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J Fd Hyg Safety Vol 5, No, pp 5~6 (00),QWTPCN QH (QQF *[IKGPG CPF 5CHGV[ Available online at http://wwwfoodhygieneorkr ù ÊÞ Ç ò Š *Â Â ã Â Ý Â öâ ýâ ÜÄÂã ÂÞ ŠÂùó Â Ü ÅöÂ Ü Â µ Â Â ý t t w, û w y yw Investigation of Norovirus Occurrence and Influence of Environmental Factors in Food Service Institutions of ChungCheong Area WooYoung Jung*, JoonHo Eom, ByeongJo Kim, MinHo Yun, InSun Ju, ChangSoo Kim, MiRa Kim, JungA Byun, YouGyoung Park, SangHyuck Son, EunMi Lee, RaeSeok Jung, MiAe Na, DongYeon Yuk, JiYeon Gang, and OkSun Heo Hazardous Substances Analysis Division, Daejeon Regional Food and Drug Administration, Bio Environmental Chemistry, Chungnam National University (Received January 8, 00/Revised March 9, 00/Accepted May 0, 00) ABSTRACT The purpose of this study was to examine the appearance of norovirus in the water for food in food service institutions and the influence of physicochemical and microbial factors of norovirus in order to work out basic data to predict the detection of norovirus Among 8 samples of water for food in food service institutions, norovirus appeared in 7 samples and the rate of appearance was 85% As for the type of norovirus, one samples contained GI type (genotype GI6) and six samples contained GII type (genotype GII, GII4, GII In the regression model of prediction of norovirus, the rate of appearance was correlated with NH N, total solids and the consumption of KMnO 4, out of such variables as NH N, total solids, the consumption of KMnO 4, depth, chloride and total colony counts, and its contribution rate for effectiveness was 7860% In order to examine the influential factor of environment upon the detection of norovirus, Pearson's correlation analysis was carried out The predictable regression formula for appearance rate of norovirus was expressed as 88 + 4677 [NH N] + 00 [total solids] + 076 [consumption of KMnO 4 ] 0009 [depth] 046 [chloride] + 0007 [total colony counts] (R = 0904, R =088, adjusted R = 0786, p < 005) The most influential factors upon the detection of norovirus were NH N, total solids and the consumption of KMnO 4 In other words, when the measured values of NH N, total solids and the consumption of KMnO 4 were higher, the possibility of appearance of norovirus increased Key words: Norovirus, physicochemical factor, microbial factor, onestep RTPCR, seminested PCR ù w zá,07 š, 000 z 54~699 xy 000 l 005 w xy r (Salmonella typhimurium) 6~%, (Vibrio parahaemolyticus) 9~6%, y s ³(Staphylococcus aureus) 67~47%, 8~570% w *Correspondence to: Woo Young Jung, Hazardous Substances Analysis Division, Daejeon Regional Food and Drug Administration, 95, Dunsandong, Seugu, Taejeon, 088, Korea Tel: +84480879, Fax: +844808790 Email: wooyoungkfda@koreakr noroviruses (genus Norovirus, family Caliciviridae) w ƒ w ) Norovirus ƒ ~4 š ( 45y) w, û ƒ k Norovirus t w RNA (ribonucleic acid)ƒ š 0, RNA (7500~7700 nt) virus 968 w Norwalk w y small round structured viruses (SRSV) ƒ noroviruses (genus Norovirus, family Caliciviridae),4) Norovirus 5 group (group I, II, IV: human, group III: bovine, group V: mouse) 5

