Journal of Bacteriology and Virology 2011. Vol. 41, No. 4 p.287 293 http://dx.doi.org/10.4167/jbv.2011.41.4.287 Original Article Genetic Characterization of Norovirus GII.3 Circulating in Korea JongIm Kim 1 and KwiSung Park 2 * 1 Department of Nursing, Woosong Information College, Daejeon; 2 Chungcheongnam-Do Institute of Health and Environmental Research, Daejeon, Korea Noroviruses (NoV) are the major viral pathogen that causes epidemic acute gastroenteritis and outbreaks of food-borne illness. The major genotypes responsible for the epidemics of NoV are GII.4 and GII.3. However, most studies of NoV have been associated with GII.4 genotype and only few studies have been done with GII.3 genotype. Here, we selected 18 GII.3 strains, which recently circulated in Korea, and determined the partial sequences of the capsid gene. Phylogenetic analysis comparing these sequences with 29 reference strains from the GenBank database was performed using the MEGA program. All NoV GII.3 strains formed 2 distinct genetic lineages and variant groups. Lineage A showed 94.1~97.6%, 90.2~94.6% nucleotide identities from lineage B and variant group, respectively. Lineage B showed 90.2~94.6% nucleotide identities from variant group. These different genetic lineages based on the phylogenetic analysis of capsid sequences imply that the circulating Korean NoV GII.3 strains have potential antigenic variation. Key Words: Gastroenteritis, Norovirus GII.3, Phylogenetic analysis, Sequence comparison 서론 바이러스성급성위장관염의주요병원체로는로타바이러스, 노로바이러스, 아데노바이러스, 아스트로바이러스등이있다 (1). 이중많은부분을차지하는것이로타바이러스와노로바이러스이며, 특히노로바이러스는성인에서도검출률이높고, 가족이나사회공동체에서집단으로감염을일으키는경우가많다 (2, 3). 노로바이러스는 1929년 Zahorsky 등에의해 winter vomiting disease로최초보고되었고, 1972년 Kapikian 등이면역전자현미경으로확인하였다 (4). 노로바이러스는분변-구강혹은구토에의한비말형성 에의해서주로전파되며, 보통 12~2월사이의겨울철에유행한다. 12~48시간의잠복기를거쳐메스꺼움, 구토, Received: August 31, 2011/ Revised: November 9, 2011 Accepted: November 14, 2011 * Corresponding author: KwiSung Park. Chungcheongnam-Do Institute of Health and Environmental Research, 44-1 Gayang2-dong, Dong-gu, Daejeon, 300-801, Korea. Phone: +82-42-620-1654, Fax: +82-42-620-1659 e-mail: kwisungp@korea.kr 설사, 복통의주요증상을나타내며때로는두통, 오한및근육통을유발하기도한다. 보통증상은 1~2일정도짧게나타나지만, 경우에따라심각한증세를보이기도한다 (5). 이바이러스는 5개의 Genogroup (G) 으로나뉘며, 이들중 GI, GII, GIV가사람을숙주로하고, GIII, GV은각각소와쥐를감염시킨다 (6). 사람에서주로문제를일으키는 GI, GII의노로바이러스는캡시드부위의다양성을근거로 GI은 14개, GII는 17개의유전자형으로분류되어있다 (7, 8). 이러한항원의다양성때문에한가지유전자형에감염되는경우에도다른형에의한재감염이일어나기도하며, 면역력은잠시동안만지속되어반복적으로재감염이일어나기도한다 (9, 10). 국내에서가장빈번하게유행하는유전자형은 GII.4형으로이것에대한전체염기서열분석을포함한분자생물학적특성의보고는종종이루어져왔으나 (11~13), GII.3형에대한분석결과는거의없는실정이다. 이에본연구에서는최근급성위장관염및집단식중독의주요병원체인노로바이러스중 GII.3형에대한부분적인캡시드유전자염기서열을결정하고, 계통분석을이용하여유전적변이를분석하였다. 287
288 JI Kim and KS Park 재료및방법바이러스 2008년부터 2010년까지순천향대학교천안병원에급성위장관염으로내원한환자로부터수거된분변중노로바이러스 GII.3형인것을대상으로하였다 (Table 1). 검체는멸균된 0.1 M PBS (phosphate buffered saline 7.2, Sigma St. Louis, MO, USA) 9 ml에분변 1 g을넣어희석한후 3,000 rpm에서 30분간원심분리하여상층액을사용하였고, 시험에사용될때까지 -70 에서보관하였다. Reverse transcription-polymerase chain reaction (RT-PCR) 노로바이러스는 semi-nested RT-PCR법을이용하여검사하였다. 전처리된상층액을 150 μl 취하고 magnetic bead (Toyobo, Osaka, Japan) 를이용하여제조사의사용방법에따라 RNA를추출하였다. 추출된 RNA는 Kojima 등에의해보고된노로바이러스에특이적인프라이머가 포함된 1st Norovirus Detection kit (GI) (Cellgenomics, Seoul, Korea) 와 1st Norovirus Detection kit (GII) (Cellgenomics, Seoul, Korea) 를이용하여각각수행하였다 (14). 추출된 RNA 5 μl를각각의키트에추가한후 47 에서 40분간 cdna를합성하고, 94 에서 30초, 54 에서 30초, 72 에서 45초동안 35회반복하여 PCR 반응을수행하였다. 그후 1차반응산물 2 μl를취하여 2nd Norovirus Detection kit (GI) (Cellgenomics, Seoul, Korea) 와 2nd Norovirus Detection kit (GII) (Cellgenomics, Seoul, Korea) 를사용하여 2차 PCR을수행하였는데, 조건은 94 에서 30초, 56 에서 30초, 72 에서 45초동안 25회반복하였다. PCR 증폭에의해생성된 DNA 산물은 1% agarose gel에서전기영동한후 EtBr (Ethidium bromide, Bioneer, Daejeon, Korea) 로염색하여확인하였다. 노로바이러스의유전형확인과계통분석염기서열분석을위하여각 PCR 산물을 QIAquick PCR purification kit (Qiagen, Hilden, Germany) 를이용하여정제하였다. 정제한 DNA는 ABI PRISM Dye terminator (Applied Table 1. Norovirus GII.3 strains isolated from patients' stool with acute gastroenteritis Isolate Gender Age Year Month Accession no. Kor08 (01-002) F 5 yr 2008 Jan JN704616 Kor08 (01-057) F 2 yr 2008 Jan JN704617 Kor08 (01-059) M under 1yr 2008 Jan JN704618 Kor08 (03-069) F 15 yr 2008 Mar JN704619 Kor08 (04-019) M 2 yr 2008 Apr JN704620 Kor08 (04-034) F 3 yr 2008 Apr JN704621 Kor08 (05-060) M 1 yr 2008 May JN704622 Kor08 (11-118) F 1 yr 2008 Nov JN704623 Kor08 (12-011) F 3 yr 2008 Dec JN704624 Kor08 (12-065) M 1 yr 2008 Dec JN704625 Kor08 (12-066) F under 1yr 2008 Dec JN704626 Kor08 (12-117) M 1 yr 2008 Dec JN704627 Kor08 (12-177) M under 1yr 2008 Dec JN704628 Kor09 (03-053) F 1 yr 2009 Mar JN704629 Kor09 (06-025) F 38 yr 2009 Jun JN704630 Kor09 (08-074) M 5 yr 2009 Aug JN704631 Kor09 (09-117) M 1 yr 2009 Sep JN704632 Kor10 (06-060) M 1 yr 2010 Jun JN704633
Genetic Characterization of Norovirus GII.3 289 Figure 1. Gel electrophoresis of PCR products of capsid region of 18 norovirus GII.3 isolates from patients with gastroenteritis; Lane M, 100 bp DNA ladder; Lane 1~18, 18 norovirus GII.3 isolates; Lane 19, negative control; Lane 20, positive control. Figure 2. Phylogenetic analysis based on a 205 bp sequence of the capsid region of Korean norovirus GII.3 strains and reference strains. Nucleotide sequences were analyzed using the neighbor-joining method.
290 JI Kim and KS Park Figure 3. Phylogenetic analysis of the capsid region of norovirus GII.3 lineages and Korea GII.4. Table 2. Identities of nucleotide and amino acid of capsid region between the lineages and variant group Lineage A Lineage B Biosystems, Foster City, CA, USA) 를사용하여 96 에서 10초, 50 에서 5초, 60 에서 4분동안 25회반복하여염기서열결정반응을수행하였다. 반응한산물은 3 M sodium acetate (ph 5.8) 와에탄올로침전시킨후 ABI 3100 Genetic Analyzer (Applied Biosystems) 를이용하여염기서열을결정하였다. 각염기서열은 National Center for Biotechnology Information의 Genbank에 JN704616~ JN704633으로등록하고 (Table 1), basic local alignment search tool (BLAST) 을이용하여가장가까운원형주와비교하여유전자형을결정하였다. 그후 GII.3형의유전자형을갖는 18개의분리주에대한염기서열은원형주들과함께 Clustal X (European Molecular Biology Laboratory, Heidelberg, Germany) 를이용하여다중정렬을수행하였다 (15, 16). 정렬된자료는 MEGA (Molecular Evolutionary Genetics Analysis, Tempe, AZ, USA) 프로그램을이용하여계통분석을실시하였다 (17). 결과 Lineage B Variants Nucleotide 94.1~97.6 90.2~94.6 Amino acid 94.1~100 95.6~100 Nucleotide - 91.7~94.1 Amino acid - 94.1~100 우리나라에유행하는노로바이러스 GII.3형에대한유전적다양성분석을위하여 2008년부터 2010년까지분리된바이러스 18주를대상으로캡시드유전자를증폭하여 313 bp의절편을확인한후, 염기서열을분석하였다 (Fig. 1). 계통분석은현재 GenBank 데이터베이스에등록되어 있는 29개의참조주를포함하여이루어졌다. 2008년도의분리주들과가장가까운참조주는 2005년일본에서분리된 "Hu/GII/OC05139/2005/JP" 주이었고, 2010년의국내분리주인 "Kor10 (06-060)" 주는 2005년도러시아에서분리된 "Hu/GII/2005/7601/Nizhny Novgorod/RUS" 와연관관계가가장높았다 (Fig. 2). 47주의노로바이러스 GII.3의계통분석결과, 2개의그룹 (Lineage A, B) 으로나누어졌으며, 국내의 18개의분리주중 16개의분리주가 Lineage B에속하였다. 2009년에분리된 "Kor09 (06-025)" 와 "Kor09 (09-117)" 주는이들과는계통학적으로연관성이낮아변이주 (Variants) 로분류되었다 (Fig. 2). 각각의클러스터에대하여염기서열의상동성비교를한결과, Lineage A는국내분리주가대다수포함된 Lineage B와 94.1~97.6% 의상동성을보였고, 변이주의그룹과는 90.2~94.6% 를나타내었다. Lineage B는변이주그룹과 91.7~94.1% 의상동성을보였다. 또한아미노산서열에대한상동성비교에서는, Lineage A는 Lineage B와 94.1~100%, 변이주그룹과는 95.6~100% 의상동성을나타내었다. 또한 Lineage B와변이주그룹과는 94.1~100% 의상동성을보였다 (Table 2). GII.3형은국내에서유행한 GII.4형의분리주들사이에 69.3~72.2% 염기서열의차이를보이며계통학적차이를나타내었다 (Fig. 3). 참조주를포함한 47주의노로바이러스 GII.3형의아미노산서열을비교한결과, 대부분이산발적으로일어나는돌연변이에의한아미노산치환이었지만, 16번위치의 D/E는돌연변이가빈번하게일어나는부위로확인되었다. 이들과국내의 GII.4형의비교에서 73.5~80.9% 의아미노산서열의차이를보이고있었다 (Fig. 4). 고찰노로바이러스는바이러스성설사질환의주요한병원
Genetic Characterization of Norovirus GII.3 291 Figure 4. Comparison of norovirus GII.3 amino acid sequences and Korea GII.4 consensus sequence in the capsid region. The box is conserved mutation site. 체로전체바이러스성설사의약 10% 를차지하고있고, 특히최근에집단발생하는식중독의대부분원인으로보고되고있다. 또한노로바이러스는환경적요인에저항성이매우큰바이러스로높은감염성과광범위한전염력을보이는데, 최근단체급식이대형화되고외식의기회가증가함에따라노로바이러스에대한감염기회가증가되고있는추세이다 (18, 19). 노로바이러스의진단은 1992년민감도와특이도가높은노로바이러스의 RT-PCR 법이개발되어세계적으로사용되고있고, 현재에는 real time RT-PCR 등이사용중이다 (20, 21). 노로바이러스의유전자형중약 75% 이상은 GII.4형으로, 대부분의노로바이러스유전학적국내연구는 GII.4형에집중되어 GII.3형을분석한자료는거의없는실정이다. 이에본연구에서는국내의노로바이러스 GII.3형을대상으로캡시드유전자에대한염기서열을결정하고, 외국에서보고된참조주들을포함하여계통분석을실시하였다. 노로바이러스 GII.3는세개의클러스터로구성되어있다고보고되어있다 (22). 클러스터 I은 1975년 ~1979년사이에유행한바이러스이고, 클러스터 II는 1987년 ~ 1995년에유행한바이러스이다. 마지막클러스터 III은 1998년 ~2006년에유행한바이러스라고분석하였다. 본연구에서의 Lineage A는클러스터 II에속하는것이며, Lineage B는클러스터 III에속하는것으로판단된다. 국내의노로바이러스연구가 2000년대초반이라는것을감안하면, 그이후에분리된노로바이러스 GII.3는세개의클러스터로구성되어있다고보고되어있다 (22). 클러스터 I은 1975년 ~1979년사이에유행한바이러스이고클러스터 III 또는 Lineage B에포함된다. 국내의분리주들중에특이한것은 2009년에분리된 "Kor09 (06-025)" 와 "Kor09 (09-117)" 으로기존에국내유행노로바이러스 GII.3형들과는 5.4~9.8% 의염기서열상차이를보였다. 이들은또한계통도에서도새로운클러스터를구성하여, 이
