMR126 G2-V (HQ425272) P[4]-V (EF077340) 48 M Seoul 2004
KMR184 G2-V (HQ425273) P[4]-V (EF077318) 23 M Daegu 2000
KMR267 G1-Ic (HQ425260) P[8]-IIIa (EF077319) 23 F Daejeon 2000
KMR294 G1-Id (HQ425261) P[6]-Ia (EF077352) 1 M Masan 2000
KMR419 G1-Id (HQ425262) P[8]-IIIa (EF077320) 4 M Gwangju 2000
KMR538 G4-Ie (HQ425289) P[8]-II (EF077331) 36 M Gangneung 2000
KMR541 G4-Ie (HQ425290) P[8]-II (EF077333) 24 M Gangneung 2000
KMR547 G2-V (HQ425274) P[4]-V (EF077349) 48 F Gangneung 2000
KMR548 G2-V (HQ425275) P[4]-V (EF077351) 36 F Gangneung 2000
KMR580 G1-Ib (HQ425263) P[8]-IIIa (EF077335) 40 M Gangneung 2000
KMR720 G9-III (HQ425292) P[6]-Ia (EF077329) 1 F Gangneung 2000
KMR733 G1-Ib (HQ425264) P[8]-IIIa (EF077339) 19 M Gangneung 2001
KMR748 G1-Id (HQ425265) P[8]-IIIa (EF077325) 18 F Gwangju 2001
KMR750 G2-V (HQ425276) P[4]-V (EF077321) 3 F Busan 2001
KMR751 G3-IIId (HQ425282) P[8]-IIIa (EF077326) 19 M Gwangju 2001
KMR757 G2-V (HQ425277) P[4]-V (EF077337) 5 M Masan 2001
KMR766 G3-IIId (HQ425283) P[8]-IIIa (EF077327) 1 M Gwangju 2001
KMR769 G2-V (HQ425278) P[4]-V (EF077322) 3 M Masan 2001
KMR773 G3-IIId (HQ425284) P[8]-IIIa (EF077328) 6 M Gwangju 2001
KMR787 G3-IIId (HQ425285) P[8]-IIIa (EF077355) 3 M Gwangju 2001
KMR792 G4-Ie (HQ425291) P[6]-Ia (EF077324) 7 F Daejeon 2001
Acc.no. = GenBank Accession Number.
isolates were associated with P[4]-V. Meanwhile, the seven G3 strains are a part of the sublineage IIId and showed 99.3%~99.5% sequence similarity to strain GER198-08. The two G3-IIId were associated with P[4]-V, and five G3-IIId were associated with P[8]-IIIa. The 16 P[4]-V including G1-Id, G2-V, and G3-IIId shared more than 97.6% nucleotide similarity, and the six G4 rotavirus strains clustered in sub-lineage Ie compared to strains KUMS00-74 has 98.7-99.4% nucleotide similarity. The four G4-Ie were associated with P[6]-Ia and two G4-Ie were associated with P[8]-II. Finally, the one G9 isolate, KMR720 showed high sequence similarity to all lineage III strains (more than 98.3%) and associated with P[6]-Ia. While the five P[6]-Ia excluding G9 isolate exhibited 98.4%~98.7% nucleotide sequence similarity to TE56, G9 isolate, KMR720 exhibited 97.1% nucleotide sequence similarity to GR846/86. While P[8]-IIIa exhibited 97.6%~ 99.4% nucleotide similarity to strains CMH032/05, P[8]-II exhibited 99.4% nucleotide sequence similarity to strain Kagawa/88-104. As a result, we confirm that a total of nine P-G genotypic isolates were identified (11 P-G subgenotypes).

4. DiscussionRotaviruses have been described as a major cause of severe diarrhea among infants and young children in

Figure 2.

Phylogenetic analysis of VP7 gene nucleotide sequences of Group A rotavirus strains from Korea between 2000 and 2004. Phylogenetic trees of alignment were constructed using the neighbor-joining method by bootstrapping with 1000 replicates, and phylogenetic distances were measured by Tajima-Nei model. Only values > 50% are given. Numbers at nodes indicate the level of bootstrap support (%). Bar represents 0.05 substitutions per nucleotide position.

EPHRP1-03-36-g002
Figure 3.

Phylogenetic analysis of VP4 gene nucleotide sequences of Group A rotavirus strains from Korea between 2000 and 2004. Phylogenetic trees of alignment were constructed using the neighbor-joining method by bootstrapping with 1000 replicates, and phylogenetic distances were measured by Tajima-Nei model. Only values > 50% are given. Numbers at nodes indicate the level of bootstrap support (%). Bar represents 0.05 substitutions per nucleotide position.

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South Korea. Epidemiologic studies worldwide have revealed that five P-G combinations, G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8], have been linked to most of the cases of rotavirus diarrhea among infants and young children worldwide. In this study, we confirmed both uncommon P-G combinations [G1P(4), G1P(6), G3P(4), G4P(6), and G9P(6)] as well as most common P-G combinations [G1P(8), G2P(4), G3P(8), and G4P(8))] There are two oral live vaccines available in Korea; Rotarix (GlaxoSmithKline, Rixensart, Belgium) is a monovalent vaccine that consists of the attenuated G1P[8] human rotavirus strain RIX4414 and has been in use in Korea since 2008. Rotateq (MERCK & CO.,INC, Pennsylvania, USA) is a pentavalent vaccine which consists of five attenuated human-bovine reassortant viruses [G1 to G4 and P(8)]; G1: human W179-bovine WC3 reassortant, G2: human SC2-bovine WC3 reassortant, G3: W178-bovine WC3 reassortant, G4: human BrB-bovine WC3 reassortant, P1A[8]: human W179-bovine WC3 reassortant. It has been available in Korea since 2007. Although these vaccines are effective on most common P-G combinations, G2P[4], G1P[8], G3P[8], G4P[8], and G9P[8], those may fail to work on the other genotypes. All things considered, development of multivalent rotavirus vaccine including new ones must be required. Therefore, this study will provide useful information for the development of effective rotavirus vaccines in the future. Also, the international relationship has been limited by the geographic location in the past, but now it has expanded all over the world, and this expansion is thought to be the cause of the change in strains.

5. ConclusionConsequently, the P-G combination genotypes of this study would serve as useful information for the development of effective rotavirus vaccines. Such P-G combinational phylogenetic study will also be necessary for international epidemiologic investigation of human rotaviruses providing novel insights into the interspecies transmission processes of rotaviruses. To strengthen our opinion, we highlight the need for continued monitoring of circulating rotavirus strains for effective prevention and vaccine development strategies.

AcknowledgmentsThis study was supported in part by a grant from the KFDA Research and Development Program on Strengthening the Safety of Biological Products.

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