The Korean Journal of Microbiology, Vol. 45, No. 2, June 2009, p. 105-111 Copyright 2009, The Microbiological Society of Korea w Hu/NLV/Gunpo/2006/KO w p 1,2 Á 1 Á 2 Á 3 Á 1 * w w w» w l s w w j Caliciviridae (family) w x w. ü RNA l 3 open reading frame (ORF)» wš, w m mw w p w. (Hu/NLV/Gunpo/2006/KO), 2 ƒ l. PCR s mw RNA 3 cdna r w w, w cdna» w. i Hu/NLV/Gunpo/2006/KO 3 ORF (ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp). 35 w, ORF1 ORF2 ORF3 w» û, p ORF2 ORF3 C- w. w m w, Hu/NLV/Gunpo/2006/KO genogroup II w, Saitama U1, Gifu'96, Mc37, Vietnam 026 j l x w. m w Hu/NLV/Gunpo/2006/KO 3 ORF», w,»» w» œw» w. Key words ý genetic relationship, Hu/NLV/Gunpo/2006/KO, norovirus, sequencing l,,», yw w w, w wù. p 5 w 700 xw (15). w ƒ w wù, y» y w y. 5 w 21 w wš, 5 w š š (2, 13). w q - (fecal-to-oral) y m w x w q, l»¾ w ùkù, ƒ w wù (8). 1972 w ƒ, j ƒ w (8). w *To whom correspondence should be addressed. Tel: 82-43-261-2863, Fax: 82-43-272-1603 E-mail: ymlee@chungbuk.ac.kr w» w. w w, (institution), ev, w, (nursing home), w, ƒ w ³, mù ƒ ùkù 24~48» z w 1~2 (5, 9). Caliciviridae (family) Norovirus (genus) (6). s ƒ k human calicivirus. calicivirusƒ mw œm l, s ƒ Caliciviridae ü calicivirus w clade x w. w, d calicivirus. Caliciviridae w 2 Lagovirus Vesivirus w w ƒ., Lagovirus rabbit hemorrhagic disease virus european brown hare syndrome virusƒ sw, Vesivirus feline calicivirusƒ sw. (buoyant density) 1.33~1.41 g/ cm 3, 27~40 nm (envelope) ƒ š, ¼ ƒ 7.5 kb ƒ 105
106 Ah-Yong Jeong et al. Kor. J. Microbiol RNA ƒ š RNA (8). RNA 3 open reading frame (ORF),, w w cis-acting elementƒ 5 3 š (1~3, 6). (nonstructural protein) g w ORF1 2C-like helicase, VPg, 3C-like cysteine protease, 3D-like RNAdependent RNA polymerase domain ƒ š w 5 cleavage siteƒ w. ORF2 58 kda e (VP1) g wš, ORF3» y» g w. w 5 genogroup (GI~GV) ù, GI, GII, GIV š, GIII GV ƒƒ (23). j k» û ùkü [, ~45% (human norovirus), ~87% (murine norovirus)]. p, human norovirus w û ùkü (1, 3, 4, 14, 19, 21, 23). Calicivirus s, human calicivirus (HuCV) š f. genogroup ü genotype. e RNA-dependent RNA polymerase w genotype w. ù ¾ s l x ü w x w mw ƒ w. w d w w» œwš, w» w, w 35 w w ³ wš w. w Hu/NLV/Gunpo/2006/KO, 2 ƒ l, l s l w. y 1g ³ 10 w w z, d w -80 o C w. RNA 200 µl l Trizol LS (Invitrogen, USA) w z RNA w. RNA 20 µl dh 2 0 g. RNA -80 o C ¾ w. cdna w s RNA 3 cdna w sw. x š j k Table 1 w. cdna w w (reverse transcription, RT) ww. t RT r(50 mm Tris-HCl; ph 8.3, 75 mm KCl, 10 mm DTT, 3 mm MgCl 2, 0.5 mm dntp) 42 o C, 50 ww. RT w first-strand cdna r l doublestranded cdna» w PCR (polymerase chain reaction) ww. t PCR 20 mm Tris-HCl (ph 8.3), 2.0 mm MgCl 2, 10 mm KCl, 6 mm (NH 4 ) 2 SO 4, 0.1% Triton X-100, 0.001% BSA, 200 µm dntps, 20 µm primers, 2.5 U Pyrobest DNA polymerase (TaKaRa, Japan) sww 100 µl ww. s v denaturation (95 o C, 30 ), annealing (60 o C, 30 ), polymerization (72 o C, 3 ) 35 j ww š, extension 72 o C 10 ww. l j» cdna r, s cdna 1% ƒ» w z, ethidium bromide w w.» BigDye terminator cycle sequencing system (Applied Biosystems, USA) w, w w 3 cdna w» ü v w f w. composite sequence ww w Hu/NLV/Gunpo/2006/KO 3 ORF w» w.» œ. Table 1. Oligonucleotides used in this study Primer Sequence (5 to 3 ) Polarity Region AF ATA TTA ATT AAG TGA ATG AAG ATG GCG TCT AA + 1-21 AR-1 ATA CGG TCC GAA CTG AGT CTC TCA TTG TAG T - 3003-3023 BF CAT GAA CAT ACA AGA CCT CT + 2500-2519 BR ATA CGG TCC GTC ACT CGA CGC CAT CTT CAT T - 5084-5104 CF AGA CAA GAG CCA ATG TTC AG + 4531-4550 CR ATA CGG TCC GTT TTT TTT TTT TTT TTT TTT - 7547-7562