54 WooYoung Jung et al, ùkü group I 4, 5) group II 7, genotype w Norovirus p t w z 4~48 z ùkù, r m z l ¼ virus ƒ š 4) ù 006 ( w wš p œ w» Áw Á»k z» m w t ),000, norovirus ) 97 004 ¾ w 789 w 8%ƒ w š 6,7) Norovirus» y w e, š Á w» w w š š 8) y ù m ƒ w e š w ù w k, w ù 9,0) ƒ w e x w ù t l norovirus y w š, norovirus, RTPCR,, ƒ y w, w z, RNA(ribonucleic acid) z, w, norovirus Ÿ w w primer w ƒ w š 5) ü w t ( t, ƒœ w ) wš ù Norovirus e š w w Norovirus y w, w ³ w ƒ v w 008 9 l 009 0 ¾ Ÿ, û sww 4 ( w, Áš w ) t 8 norovirus Áyw, w ƒ e w w mw norovirus y ³ wš w mw t w norovirus wš, norovirus w» w š ƒ ( RNA PCR» sequencing) w» x wš r w yw w norovirus l y wš w w Ì Ý ßá Ý Ì t Áyw w ye (Tedia, Ohio, USA), y (Sigma Chemical, Steinheim, Germany), 8% (Wako, Tokyo, Japan), j T (Sigma Chemical), (Samchun, Pyeongtaek, Korea), y Á6 y (Sigma Chemical), p (Yakuri, Osaka, Japan), k e (Sigma Chemical), p lp p ùp (Sigma Chemical), y (Duksan, Ansan, Korea), ùp (Kanto, Tokyo, Japan), e (Kanto), yg pá6 y (Wako), x p (Junsei, Tokyo, Japan), sodium thiosulfate (Wako), ùp (Sigma Chemical), j e (Hayashi, Osaka, Japan), (Kojima, Sayama, Japan) w š, w standard plate count agar (Oxoid LTD, Basingstoke, Hampshire, England), ECMUG (Oxoid Ltd), lactose broth (Oxoid Ltd), BGLB (BD, Le pont de claix, France), EMB (Oxoid Ltd), Bile esculin azide agar (Remel Ltd, Lenexa, USA), asparazine agar (Difco Ltd, Le Pont de Clax, France), acetamide slant agar medium (Difco Ltd), trypcase soy broth (Millipore, Molsheim, France), selenite broth base (Oxoid Ltd), bismuth sulphite agar (Oxoid Ltd), w š»» (BD, Le Pont Claix, France), QIAGENViral RNA Mini Kits (Qiagen, Hilden, Germany), step RTPCR enzyme mix, step RTPCR buffer mix water DEPC treated (Gendepot, Texas, USA), G F M, G F R R F F R M G R M (Bioneer, Taejeon, Korea), Tag polymerase, 0 Tag buffer dntpmix (Solgent, Taejeon, Korea) w ý 008 9 l 009 0 ¾ Ÿ, û sww 4 t 8 w w (Fig ) yw w w w x w» w 76 L t w z L ³»(Y&K, Ansung, Korea) s w x w, t,500~,800 L MDS Cartridge(M, Saint Paul, USA) s w x w» þ (Windex, Seoul, Korea) w w w, ƒ norovirus s p w š, t Norovirus x [ t t š 00768y(007 0 8)] w 6)

Investigation of Norovirus Occurrence and Influence of Environmental Factors 55 Fig Sampling stations in ChungCheong area The detection rate of norovirus in food service institutions, which using ground water in Chungcheong area (Daejeon Metropolitan City, ChungchengnamDo and ChungchengbukDo), was examined over the period from September 008 to October 009 ÚÐ Ý ÜôÚ æö (NH N), (NO N), e (consumption of KMnO 4 ), šx (total solids), k (turbidity), (color), (hardness), (chloride), (ph) œ x [y š 00746y(007 0 0)] w ƒ v w w,, k norovirus w x d w x v w,, e,,,, w x d w» w, w w ³ (total colony counts), ³(fecal coliforms), ³ (total coliforms), ³(fecal Streptococcus), ³(Pseudomonas aeruginosa), (Salmonella typhimurium) w ³ z þ k(0~0 o C) w ww Norovirus Áyw, w ƒ e w» w norovirus y w w Pearson w š, w w, norovirus dw» w z w x d w æö Fig (Liquid filteration MDS 000, Saint Paul, USA), (J0I, Beckman coulter, Indienapols, USA), (Qiacube, Qiagen, Hilden, Switzerland), PCR (Thermocycler, Bioneer, Gottingen, Germany)» (MULTINA MCE0, Shimadzu, Tokyo, Japan) Norovirus w w RTPCR (reverse transcriptionpolymerase chain reaction) PCR w x ww onestep RT PCR w š, PCR v å seminested PCR w primers sequences Table Onestep RTPCR seminested PCR ww z 5µl automatic DNA/RNA analyser (MULTINA MCE0, Shimadzu, Tokyo, Japan) w w Onestep RTPCR GI GII 45 C o 0, 94 C 5 DNA o jš, 94 o C 0, 55 o C 0, 7 o C 0 z w 5z k z, 7 C 7 o w, seminested PCR GI GII 94 C 5 DNA o jš 94 o C 0, 55 o C 0, 7 o C 0 z w 5z k z 7 C 7 o w Agarose gel (Junsei, Tokyo, Japan) bp (base pair) ƒ GIx norovirus, 0 bp (base pair) ƒ GIIx norovirus y w Norovirus ƒ y y w agarose gel (Junsei, Tokyo, Japan) w w PCR w z sequencing»(70xl, Applied biosystems, Lincoln Centre Drive Foster, USA) xw q w Ultrahigh resolution FESEM (Scanning Electron Microscopy) t l norovirus ultrahigh resolution FESEM (Hitachi, S4800, Minatoku, Tokyo, Japan) w» w 5mm 7mm p w z norovirus w Norovirus t l MDS vl(m, Saint Paul, USA) w z w» w ioncoater (Coxem, KICA, Daejeon, Korea) w ~5 nm gold coating w, w r l v (Nisshin, Toryo, Japan) w Ultrahigh resolution FESEM (Scanning Electron Microscopy) w t xk x x (Hitachi, S4800, Minatoku, Tokyo, Japan) w 5 nm 0 nm ü norovirus d w ßá w norovirus Áyw, w ƒ e w» w norovirus y w w w Pearson w š, w w, norovirus dw» w z w x d w m SPSS ver 70 (SPSS Inc, USA) w

56 WooYoung Jung et al Fig Schematic diagram of norovirus analysis Flow sheet summarizing the procedure of onestep RTPCR and seminested PCR for norovirus analysis of the water Table Primers and sequences Genogroup Primer Sequence (5' ') Application I II GIFM CTG CCC GAA TTY GTA AAT GAT GAT Onestep RT PCR GIRM CCA ACC CAR CCA TTR TAC ATY TG Onestep RT PCR / Seminested PCR/ Sequencing GIF ATG ATG ATG GCG TCT AAG GAC GC Seminested PCR / Sequencing GIIFM GGG AGG GCG ATC GCA ATC T Onestep RT PCR GIIRM CCR CCI GCA TRI CCR TTR TAC AT Onestep RT PCR / Seminested PCR/ Sequencing GIIFM TTG TGA ATG AAG ATG GCG TCG ART Seminested PCR / Sequencing

Investigation of Norovirus Occurrence and Influence of Environmental Factors 57 Fig Result of onestep RTPCR and seminested PCR (GI type of norovirus) Lane X: marker, Lane NC: negative control, Lane PC: positive control (Type: GI6), Lane BL: blank, Lane A: detected sample (red dotted circle), Lanes BCDEFG: not detected samples <Left> Electrophoresis of norovirus <Right> Rf of electrophoresis ŠŒ Ý ý Norovirus æö 008 9 l 009 0 ¾ Ÿ, û sww «( w, š w ) t 8 y w norovirus w 7 norovirusƒ, 85% w norovirus (Fig ) GI type (genotype GI6), 6 (Fig 4) GII type (genotype GII, GII4, GII q Gong 5) norovirus x s w» w 005 l 007 ¾ w norovirus 0 x w GI x, GII x 4 6 x š w Norovirus genotype GI 4 type, GII 7 type genotype, norovirus genotype ü 5) GI GII genotype GI x 06%, GII x 994% GII genotype w y ü 7) 999~004 Chiba x8), 9) ùkù GII genotype w ùkù ew w t norovirus norovirus genotype GI genotype 4%, GII genotype 857% ùkû t l norovirus xkw p q Fig 4 Result of Onestep RTPCR and Seminested PCR (GII type of norovirus) Lane X: marker, Lane NC: negative control, Lane PC: positive control (Type: GII4), Lane BL: blank, Lane A: not detected sample, Lanes BCDEFG: detected samples (dotted circle) <Left> Electrophoresis of norovirus <Right> Rf of electrophoresis Fig 5 Norovirus (red dotted