한국에서 분리된 콕사키 바이러스 B3 cDNA의 클로닝 및 전체 염기서열 분석

정윤석(Yoon Suk Jung),김기순(Gee Sun Kim),박정구(Jung Goo Park),이윤성(Yoon Sung Lee),신수연(Soo Yeon Shin),천두성(Doo Sung Chun),지영미(Young Mi Jee),김문보(Mun Bo Kim),나병국(Byung Gook Na),윤재득(Jae Deuk Yoon),이광호(Gwang Ho Lee 대한바이러스학회 2000 Journal of Bacteriology and Virology Vol.30 No.1

We have determined and analyzed the full-length cDNA sequence of a coxsackievirus B3 (CVB3) Korean isolate (CVB3-Korea/97) which has been known as a general human pathogen. The whole genome contains 7,400 nucleotides and has a single large open reading frame with 6,555 nucleotides that encodes a potential polyprotein precursor of 2,185 amino acids. The genome also contains a 5 non-coding region (NCR) of 741 bases and a 3' NCR of 104 bases followed by poly(A) tail. Sequence homologies of nucleotides and deduced amino acids between the CVB3-Korea/97 strain and the prototype (Nancy strain) were 81.7% and 91.5%, respectively. The genes encoding the functional proteins including viral protease and RNA dependent RNA polymerase showed higher homology than those encoding the structural proteins. We have further analyzed the sequences of 5' NCR, VP1 and VP2 of CVB3-Korea/97, which are known as cardiovirulent determining factors at the nucleotide and amino acid levels. Although the CVB 3-Korea/97 strain was isolated from an aseptic meningitis patient without cardiomyopathy, its 234th nucleotide and 165th amino acid were uracil and Asn as same as those of other cardiovirulent strains one. However, the 155th amino acid of VP1, which closely associated with cardiovirulence, was replaced with Arg by single nucleotide substitution from A2916 to T2916. Moreover, additional amino acid substitutions were observed in the flanking region of Asp155. Taken together, amino acid(s) substitution in VP1 may play a critical role in determining cardiovirulence of the CVB3- Korea/97 strain rather than individual nucleotide replacements in the 5' NCR and/or an amino acid substitution in VP2.

국내 소아로부터 분리된 장바이러스(Enterovirus)의 5'-Noncoding Region의 Sequencing 분석

정민아,류정우,김동수,윤재득,김기순,이윤성,Chung, Min A,Lou, Chung Woo,Kim, Dong Soo,Yun, Jae Deuk,Kim, Ki Soon,Lee, Yoon Sung 대한소아감염학회 1999 Pediatric Infection and Vaccine Vol.6 No.1