Vol. 45, No. 2 NLV w 107 Table 2. Noroviruses used in this study Virus strain Place and year of isolation GenBank assession no. SzUG1 Japan, 1997-1999? AB039774 Saitama U1 Japan, 1997-1999? AB039775 Saitama U3 Japan, 1997 AB039776 Saitama U4 Japan, 1997 AB039777 Saitama U16 Japan, 1997 AB039778 Saitama U17 Japan, 1997 AB039779 Saitama U25 Japan, 1997-1999? AB039780 Saitama U18 Japan, 1997-1999? AB039781 Saitama U201 Japan, 1998 AB039782 Gifu 96 Japan, IU AB045603 WUG1 Japan, 2000 AB081723 Hu/Gl/otofuke/1979/JP Japan, 1979 AB187514 Chiba (Hu/NLV/Chiba 407/1987/JP) Japan, 1987 AB042808 Hu/Chiba/04-1050/2005/JP) Japan, 2005 AB220921 BS5 IU AF093797 Vietnam 026 Vietnam, IU AF504671 Hu/NLV/GII/MD145-12/1987/US USA, 1987 AY032605 Snow Mountain USA, IU AY134748 Mc37 Thailand, IU AY237415 Hu/NLV/GII/Langen1061/2002/DE Germany, 2002 AY485642 Hu/NLV/Oxford/B5S22/2003/UK UK, 2003 AY581254 Hu/NLV/Oxford/B4S2/2002/UK UK, 2002 AY587983 Hu/NLV/Oxford/B4S5/2002/UK UK, 2002 AY587984 Hu/NLV/Oxford/B4S6/2002/UK UK, 2002 AY587985 Hu/NLV/Oxford/B4S4/2002/UK UK, 2002 AY587986 Hu/NLV/Oxford/B4S7/2002/UK UK, 2002 AY587987 Hu/NLV/Oxford/B4S1/2002/UK UK, 2002 AY587988 Hu/NLV/Oxford/B2S16/2002/UK UK, 2002 AY587989 Hu/NLV/Dresden174/pUS-NorII/1997/GE Germany, 1997 AY741811 Hu/NLV/GII/Neustrelitz260/2000/DE Germany, 2000 AY772730 Hu/Guangzhou/NVgz01/CHN China, IU DQ369797 Southampton UK, IU L07418 Norwalk(M87661) IU M87661 Norwalk(NC_001959) USA, IU NC_001959 Lordsdale IU X86557 HU/NLV/Seoul/2006/KO Korea, 2006 This study IUS information unavailable Multiple sequence alignment phylogenetic analysis Multiple sequence alignment phylogenetic analysis w Hu/NLV/Gunpo/2006/KO sww GenBank z 35» w. w w GenBank accession no. Table 2 w. w w, CLUSTAL X (22) v w default w multiple sequence alignment
108 Ah-Yong Jeong et al. Kor. J. Microbiol ww. q l e l neighbor-joining method (18) w m w. w TREEVIEW software (16) w m ƒyw. w Hu/NLV/Gunpo/2006/KO» 2006» s w y RNA w» w w, Hu/NLV/Gunpo/2006/KO w. 5 3 w» w» w, 3 cdna r sw. cdna w PCR s š j k Table 1 w. w 3 kb j» s cdna» w.» r», DNA strider v w wù composite sequence w.», ü Hu/NLV/Gunpo/2006/KO 3 ORF ƒƒ 5,100 bp (ORF1), 1,647 bp (ORF2), 765 bp (ORF3). w ¾ ƒ 3 poly(a) tail óù y w. w Hu/NLV/Gunpo/2006/KO w p Multiple sequence alignment mw Hu/NLV/Gunpo/2006/KO sww ü 36 (Table 2) consensus sequence wš,» w. mw 3 ORF ORF2 ORF3ƒ» yƒ j (Fig. 1). p, ORF2 C- ƒ virus-like particle 3D mw ƒ Á e š (17)., ORF1 ORF2 ùkü (Fig. 1). w ORF2 upstream sequenceƒ packaging ù y g wš ew (13). w, consensus sequence 5,071 bp~5,090 bp 20 j k, 5,103 bp~5,123 bp 2 w 20 j k ƒ ew w (Fig. 2). ƒ š RNA-dependent RNA polymerase C- PCR s v w». w Hu/NLV/Gunpo/2006/KO w m w Hu/NLV/Gunpo/2006/KO 35 w w» w,» m multiple sequence alignment ww, ƒ š neighbor joining method (18) w Fig. 1. Nucleotide variability of the ORF1 (A), ORF2 (B), and ORF3 (C) of 36 norovirus genomes. The number of nucleotide differences was plotted at each residue throughout the 36 norovirus genomes after multiple sequence alignments.
Vol. 45, No. 2 NLV 한국분리주 109 Fig. 2. Nucleotide sequence alignment of the conserved region in the C-terminus of ORF1 of the 28 fully sequenced GII norovirus strains including Hu/NLV/Gunpo/2006/KO. Two highly conserved regions were shown in dotted boxes. Fig. 3. A phylogenetic tree based on the 36 fully sequenced human noroviruses including Hu/NLV/Gunpo/2006/KO. The multiple sequence alignment was obtained by CLUSTAL X, and the phylogenetic tree was constructed by the neighbor-joining method. The scale bar represents the number of nucleotide substitutions per site.
110 Ah-Yong Jeong et al. Kor. J. Microbiol m w. w m 2 j j l(cluster) x w (Fig. 3). ¾ ƒ 2 genogroup (GI GII) ew (1, 3, 14, 19, 21). GI j l Hu/GI/Otofuke/1979/JP, BS5, Chiba, SzUG1, Southampton, WUG1, Norwalk (NC_001959), Norwalk (M87661) sww 8 ƒ sw. GII j l 2 cladeƒ x. 2 clade wù Saitama U25, U3, U4, U16, U17 swwš, wù Hu/NLV/Gunpo/2006/KO w Saitama U1, Gifu'96, Mc 37, Vietnam 026 sw. Cladeƒ ù,, ƒ j wù clade x w,,, pû, w ƒ clade x w w w. š, 2 ƒ l w Hu/NLV/Gunpo/2006/KO» w. l» m» 35 wš w m w, Hu/NLV/Gunpo/2006/KOƒ GII w Mc37, Vietnam 026, Saitama U1, Gifu'96 ƒ w ùkü. w, multiple sequence alignment ww j k ORF1 ORF2 ORF3 w ùkü. ORF1 ƒ packaging signal y transcription start signal g wš w ew, ƒ w e ƒ. ORF1 RNA-dependent RNA polymerase s ƒ w (12). ORF2ƒ g w e N- w w N/S ORF1 û ùkü. š e C- w w P2 ƒ û ù kü., human immunodeficiency virus (HIV) V3 loop (7) hepatitis C virus (HCV) E2/hypervariable (10) j k d ù kü. HIV HCV t, w wš vw (11, 20). w v e P2 w w w. P2 t y wš w w w w w ƒ. mw Hu/NLV/Gunpo/2006/KO»» w» w» y». w, w w w w w. yw, w, w, x w ƒ ü s l w w. 2007 w w w. š x 1. Ando, T., S.S. Monroe, J.R. Gentsch, Q. Jin, D.C. Lewis, and R.I. Glass. 1995. Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-pcr and southern hybridization. J. Clin. Microbiol. 33, 64-71. 2. Glass, R.I., J. Noel, T. Ando, R. Fankhauser, G. Belliot, A. Mounts, U.D. Parashar, J.S. Bresee, and S.S. Monroe. 2000. The epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics. J. Infect. Dis. 181, 254-261. 3. Green, K.Y., T. Ando, M.S. Balayan, T. Berke, I.N. Clarke, M.K. Estes, D.O. Matson, S. Nakata, J.D. Neill, M.J. Studdert, and H-J. Thiel. 2000. Taxonomy of the Caliciviruses. J. Infect. Dis. 181, 322-330. 4. Green, S.M., K.E. Dingle, P.R. Lambden, E.O. Caul, C.R. Ashley, and I.N. Clarke. 1994. Human enteric Caliciviridae: a new prevalent small round-structured virus group defined by RNA-dependent RNA polymerase and capsid diversity. J. Gen. Virol. 75, 1883-1888. 5. Greenberg, H.B., J. Valdesuso, R.H. Yoken, E. Gangarosa, W. Gary, R.G. Wyatt, T. Konno, H. Suzuki, R.M. Chanock, and A.Z. Kapikian. 1979 Role of Norwalk virus in outbreaks of nonbacterial gastroenteritis. J. Infect. Dis. 139, 564-568. 6. Hardy, M.E., S.F. Kramer, J.J. Treanor, and M.K. Estes. 1997. Human calicivirus genogroup II capsid sequence diversity revealed by analyses of the prototype Snow Mountain agent. Arch. Virol. 142, 1469-1479. 7. Holmes, E.C., L.Q. Zhang, P. Simmonds, C.A. Ludlam, and A.J. Brown. 1992. Convergent and divergent sequence evolution in the surface envelope glycoprotein of human immunodeficiency virus type 1 within a single infected patient. Proc. Natl. Acad. Sci. USA 89, 4835-4839. 8. Kapikian, A.Z., R.G. Wyatt, R. Dolin, T.S. Thornhill, A.R. Kalica, and R.M. Chanock. 1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10, 1075-1081.
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