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1 Journal of Bacteriology and Virology Vol. 40, No. 4 p DOI /jbv Original Article Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA Hyung Tae Lee 1, Kyoung Hwa Jung 1, Ji Hyun Park 1, Gun-Woo Ha 2, Jin-Sik Oh 2, Youn-Kyoung Oh 2 and Young Gyu Chai 1 * 1 Division of Molecular and Life Sciences, Hanyang University, Ansan, Korea, 2 Bionote, Inc., Suwon, Korea Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever ( and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA. Key Words: Avian influenza, H9 subtype, Nonstructural protein 1 (NS1), Synthetic peptide-based ELISA 서론 독감바이러스 (influenza virus) 는사람이나동물에게감염되어매우치명적인피해를주는병원체중의하나로현재전세계적으로널리퍼져있으며, 특히독감바이러스 A와 B가급성바이러스성호흡기질환의주된원인체이다. 조류독감 (avian influenza, AI) 바이러스는아형에따라고병원성과저병원성으로분류할수있다. 고병원성 AI 바이러스 (H5 또는 H7 아형 ) 는감염확률이매우높 고그로인한치사율도높기때문에사람을포함해서많 Received: July 12, 2010/ Revised: September 28, 2010 Accepted: September 29, 2010 * Corresponding author: Young Gyu Chai Ph.D. Division of Molecular and Life Sciences, Hanyang University, Ansan, , Korea. Phone: , Fax: ygchai@hanyang.ac.kr 은동물들에게치명적이다 (1~3). 1997년부터아시아, 유럽국가에서고병원성 AI 바이러스에감염되었다는보고가있다. 2003~2004년에는한국을포함하여많은아시아국가에서가금류에 H5N1 고병원성 AI 바이러스에의한감염이보고되었다 (4~6). 저병원성 AI 바이러스 (H7 또는 H9 아형 ) 는고병원성 AI 바이러스와는달리사람이나동물에게치명적이지는않지만이바이러스에감염된가금류는가벼운호흡기질환을앓게되며, 달걀의생산이줄어들게됨으로써경제적인피해를입힌다 (7). 1998년부터우리나라를포함하여중앙아시아여러국가에서저병원성 AI 바이러스에감염되었다는보고가증가하고있는데, 그중에이란과파키스탄에서는상업용으로판매하는닭이저병원성 AI 바이러스를전염시키는원인이라는보고가있다 (8, 9). 또한저병원성 AI 바이러스에포함되는 H9N2 바이러스는아시아지역의풍토병으로알려져있으며, 홍콩과중국에서사람에감염되었다는것이보고되었으며 (10, 11), 207

2 208 HT Lee, et al. 시간이지날수록 H9N2 바이러스가감염할수있는숙주범위가넓어지고사람을포함하는다른종에게보다쉽게전파될수있는가능성을갖고있다 (12). 최근한국을비롯한동아시아국가에서는 AI 바이러스감염에의한보고가증가되어피해가확산되고있으므로국제수역사무국 (Office International des Epizooties; OIE) 에서는 AI 바이러스에의한감염을 List A 질병으로분류하였고, 국내에서는제1종가축전염병으로분류하고있다. 병원성조류독감이발생한경우에는우리나라를포함하여전세계의대부분국가들이살처분을하고있으며, 발생한국가에서는양계산물을수출할수없게된다. 이러한피해를막기위해한국을포함하는제한된숫자의국가에서는백신접종을하고있다. 하지만, 조류독감이발생하는경우현존하는혈청검사를통하여백신을접종한개체와자연적으로감염된개체를구분할수없기때문에모든닭을살처분할수밖에없는문제점을가지고있다 (7, 13, 14). 