목 적 : 무균성뇌막염은 소아에서 주로 발생하는 질환으로, 흔히 장 바이러스에 의해 초래 된다. 저자들은 그동안 우리나라에서 분리된 무균성뇌막염 원인바이러스의 5'-NCR에 대한 sequencing을 통하여 prototype과의 homology를 비교하고 이 연구를 진단에 이용하기 위한 기초자료로 이용하기 위하여 본 연구를 시도하였다. 방 법 : 과거 4년간 우리나라에서 분리한 장바이러스 Coxsackie B1, Echovirus 3, 7, 9, 30 을 이용하여 RNA를 분리하고 RT-PCR을 이용하여 DNA를 합성한 후, direct sequencing을 이용하여 WHO에서 얻은 prototype과 homology를 비교하였다. 결 과 : 1) PCR product는 155bp와 440bp부위에 특징적인 띠를 관찰할 수 있었다. 2) 155bp에 관한 sequencing homology를 보면 prototype의 Coxsackie virus와 echovirus 는 92.1%의 homology를 보였다. 3) 환자에서 분리한 Coxsackie B1은 prototype과 94.1%의 homology를 보였다. 4) 환자에서 분리한 Echovirus 3은 92.8%, Echovirus 7은 92.8%, Echovirus 9는 94.1%, Echovirus 30은 82.9%의 homology를 보였다. 결 론 : 장바이러스의 5'-NCR은 homology가 높아서 진단에 이용하기에 좋으며 이 부위를 통한 typing을 위해서는 더 긴 부위를 sequencing할 필요가 있다. Purpose : Meningitis is an inflammation of meninges by various kinds of organisms. Almost 85% of aseptic meningitis is caused by Enterovirus. This study was done to detect the causative virus of those with aseptic meningitis through sequencing the 5'-noncoding region to compare prototype and homology. Methods : RNA was extracted from Coxsackie Bl, Echovirus 3, 7, 9, 30. DNA was synthesized by RT-PCR and we compared homology with prototype from WHO by direct sequencing. Results : 1) PCR products from these viruses showed same bands of 155 bp and 440 bp on gel electrophoresis. 2) Coxsackievirus and Echovirus 11 prototype sequences were compared, which showed 12 bp changes with 92.1%. 3) Coxsackievirus B1 from a patient showed 94.1% homology when compared with prototype. 4) Echovirus 3 showed 92.8%, echovirus 7 92.8%, echovirus 30 82.9% homology. Conclusion : 5'-NCR of enterovirus has high homology which was good for use of diagnosis and more long sequencing requires for typing of viruses.

Development of a simple and sensitive method to detect enteric viruses from oysters

정은영,제회복,전홍기,윤재득,지영미,천두성,조해월,장경립,Chung, Eun-Young,Je, Hee-Bok,Jun, Hong-Ki,Yoon, Jae-Deuk,Jee, Young-Mee,Cheon, Doo-Sung,Cho, Hae-Wol,Jang, Kyung-Lib Korean Society of Life Science 2002 생명과학회지 Vol.12 No.1

Development of a rapid method possessing the requisite sensitivity and specificity for virus monitoring is necessary for protection of the shellfish-consuming public. Oysters tissue usually contains virus particles in relatively small concentrations along with various other substances that can interfere with detection steps. Therefore, the critical point concerning the detection of viruses in shellfish tissues resides in the processing of samples. The current study demonstrated the possibility of purifying small amounts of virus particles at the interface of a 10/50% sucrose gradient after a single round of sucrose gradient ultracentrifugation. We could detect HAV and poliovirus simultaneously from oyster tissues by using two different sets of primer. Furthermore, the method showed a high level of virus recovery rate (>95%) as determined by plaque assays of the final samples. Taken the advantages of the simple and sensitive methods, it was possible to detect 2 pfu of HAV in 5 g of oyster digestive tissues within 24h. 굴 소비자의 보호를 위해서는 매우 민감하고 특이적으로 바이러스를 모니터링할 수 있는 신속진단법의 개발이 필수적이다. 굴 조직은 흔히 비교적 소량의 바이러스와 검출단계를 방해할 수 있는 다른 물질들을 함께 함유한다. 따라서 굴으로부터 바이러스의 검출에서 가장 중요한 과정은 시료의 가공단계이다. 본 연구에 의하면 한번의 sucrose 구배 초원심분리에 의하여 10%와 50% 사이에서 소량의 바이러스를 분리할 수 있음을 제시하였다. 우리는 두 종류의 primer 세트를 이용하여 HAV와 poliovirus를 동시에 굴 조직으로부터 검출할 수 있었다. 또한, 이 방법은 높은(>95%) 바이러스 회수율을 나타내었다. 이 방법은 24시간이내에 5 g의 굴조직으로부터 2 pfu의 HAV를 검출할 수 있을 정도로 신속하고 민감한 검사법이다.

B형 간염 예방 접종 실태 평가를 위한 혈청 역학적 연구 -경기도 지역 초등학생을 대상으로-

이석용,최보율,신영전,방금녀,기모란,박항배,윤재득,이계철,윤배중,Lee, Suk-Yong,Choi, Boyoul,Shin, Young-Jeon,Bang, Keum-Nie,Ki, Moran,Park, Hung-Bae,Yoon, Jae-Deuk,Lee, Kye-Chol,Youn, Bae-Joong 대한소아감염학회 1997 Pediatric Infection and Vaccine Vol.4 No.2

Purpose: To evaluate the hepatitis B vaccination program which has been conducted since 1980, Korea. Methods: This study was carry out self reported questionnaire and serologic test covering 2,072 elementary school students who were born between 1980 and 1987, selected by cluster sampling. The HBV serologic markers (HBsAg, Anti-HBs and Anti-HBc) were tested by radioimmunoassay (RIA). The contents of questionnaire include demographic data of students and parents, vaccination status, vaccination frequency, vaccination age, past history of mother's HBV test. Results: 1) The HBsAg positive rates by sex showed 3.7% for male and 2.7% for female, representing an average rate of 3.4%. The HBsAg positive rates by age group showed 5.6% for 13 years and l.5% for 6 years, representing a tendency of lowering rate as ages being younger. 2) The pre-natal HBV test rate was 10.2%, while post-natal HBV test rate was 42.5%. The test showed that the parents' educational level being higher than others, the pre- and post-natal HBV test showed higher rates. In case the fathers occupation being office worker, the post-natal HBV test showed a higher rate compared with other occupation. 3) Overall vaccination rate was 82.6%, complete vaccination rate 69.8%, booster injection rate 42.8%. The vaccination rate, complete vaccination rate and booster injection rate increased as the age being younger. If the educational level of parents were higher, the vaccination rate, complete vaccination rate and booster injection rate showed higher rates. Younger students showed younger vaccination age, and higher educational background of family showed younger vaccination ages. 4. With regard to positive rate of HBV markers by vaccination age, HBsAg and Anti-HBc positive rate showed higher degrees in the following order; preschool age (1-6 years), school age (6-13 years) and infancy (0-1 year). Anti-HBs positive rate was increased as the frequency of vaccination increased. Five years after initial complete vaccination, minimum protective rate was lowest, 69.6%. 목 적 : 초등학생의 성별, 연령별, 예방 접종률, 접종 완성률, 접종 시기를 비교하고, 간염 표지자 양성률을 검토하여 경기도 지역에서 과거 15년 동안 영아, 유아 및 초등학생을 대상으로 병의원, 보건소 및 학교 등에서 실시한 B형 간염 예방 접종 실태를 파악하고자 하였다. 방 법 : 1993년에 경기도 지역 초등학교 학생들을 집락 추출(cluster sampling)하여 실시하였다. 학교마다 각 학년별로 한 학급을 선정하여 총 2,072 명을 대상으로 혈청학적 검사와 설문 조사를 실시하였다. 결 과 : 1) 성별 HBsAg 양성률은 남자 3.7%, 여자 2.7%였고, 평균 3.4%였다. 연령별 HBsAg 양성률은 7세 3.5%, 12세 5.2%로 연령이 증가함에 따라 높아지는 경향을 보였다. 2) 어머니들의 B형 간염 출산 전 수검율은 10.2%, 출산 후 수검율은 42.5% 였다. 부모의 학력이 높을수록, 아버지의 직업이 사무직인 경우에 출산 전, 출산 후 수검율이 높았다. 3) 전체 예방 접종률은 82.6%, 접종 완성률은 69.8%, 추가 접종률은 42.8%였다. 연령이 증가함에 따라 예방 접종률, 접종 완성률, 추가 접종률이 모두 감소하였다. 부모의 학력이 높을 수록 예방 접종률, 접종 완성률, 추가 접종률이 증가하였다. 연령이 어릴수록 1 세 미만에 접종한 비율이 증가하였고, 7세 이상에 접종한 비율은 감소하였다. 부모의 학력이 높을수록, 아버지의 직업이 사무직인 경우에 1세 미만에 접종한 비율이 높았다. 4) 예방 접종 시기에 따른 간염 표지자 양성률에서 HBsAg과 Anti-HBc 양성률은 1~6세, 7세 이후, 1 세 미만의 순서로 높았다. 접종회수가 증가함에 따라 Anti-HBs 양성률은 유의하게 증가하였다. 기본 3회 접종 후 5년째 된 군에서 Anti-HBs 양성률은 85.7%, 유효 역가율은 69.6% 로 가장 낮았다. 결 론 : 이상의 결과를 통하여 전체 접종률은 82.6%, 접종 완성률은 69.8% 로 아직 낮은 수준으로 나타났다. 특히 부모의 학력이 낮고 아버지 직업의 사회적 지위가 낮은 계층은 출산 전 출산 후 산모의 HBV 수검율, 자녀의 백신 접종률, 접종 완성률이 모두 낮은 것으로 보아 의료 기관의 접근도가 떨어지고 예방 접종의 중요성에 대한 인식도 낮은 것으로 생각된다. 앞으로 B 형 간염 예방 접종 사업은 지속적인 보건 교육과 함께 이러한 사회적 지위가 낮은 계층 자녀의 접종률을 높이는 특별 관리 대책이 필요하리라 생각된다.