292 JI Kim and KS Park 번분석에서변이주라고지정하였다. 국내의 GII.3형의노로바이러스와 GII.4형과는 27.8~30.7% 의염기서열의차이를보였고, 19.1~26.5% 의아미노산서열의차이를보여유전적으로독립적인것을확인할수있었다. 보고된참조주들을포함한아미노산서열을비교한결과, 돌연변이는산발적으로여러부분에서일어나는것을확인할수있었다. 특히 16번위치에아미노산이 D와 E가공존하여나타나는것은이부위가빈번하게돌연변이가나타나는부분이라는것을생각할수있다. RNA 바이러스의돌연변이발생률은매우높은데, 이는바이러스의 RNA 게놈복제를담당하는 RNA 의존성 RNA 중합효소가교정기능을갖지않기때문이다 (23). 결론적으로본연구는국내의노로바이러스 GII.3형에대한캡시드유전자염기서열을결정하였고, 계통분석을실시하여유전적변이를분석하였다. Acknowledgement We thank Dr. Park JoonSoo and Choi YoungJin for kindly providing us with the specimens used for detection of Norovirus. 참고문헌 1) Glass RI, Bresee J, Jiang B, Gentsch J, Ando T, Fankhauser R, et al. Gastroenteritis viruses: an overview. Novartis Found Symp 2001;238:5-19. 2) Chikhi-Brachet R, Bon F, Toubiana L, Pothier P, Nicolas JC, Flahault A, et al. Virus Diversity in a Winter Epidemic of Acute Diarrhea in France. J Clin Microbiol 2002;40:4266-72. 3) Dey SK, Nguyen TA, Phan TG, Nishio O, Salim AF, Rahman M, et al. Molecular and epidemiological trend of norovirus associated gastroenteritis in Dhaka City, Bangladesh. J Clin Virol 2007;40:218-23. 4) Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, Chanock RM. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J Virol 1972;10:1075-81. 5) Kaplan JE, Gary GW, Baron RC, Singh N, Schonberger LB, Feldman R, et al. Epidemiology of Norwalk gastroenteritis and the role of Norwalk virus in outbreaks of acute nonbacterial gastroenteritis. Ann Intern Med 1982;96:756-61. 6) Vinjé J, Hamidjaja RA, Sobsey MD. Development and appli- cation of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J Virol Methods 2004;116:109-17. 7) Ambert-Balay K, Bon F, Le Guyader F, Pothier P, Kohli E. Characterization of new recombinant noroviruses. J Clin Microbiol 2005;43:5179-86. 8) Medici MC, Martinelli M, Abelli LA, Ruggeri FM, Di Bartolo I, Arcangeletti MC, et al. Molecular epidemiology of norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy. J Med Virol 2006;78:1486-92. 9) Gallimore CI, Cubitt D, du Plessis N, Gray JJ. Asymptomatic and symptomatic excretion of noroviruses during a hospital outbreak of gastroenteritis. J Clin Microbiol 2004;42:2271-4. 10) Lambden PR, Caul EO, Ashley CR, Clarke IN. Sequence and genome organization of a human small round-structured (Norwalk-like) virus. Science 1993;259:516-9. 11) Park KS, Jeong HS, Baek KA, Lee CG, Park SM, Park JS, et al. Genetic analysis of norovirus GII.4 variants circulating in Korea in 2008. Arch Virol 2010;155:635-41. 12) Park JW, Lee SG, Lee YM, Jheong WH, Ryu S, Paik SY. Full sequence analysis and characterization of the South Korean Norovirus GII-4 variant CUK-3. Virol J 2011;8:167. 13) Jeong AY, Yun SI, Jee YM, Kang YS, Lee YM. Molecular characterization of Korean isolate of human norovirus, the Hu/NLV/Gunpo/2006/KO Strain. Korean J Microbiol 2009; 45:105-11. 14) Kojima S, Kageyama T, Fukushi S, Hoshino FB, Shinohara M, Uchida K, et al. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J Virol Methods 2002;100:107-14. 15) Thompson JD, Higgins DG, Gibson TJ. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673-80. 16) Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4: 406-25. 17) Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9. 18) Kim SH, Cheon DS, Kim JH, Lee DH, Jheong WH, Heo YJ, et al. Outbreaks of gastroenteritis that occurred during school excursions in Korea were associated with several waterborne strains of norovirus. J Clin Microbiol 2005;43:4836-9.
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