독감 A 바이러스표면의당단백질인헤마글루티닌 (hemagglutinin: HA), 뉴라미니다제 (neuraminidase: NA) 단백질에대한항원성 (antigenicity) 에따라다양한아형 (subtype) 으로구분되며, 현재 H1-H16과 N1-N9이알려져있다 (15). 독감 A 바이러스는 RNA 게놈을가지고있으며, HA, NA, nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acid (PA), polymerase basic 1 (PB1), polymerase basic 1 - F2 (PB1-F2) 그리고 polymerase basic 2 (PB2) 라는단백질을발현한다 (16). 이들중 NS1은바이러스가숙주에감염되었을시발현되어 RNA에결합을하는부분과다른단백질들과상호작용을하는부분을가지고있는단백질이다 (17). 즉, 바이러스가숙주에감염되었을때숙주의여러많은 RNA, 단백질들과상호작용하여바이러스가복제하는데중요한역할을하고숙주의면역반응을억제하는등여러기능을한다 (15, 18). NS1은바이러스가숙주세포에감염되고증식되는경우에만감염된개체에서발현되기때문에 NS1 단백질의존재유무는백신을접종한개체와바이러스에감염된개체를구별할수있는표지자로사용될수있다 (7, 19). 본연구에서는 AI 바이러스에감염된닭과백신을접종한닭을구별하기위해감염된개체에서높은발현양 을보이는 NS1을이용해한국에서발생한 AI 바이러스인 H9N2의 NS1 단백질의에피토프를 MultAlin과 PVS 프로그램으로계산하여예상하였고, 예상된에피토프의펩타이드를합성하여 enzyme-linked immunosorbent assay (ELISA) 방법을이용하여 AI 바이러스에감염된닭과백신을접종한닭의혈청과의반응을관찰하였다. 닭혈청 재료및방법 총 61개 ( 감염되지않은 26개, 백신을접종한 26개, 감염된 9개 ) 의닭혈청을 Bionote 사 (Suwon, Korea) 로부터얻었다. 특정질병부재 (specific pathogen free: SPF) 닭의혈청은 5주령부터 70주령까지의닭에서혈액을추출하여분리하였다. 백신을접종한닭혈청은 18주령, 40주령의닭에백신을접종한후혈액을추출하였다. 감염된닭의혈청은 6~7주령닭에게 H9N2 바이러스를접종한후에혈액을추출하였다. 얻은혈청을 15,000 g에서 10분동안원심분리하여불순물을제거한후사용전까지 4 에서보관하였다. 펩타이드합성 한국에서발생한 AI 바이러스인 H9N2의 NS1 단백질서열을확보하여 MultAlin ( Table 1. GenBank accession number of NS1 proteins in Korea avian influenza NS1 protein of Korea A/Chicken/Korea/ /96 (H9N2) A/Chicken/Korea/S21/2004 (H9N2) A/Korea/KBNP-0028/2000 (H9N2) A/Chicken/Korea/AI-96004/1996 (H9N2) A/Chicken/Korea/MS96-CE6/1996 (H9N2) A/Chicken/Korea/38349-p96323/96 (H9N2) A/Chicken/Korea/99029/99 (H9N2) A/Chicken/Korea/S21/2004 (H9N2) A/Chicken/Korea/S1/2003 (H9N2) A/Chicken/Korea/S27/04 (H9N2) A/Chicken/Korea/S25/04 (H9N2) A/Chicken/Korea/S24/04 (H9N2) GenBank accession number AAD52955 ACD37776 ABQ57383 ACZ47501 ACZ47499 AAD52953 AAQ05002 ACD37766 AAV65821 ABC48833 ABC48823 ABC48813

Discrimination of AI Viral Infection 209 multalin/multalin.html) 으로정렬하였고, 예상에피토프는 PVS 프로그램 (http://imed.med.ucm.es/pvs/, Protein Variability Sever) 으로계산하였다.