2002년 울산지역에서 유행한 무균성 뇌막염에 대한 임상적 고찰

김치관(Chi Kwan Kim),하태영(Tae Young Ha),이준화(Jun Hwa Lee),윤재득(Jae Deuk Yoon),김영돈(Young Don Kim),지영미(Young Mee Jee),박상규(Sang Kyu Park),정진영(Jin Young Jung) 대한소아신경학회 2003 대한소아신경학회지 Vol.11 No.2

목 적 : 2002년 울산지역에서는 무균성 뇌막염이 급속히 유행하여 많은 소아들이 입원하여 치료하였다. 이에 저자들은 울산에서 유행한 무균성 뇌막염의 임상 양상과 검사소견 및 원인을 파악하고자 본 연구를 시행하였다. 방 법 : 2002년 5월부터 7월까지 울산대학교병원 소아과에 입원하여 무균성 뇌막염으로 진단받고 치료한 371명의 환자를 대상으로 성별, 나이, 임상증상에 대해 조사하였고 말초혈액 검사, 뇌척수액 검사를 시행하였으며 바이러스 배양검사는 뇌척수액, 분변을 채취하여 실시하였다. 결 과 : 1) 성별 분포는 남아 242례(65.2%), 여아 129례(34.8%)로 남녀비는 1.9:1로 남아에서 더 호발하였다. 2) 연령별 분포는 0-1세 미만군이 14례(3.8%), 1-5세군이 155례(41.8%), 6-10세군이 115례(31.0%), 11-15세군이 87례(23.5%)로 1-5세군에서 가장 많이 발생하였으며 평균 연령은 6.8±5.1세였다. 3) 임상증상은 발열 346례(93.3%), 두통 308례(83.0%), 구토 292례(78.7%), 복통 91례(24.5%) 순이었고 경부강직은 60례(16.2%)에서 보였으며 발열의 기간은 평균 3.2±1.8일이었다. 4) 말초혈액 검사상 백혈구 수는 평균 10,355±4,800/mm³였으며 CRP는 평균 0.96±1.67 mg/dL였다. 5) 뇌척수액 검사상 백혈수 수는 6-4,830/mm³으로 평균 212±385/mm³였으며 500/mm³ 미만이 88.4%로 대부분을 차지하였으며 다핵구가 평균 29.3±28.9%, 림프구가 평균 55.2±28.8%였다. 뇌척수액의 평균 단백은 37.0±18.9 mg/dL, 당은 68.3±17.6 mg/dL였다. 6) 장바이러스 배양 검사상 바이러스가 분리된 경우는 총 371례 중 67례(18.1%)였다. 이중 분변에서는 98례 중 39례(39.8%), 뇌척수액에서는 371례 중 34례(9.2%)에서 분리되었으며 배양 검사상 분리된 바이러스는 echovirus 6형이 10례, echovirus 9형이 15례, echovirus 11형이 4례, echovirus 13형이 18례, 분류되지 않은 enterovirus가 20례였다. 결 론 : 2002년 울산지역에서 유행한 무균성 뇌막염은 임상 양상이나 뇌척수액 검사소견 등이 지금까지 장바이러스에 의한 뇌막염에 대한 보고와 유사하였으며 원인 바이러스는 echovirus 6, echovirus 9, echovirus 11, echovirus 13, unclassified enterovirus 등이었다. Purpose : There occurred an explosive outbreak of aseptic meningitis in Ulsan, in the year of 2002. In this study, we report clinical manifestations, laboratory data and causative viruses of the outbreak. Methods : Were assessed 371 children with aseptic meningitis who were admitted to the Department of Pediatrics, Ulsan University Hospital in Ulsan between May and July, 2002. We reviewed the sex, age, clinical manifestations, peripheral blood examinations and CSF examinations of the patients. Virus isolation and serotype identification were performed by cell cultures of cerebrospinal fluid and stool. Results : The ratio of males to females was 1.9:1. The majority of aseptic meningitis occurred in children between 1 and 5 years of age with a mean age of 6.8±5.1 years. The clinical manifestations was fever, headache, vomiting, and abdominal pain and 16.2% of the patients developed neck stiffness. The mean duration of fever was 3.2 days. The mean value of peripheral