circle) cling to the surface of MDS filter (HM mode 50,000 ) Ultrahigh Resolution FESEM (Hitachi, S4800, Minatoku, Tokyo, Japan) Resolution: 0 nm ( kv, WD = 5 mm, normal mode) Magnification: HM mode 50,000 Speciman stage (type I): X = 050 mm, Y = 050 mm, Z = 50 mm, T = 5 to + 70 o, R = 60 o Maximum sample size = 00 mm dia w» w ultrahigh resolution FESEM (Hitachi, S 4800, Minatoku, Tokyo, Japan) MDS vl(m, Saint Paul, USA) k norovirus d w, norovirus j» 0 nm ü ùkû (Fig 5) norovirus ƒ 0 small round structured viruses (SRSV) xk j»ƒ 0 nm ew,4) m w norovirus s w MDS vl (M, Saint Paul, USA) Norovirus ƒ xk y w t norovirusƒ

58 WooYoung Jung et al Table Physicochemical results of drinking water in food service institutions Item Standard ) Unit Min Max Mean ± SD ) Exceedances (%) NH N Not more than 05 mg/ml ND 0 00 ± 004 000 NO N Not more than 0 mg/ml 00 580 44 ± 98 000 Consumption of KMnO 4 Not more than 0 mg/ml ND 79 07 ± 088 000 Total solids Not more than 500 mg/ml 4400 000 890 ± 6680 000 Turbidity Not more than NTU ND 58 040 ± 08 40 Color Not more than 5 Degree 00 500 70 ± 80 40 Hardness Not more than 00 mg/ml 470 00 9440 ± 950 000 ph 58~85 600 80 690 ± 050 000 Chloride Not more than 50 mg/ml ND 00 00 ± 800 000 Value are Min, Max and Mean ± SD, n=8 ) Standard: NIER (National Institute of Environmental Research) standard of drinking water ND: Not detected ) SD: Standard deviation Table Microbial results of drinking water in food service institutions Item Standard ) Unit Min Max Mean ± SD ) Exceedances (%) Total colony counts Not more than 00 CFU/mL NG 000 700 ± 4900 70 Total coliforms Negative CFU/00 ml NG 000 Fecal coliforms Negative CFU/00 ml NG 000 Fecal Streptococcus Negative CFU NG 000 P aeruginosa Negative CFU NG 000 S typhimurium Negative CFU NG 000 Bacterial counts were presented as CFU/mL Value are Min, Max and Mean ± SD, n = 8 ) Standard: NIER (National Institute of Environmental Research) standard of drinking water NG: Negative, ) SD: Standard deviation w t v š, norovirus w w» w» kj, x» v w USEPA (united states environmental protection agency) w w» w pilot scale v w 0) ý ÚÐ æö t yw s³ 690, 70, k 040 NTU (nepthelometric turbidity unit), 00 mg/ml, 44 mg/ml, e 07 mg/ml, šx 890 mg/ml, 9440 mg/ml, 00 mg/ml» w w, k ƒƒ 40% ùkû (Table, k ƒ» w (filter ) v w Song w ¾ p s³ 690 ¾ ƒ, 0) ¾ ƒ ùk ù š w t ƒ w ¾, w e» š w t yw,,, ³, e ¾ ƒ ùkû,» w Song ew ý ÜôÚ æö Chung ³, ³, ³ š ³, ³ y š w w, p y ƒ w, 5 o C w û p š w, ³» (sediment) ƒ ù ³ ƒ y w š w t w ³, ³, ³, ³

Investigation of Norovirus Occurrence and Influence of Environmental Factors 59 Table 4 Comparison of positive samples and negative samples identified by KFDA method with norovirus water quality factors Item Unit Positive Samples(n=7) Negative Samples(n=75) Min Max Mean ± SD ) Min Max Mean ± SD ) ph 700 750 70 ± 00 600 80 690 ± 050 Color Degree 00 00 0 ± 040 00 500 90 ± 40 Turbidity NTU 058 086 069 ± 0 ND ) 58 04 ± 089 NH N mg/ml 005 00 008 ± 00 ND 0 00 ± 00 NO N mg/ml 00 690 454 ± 8 090 580 49 ± 05 Consumption of KMnO 4 mg/ml 5 7 05 ± 06 ND 79 055 ± 088 Total solids mg/ml 5070 000 870 ± 80 4400 900 6900 ± 540 Hardness mg/ml 470 900 9080 ± 4890 4800 00 970 ± 500 Chloride mg/ml 400 00 60 ± 0 ND 00 40 ± 800 Total colony counts CFU/mL 000 500 700 ± 000 NG 000 000 ± 4900 Fecal coliforms CFU/00 ml NG NG Total coliforms CFU/00 ml NG NG Fecal Streptococcus CFU NG NG P aeruginosa CFU NG NG S typhimurium CFU NG NG ) SD: Standard deviation, NG: Negative, ) ND: Not detected ü 000 CFU/mL» 70% ùkû (Table ) t sƒw ³, ³ ³, ƒ t ³ ³ ƒ» w, w w w sœ(abandoned well), e v» Norovirus ˆ æ»ï Š æö Ko 0) (5 ea) (8 ea) yw p w j ƒ ù ³, šx s ³ s³ Table 4 norovirus (7 ea) (75 ea) yw p w j ƒ š, ³, šx s³ s³, Ko 0) e w w, k, w norovirus s³ s³ Norovirus s³ 70, 0, k 069 NTU, 008 mg/ml, 454 mg/ml, e 05 mg/ml, šx 870 mg/ ml, 9080 mg/ml, 60 mg/ml, ³ 700 CFU/mL ³, ³, ³, ³, ³ ùkü Norovirus s³ 690, 90, k 04 NTU, 00 mg/ml, 49 mg/ml, e 055 mg/ml, šx 6900 mg/ml, 970 mg/ml, 40 mg/ml, ³ 000 CFU/mL ³, ³, ³, ³, ùkü æö norovirus Lee 4) w k ƒ ƒw, w e š w š, Stetler 5) šw k w ƒ š w, Keswick 6)» k ƒ ƒw ƒ w š w Norovirus w y w» w Pearson w, norovirus ƒ w e, šx, e š,, šx, e norovirus ƒ ƒ w ùkü z mw norovirus x d (Table 5) Norovirus = 88 + 4677 [NH N] + 00 [total solids] + 076 [consumption of KMnO 4 ] 0009 [depth] 046 [chloride] + 0007 [total colony counts] (R = 0904, R =088, adjusted R = 0786, p < 005) Norovirus x k,

60 WooYoung Jung et al Table 5 Pearson correlation between norovirus, total colony counts and water parameters Item Depth NH N NO N CMn TS ) Tb 4) Color Hard 5) ph TCC ) Chloride Depth 000 NH N 04 000 NO N 000 055 ** 000 CMn 00 08 06 000 TS ) 097 0509 ** 09 00 * 000 Tb 4) 008 0055 000 0009 0098 000 Color 0097 006 0048 008 00 0978 ** 000 Hard 5) 009 009 009 0055 045 ** 055 0 * 000 ph 000 000 046 ** 09 * 096 0007 00 075 000 TCC ) 09 040 ** 09 005 0046 06 007 05 * 007 000 Chloride 004 05 07 * 0045 008 06 0009 00 07 054 000 NV 6) 079 * 079 ** 000 045 ** 0674 ** 066 007 004 006 0 * 085 ** ) TS: total solids, CMn: consumption of KMnO4, ) TCC, total colony counts, 4) Tb: turbidity, 5) Hard: hardness, 6) NV: norovirus * Marked correlations are significant at p < 005 ** Marked correlations are significant at p <00 Table 6 Regression summary for dependent variable Item Unstandardized coefficients B Std error of B Beta Standardized coefficients t pvalue R adjusted R Constant 88 9 56 06 088 0786 NH N 4677 058 094 405 0000 TS ) 00 0005 040 4498 0000 NV 4) CMn 076 06 076 7 000 Depth 0009 0008 0088 8 067 Chloride 046 006 006 4040 0000 TCC ) 0007 0006 0094 45 060 ) TS: total solids, CMn: consumption of KMnO4, ) TCC: total colony counts 4) NV: norovirus The predictable regression formula for appearance rate of norovirus was expressed as 88 + 4677 [NH N] + 00 [TS ) ] + 076 [CMn ] 0009 [depth] 046 [chloride] + 0007 [TCC ) ] (R = 0904, R = 088, adjusted R = 0786, p < 005) šx, e, ¾ (depth),, ³ w, e š z» z ùkü» 7860% sƒ (Table 6) Norovirus w w ƒ ~4 š ( 45y) û ƒ k 5,78) ƒ v w «ü w w t 8 norovirus w 7, 85% w w Norovirusƒ w,, y ƒ v w Norovirus w, šx, e ùkû Norovirus p w j ƒ š, k,, ³, šx w norovirus s³ s³ ùkü Norovirus y l mw» w» Ù Choi, WS, Go, GP, Park, GJ, Moon, AR, Lee, HJ, Kim, MS and Kim, HY: Development of norovirus detection method from food The Annual Report of KFDA,, (007)

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