3 Discrimination of AI Viral Infection 209 multalin/multalin.html) 으로정렬하였고, 예상에피토프는 PVS 프로그램 ( Protein Variability Sever) 으로계산하였다. 즉, 1996년부터 2004년까지한국에서발생한 H9N2의 NS1서열 12개를 Gen- Bank ( 를통해서얻었으며 (Table 1), MultAlin을이용하여모든서열을정렬하였다 (20). PVS를사용하여분석한 NS1 서열에서면 Table 2. List of synthesized peptide of NS1protein Synthesized peptide sequence Location in NS1 protein NS1-1 RLRRDQKSL 35~43 NS1-2 AIMDKNITL 122~130 NS1-3 TLKANFSVI 129~137 NS1-4 EDVKNAIGV 172~180 NS1-5 KRKMARTIESEV 219~230 NS1-1 ~ NS1-4 peptides were predicted by PVS (Protein Variability Sever, NS1-5 peptide was referred from reports of Dundon et al (5, 21) 역항체들이결합할수있는에피토프부분을선별하였다. 얻은 4 펩타이드 (9 mer) 와 Dundon 등 (5, 21) 의연구에서사용하였던 NS1 서열중에 C 말단부분 (12 mer) 을참조하여한국에서발견된서열에서다섯부분 (RLRRDQKSL, AIMDKNITL, TLKANFSVI, EDVKNAIGV, KRKMARTIESEV) 을고안하였다 (Table 2). 고안하여선별한총 5부분의펩타이드를합성하였다 (Cosmo Co., Seoul, Korea). ELISA Table 2의펩타이드를 coating buffer (15 mm Na 2 CO 3, 35 mm NaHCO 3, 0.2 g/l NaN 3, ph 9.6) 에 1 μg/100 μl 농도로희석시킨후, 96 well maxisorp immuno plate (Nunc, Roskilde, Denmark) 에각웰당 100 μl씩분주하여하룻밤동안 4 에서 coating하였다. Coating buffer를제거한후인산염완충생리식염수 (PBS)/0.5% Tween 20/5% 소혈청알부민 (BSA) 으로 37 에서 1시간동안 blocking하였다. Blocking buffer를 PBS/0.5% Tween 20으로 4회씻어서제거한후 Figure 1. Multiple sequence alignment of NS1 proteins from avian influenza virus reported in Korea using Multalin. The NS1 protein sequences were obtained from GenBank. Multiple sequence alignment analysis of NS1 proteins using Multalin displayed 84% homology.

4 210 HT Lee, et al. 1차항체로는닭혈청을희석해서 1시간동안상온에서반응하였다. 반응후 4회세척하고 HRP (horseradish peroxidase) 가결합되어있는 goat-anti-chicken IgG (H+L) (BioFX laboratories, Owings Mills, MD, USA) 를사용하여 1시간동안상온에서반응하였다. 펩타이드를이용한 ELISA 실험에가장적합한혈청과 2차항체의비율을결정하기위해서혈청과 2차항체의희석비율 ( 혈청 : 1/100, 1/1000, 1/3000, 1/5000, 1/10000, 2차항체 : 1/5000, 1/10000) 을각각다르게해서실험을하였다. NS1-5 펩타이드를사용하여얻은결과를통해서혈청은 1/100, 2차항체는 1/5000의비율로 PBS/0.5% Tween 20/5% BSA buffer 에희석하는최적조건을결정하였다. 2차항체를제거후에 TMB (3,3', 5,5'-tetramethylbenzidine)-ELISA (Thermo scientific, Rockford, IL, USA) 용액을첨가하고 450 nm 파장에서흡광도를측정하였다. 측정결과값으로평균흡광도 + 2 X ( 표준편차 ) 공식을이용하여 cut-off 값을계 A 산하였다. 만약흡광도값이 cut-off 값보다높을경우그혈청은바이러스에감염된것으로결정하였다 (7). 결과 NS1 서열분석및에피토프예측 NS1 서열중에서면역항체가결합할수있는에피토프를예측하기위해한국에서발생한 H9N2 바이러스 NS1 서열을분석하였다 (Fig. 1). NCBI에서얻은 NS1 서열 12개를이용해정렬한결과모든서열간에 84% 의상동성을가진다는것을확인하였다. 이들서열을이용하여면역항체가결합할수있는 4개 (NS1-1, NS1-2, NS1-3, NS1-4) 의에피토프를예측해내었다. Dundon 등 (5, 21) 의연구에서사용하였던 NS1 단백질의 C 말단부분을참조하여한국에서발견되었던 NS1의 C 말단부분의펩타이드 (NS1-5) 서열을선별하여합성하였다 (Table 2). B C D Figure 2. Detection of anti-ns1 antibodies by synthetic peptide-based ELISA in chicken sera. (A) NS1-1, (B) NS1-2, (C) NS1-3, and (D) NS1-4. Dashed horizontal line represents the cut-off value calculated by adding an OD 450 nm mean value and two standard deviations (SD). The cut-off values for the test are (A) 1.122, (B) 1.127, (C) 0.601, and (D) Serum sample is positive when OD value is greater than the line.