blood leukocytes was 10,355±4,800/mm³ and the mean value of CRP was 0.96±1.67 mg/dL. Initial CSF findings revealed leukocytes 212±385/mm³, protein 37.0±18.9 mg/dL and glucose 68.3±17.6 mg/dL. Viruses were isolated 67(18.1%) out of 371 patients and the serotypes were echovirus 6 in 10 cases, echovirus 9 in 15 cases, echovirus 11 in 4 cases, echovirus 13 in 18 cases, and unclassified enterovirus in 20 cases. Conclusion : Aseptic meningitis was prevalent in Ulsan, in the year of 2002. The clinical manifestations and the laboratory findings of the patients were compatible with those of previous reports. The causative viruses were echovirus 6, echovirus 9, echovirus 11, echovirus 13 and unclassified enterovirus.

원주지역 설사 환자에서 분리한 Small Round Structured Viruses ( SRSV ) 염기서열 분석

지영미(Young Mi Jee),김기순(Gee Sun Kim),천두성(Doo Sung Chun),박정구(Jung Goo Park),강영화(Young Hwa Kang),정윤석(Yoon Suk Jung),고운영(Woon Young Ko),신영학(Young Hak Shin),윤재득(Jae Deuk Yoon) 대한바이러스학회 1999 Journal of Bacteriology and Virology Vol.29 No.4

Small round structured viruses (SRSV) are the major ethological agents which can cause outbreaks of non-bacterial gastroenteritis or food poisoning both in children and adults. The classification of family Caliciviridae to which SRSV belong, is based on the genome encoding three open reading frames. The rotavirus is another major pathogen which causes diarrhea in young children. We examined stool specimens obtained from diarrheal patients in Wonju from which bacterial pathogens were not found. To detect causative viruses from stool specimens of patients, reverse transcription (RT)-polymerase chain reaction (PCR) or nested PCR using rotavirus or SRSV specific primers was performed. In this study, RT-nested PCR procedure which can amplify a 330 bp fragment derived from RNA dependent RNA polymerase (RDRP) region within ORF1 was applied for the detection of SRSV. For the detection of rotaviruses, a 877 bp fragment from the VP4 region of rotavirus genome was amplified. As a result, rotavirus was not detected while SRSV sequences were detected from one out of five specimens. The nucleotide and amino acid sequences of the Wonju isolate were compared with other 6 Korean isolates which have been isolated and sequenced in our laboratory. Sequence analysis revealed that the Wonju isolate was rather distinct from other Korean isolates: the Wonju isolate was closer to genogroup I of SRSV while other 6 Korean isolates belonged to genogroup II.

부산지역 무균성 뇌막염 원인 바이러스의 분리 및 동정 : 1998년을 중심으로

김영희,정영기,김기순,지영미,윤재득,김병준,구평태,민상기,정구영,김만수,조경순 동의대학교 기초과학연구소 2000 基礎科學硏究論文集 Vol.10 No.1

The incidence of aseptic meningitis infection is ensuing and threatening the health of children. Enteroviruses are the major agents of aseptic meningitis and identification of virus has been a clue to diagnosis and epidemiology. The outbreak of aseptic meningitis occurred in Pusan, 1998. Patients were concentrated from April through November. Children were more susceptible than adults. Among 306 cases of specimens from stool, throat swab tested, only 7.2% were positive on virus isolation, 12 cases from stool and 10 from throat, respectively. All isolated 7 serotypes of viruses represented cytopathic effect on cultured cells. Three types of echovirus 6, 25, 30 and coxsackievirus B2, B3, B4, B6 were identified by neutralizing anti body test. Isolated coxsackievirus and echovirus were observed by an electron microscope with negative staining.

강원도 원주시 초등학생 Small Round Structured Virus (SRSV) 유행발생 보고

고운영,신영학,유정식,지영미,김기순,윤재득 대한감염학회 2001 감염 Vol.33 No.3

Small Round Structured Virus (SRSV) is a common cause of non-bacterial acute gastroenteritis in developed countries. In 30 June 1999, a large number of gastroenteritis cases occurred among elementary school children in Wonju province, who had attended the summer camp from 28 to 30 June 1999. We interviewed those students and performed both bacteriological and virological examinations. The reported cases occurred from 30 June, peaked on 1 July and decreased from 2 July, which suggested the point source outbreak pattern. The attack rate was 22.5% according to the clinical case definition which is defined as those who have more than two symptoms among; vomiting, abdominal pain and diarrhea. The most common symptom was diarrhea (48.0%), followed by abdominal pain (38.1%) and vomiting (17.1%). The duration of diarrhea was one to two days. Among the 352 stool examinations, all of the following organisms, Salmonella spp, Shigella app. and EHEC O157, were negative, and in virological examinations of 5 stool specimens for rotavirus, enterovirus, and SRSV, one specimen was positive for SRSV. Sequence analysis showed that detected SRSV was closely related to genogroup I. We concluded that SRSV might be the possible cause of this outbreak (Korean J Infect Dis 33:210∼213, 2001)

부산지역 무균성 뇌막염 원인 바이러스의 분리 및 동정 : 1998 년을 중심으로

조경순,김만수,정구영,민상기,구평태,김병준,윤재득,지영미,김기순,김영희,정영기 한국환경과학회 1999 한국환경과학회지 Vol.8 No.2

The incidence of aseptic meningitis infection is ensuing and threatening the health of children. Enteroviruses are the major agents of aseptic meningitis and identification of virus has been a clue to diagnosis and epidemiology. The outbreak of aseptic meningitis occurred in Pusan, 1998. Patients were concentrated from April through November. Children were more susceptible than adults. Among 306 cases of specimens from stool, throat swab tested, only 7.2% were positive on virus isolation, 12 cases from stool and 10 from throat, respectively. All isolated 7 serotypes of viruses represented cytopathic effect on cultured cells. Three types of echovirus 6, 25, 30 and coxsackievirus B2, B3, B4, B6 were identified by neutralizing antibody test. Isolated coxsackievirus and echovirus were observed by an electron microscope with negative staining.