5 Discrimination of AI Viral Infection 211 고찰 Figure 3. Detection of anti-ns1 antibodies by synthetic NS1-5 peptide-based ELISA in chicken sera. Dashed horizontal line represents the cut-off value calculated by adding an OD 450 nm mean value and two standard deviations (SD). The cut-off value for the test is Serum sample is positive when OD value is above line. Enzyme-linked immunosorbent assay 합성한펩타이드를이용한 ELISA 실험에가장적합한혈청과 2차항체의비율을결정하기위해서혈청과 2차항체의희석비율을각각다르게해서실험을하였다. NS1-5의펩타이드를사용하여얻은결과를통해서혈청은 1/100, 2차항체는 1/5000의비율로희석하는최적조건을결정하였다 ( 실험결과미제시 ). 펩타이드서열분석을통해서얻은 5개의펩타이드 (1 μg/well) 를이용하여 ELISA를수행한후에감염되지않은닭혈청을이용한결과값으로 cut-off 값을계산하였다 (NS1-1 : 1.122, NS1-2 : 1.127, NS1-3 : 0.601, NS1-4 : 1.787, NS1-5 : 1.257). ELISA 결과값이 cut-off 값보다높으면바이러스에감염이되었다는것으로확인할수있는데, 4개의 NS1-1 ~ 4 펩타이드를이용하여얻은값으로평균값을구해서비교한결과 SPF 닭 (NS1-1 : , NS1-2 : , NS1-3: , NS1-4 : ) 과백신을접종한닭 (NS1-1 : , NS1-2 : , NS1-3 : , NS1-4 : ), 감염된닭의혈청 (NS1-1 : , NS1-2 : , NS1-3 : , NS1-4 : ) 에서 cut-off 값보다낮은것을관찰할수있었다 (Fig. 2). 하지만 NS1-5 펩타이드서열을이용한실험결과에서는 SPF 닭 (NS1-5 : ) 과백신을접종한닭 (NS1-5 : ) 에서는 cut-off 값보다낮지만감염된닭 (NS1-5 : ) 에서는얻은결과값이 cut-off 값보다높은것을확인하였다 (Fig. 3). 전세계적으로광범위한지역에퍼져많은피해를주고있는병원체중에하나인독감바이러스는바이러스표면을이루고있는 HA, NA 단백질에따라고병원성 (H5 또는 H7 아형 ) 과저병원성 (H7 또는 H9 아형 ) 으로분류한다 (7). 특히한국에서는저병원성 H9N2 바이러스에의해가금류사업에많은피해를입기때문에최근 AI 바이러스감염에의한피해를줄이고자사육하는닭에게백신을처리하고있다. 하지만, 현존하는기술로는 AI 바이러스에감염된조류와 AI 백신을접종한조류를구분하지못하기때문에조류독감이발생하는경우백신을접종한개체와자연적으로감염된개체의모든닭을살처분하고있다. AI 바이러스가조류에감염되면 NS1 단백질이발현되어숙주의여러많은 RNA, 단백질들과상호작용하여바이러스가복제하는데도움을주며숙주의면역반응을억제하여 AI 바이러스의증식을돕게된다. 이러한 NS1 단백질의유무를점검하여 AI 바이러스의감염여부를구별하고자하였다 (7). NS1 단백질을이용한 Dundon 등 (5, 21) 의이전연구에서보고한바에따르면, NS1 전체단백질을사용하거나혹은부분펩타이드로서 KRYMARRVESEV 혹은 KRKMARTIESEV를사용하였다. 본연구에서는한국 AI 바이러스의 NS1 서열 12개를 GenBank에서얻은후 MultAlin을이용해정렬하여모든서열간에 84% 의상동성을가진다는것을확인하였다. 한국 AI 바이러스 NS1의상동성이있는부분에서에피토프 4부분 (NS1-1, NS1-2, NS1-3, NS1-4) 과 Dundon 등 (5, 21) 의연구에서사용하였던 NS1의 C 말단끝부분 (KRKMARTIESEV) 을포함하는총 5개의펩타이드서열을선별하여합성하였다. 합성된각각의펩타이드를이용하여 ELISA를수행한결과 NS1-1 ~ NS1-4 펩타이드에서는 SPF 닭혈청을이용하여계산한 cut-off 값보다낮은값을보여 SPF 닭과백신을접종한닭, 그리고바이러스에감염된닭사이를구별할수없었다. 그러나 C 말단끝부분인 NS1-5 펩타이드는오직바이러스에감염된닭에서얻은결과값에서 cut-off 값보다높은값을보여 SPF 닭과백신을접종한닭, 그리고바이러스에감염된닭사이를구별할수있었다. 결과적으로한국산 AI 바이러스의에피토프예상지역

6 212 HT Lee, et al. 을포함하는 NS1 단백질펩타이드 5개의서열을가지고 ELISA 실험을수행한본연구에서는에피토프를예상하여합성한 4개의펩타이드서열을이용하여 H9N2 바이러스에의한감염여부를판단하는것은적합하지않은것을확인하였다. 본연구는한국에서발견되었던 NS1 단백질의펩타이드서열을가지고연구를수행하였다는것과이전에사용되지않았던펩타이드서열을사용한것에의미가있고, NS1-5 펩타이드의서열을통해완벽하지는않지만백신처리한닭과감염된닭을구별하는데이용할수있다는것을확인하였다. 따라서본연구의결과는 NS1 단백질을이용한새로운에피토프펩타이드를고안하여 AI 바이러스에감염된닭과백신처리닭을특이적으로판단할수있는방법을찾아내는데기초자료가되리라생각한다. 참고문헌 1) Yuen KY, Chan PK, Peiris M, Tsang DN, Que TL, Shortridge KF, et al. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 1998;351: ) Swayne DE, Suarez DL. Highly pathogenic avian influenza. Rev Sci Tech 2000;19: ) Tumpey TM, Alvarez R, Swayne DE, Suarez DL. Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus. J Clin Microbiol 2005;43: ) Capua I, Alexander DJ. Avian influenza: recent developments. Avian Pathol 2004;33: ) Dundon WG, Milani A, Cattoli G, Capua I. Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry. Virus Res 2006;119: ) Webster RG, Peiris M, Chen H, Guan Y. H5N1 outbreaks and enzootic influenza. Emerg Infect Dis 2006;12:3-8. 7) Zhao S, Jin M, Li H, Tan Y, Wang G, Zhang R, et al. Detection of antibodies to the nonstructural protein (NS1) of avian influenza viruses allows distinction between vaccinated and infected chickens. Avian Dis 2005;49: ) Banks J, Speidel EC, Harris PA, Alexander DJ. Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype. Avian Pathol 2000;29: ) Alexander DJ. Report on avian influenza in the Eastern Hemisphere during 1997~2002. Avian Dis 2003;47: ) Guo YJ, Krauss S, Senne DA, Mo IP, Lo KS, Xiong XP, et al. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 2000;267: ) Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, et al. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci U S A 2000;97: ) Horimoto T, Kawaoka Y. Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 2005;3: ) Lambrecht B, Steensels M, Van Borm S, Meulemans G, van den Berg T. Development of an M2e-specific enzyme-linked immunosorbent assay for differentiating infected from vaccinated animals. Avian Dis 2007;51: ) Jeong OM, Kim MC, Kang HM, Ha GW, Oh JS, Yoo JE, et al. Validation of egg yolk antibody based C-ELISA for avian influenza surveillance in breeder duck. Vet Microbiol 2010; 144: ) Hale BG, Randall RE, Ortín J, Jackson D. The multifunctional NS1 protein of influenza A viruses. J Gen Virol 2008;89: ) Ghedin E, Sengamalay NA, Shumway M, Zaborsky J, Feldblyum T, Subbu V, et al. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature 2005;437: ) Wang W, Riedel K, Lynch P, Chien CY, Montelione GT, Krug RM. RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 1999;5: ) Basler CF, Aguilar PV. Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses. Antiviral Res 2008;79: ) Krug RM, Etkind PR. Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK cells. Virology 1973;56: ) Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 1988;16: ) Dundon WG, Maniero S, Toffan A, Capua I, Cattoli G. Appearance of serum antibodies against the avian influenza nonstructural 1 protein in experimentally infected chickens and turkeys. Avian Dis 2007;51:

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012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar