General - Primer를 이용한 무균성뇌막염 원인 바이러스 분석

김문보,김기순,배유병,송철용,윤재득,이광호,신학균 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.2

Aseptic meningits, an acute inflammation of the meninges, is a common illness during childhood. Virus is the most important cause of aseptic meningitis. Especially enterovirus causes approximately above 85% of all cases of aseptic meningitis. In 1993, there was a big epidemic of aseptic meningitis by ECHO 9 and ECHO 30 viruses. And ECHO 3 virus was isolated as a causative agent of aseptic meningitis in 1994. This study was aimed to detect the causative agent of aseptic meningitis in 1995 and to analyze the 5'-noncoding region which was used to detect virus. Virus was isolated from 87 stools and cerebrospinal fluid specimens of the patients by cultured RD and Hep-2 cell. Neutralizing antibody tests using enterovirus serum pool were performed on the specimens with cytopathic effect. 3 of ECHO 7 viruses and 5 of Coxsackie B3 viruses were isolated from stool specimens and 1 of ECHO 7 and Coxsackie B3 mixed type was confirmed from cerebrospinal fluid specimens. RNA was isolated from the culture supernatants of infected cells and general primers were selected in highly conserved part of the 5'-noncoding region of the enteroviral genome for RT-PCR. PCR product from this virus showed a 152bp band on gel electrophoresis. Sequence of obtained DNA was compared with prototype sequences by accessing to the Genebank database. 5'-noncoding region of isolated Coxsackie B3 virus, which has point mutations in nu.cleotide sequence positions 493, 497, 502, 523, was closely related to that of polio virus type 1, Mahoney strain. In case of isolated ECHO 7 virus, nucleotide has been changed from cytosine to thymine at position 581 and from thymine to cytosine at position 583. We concluded the causative agents of the outbreak of aseptic meningitis during June to July in 1995 were both ECHO 7 and Coxsackie B3 virus, and the primer used in this study could allow a rapid diagnosis of enteroviruses by PCR.

한국인에서 분리된 C형 간염바이러스 단백분해효소의 발현

김문보,송철용 대한바이러스학회 1993 Journal of Bacteriology and Virology Vol.23 No.2

Hepatitis C virus(HCV) is the major causative agent of post transfusion non-A, non-B hepatitis which is often liver disease and developed into the malignant disease such as liver cirrhosis and hepato cellular carcinoma. To find out antigenic proteolytic enzyrne of HCV, an experiment to express proteinase of HCV was carried out by PCR using isolated DNA from sera of HCV carriers. And the nucleotide and deduced amino acid sequence of noncoding and core region of HCV were also analyzed by DNA star software. The nucleotide and deduced arnino acid sequence of noncoding and core region of HCV genome isolated in Korea showed a sequence homology at the nucleotide level of about 67.5% with the Japanese HCV prototype, and about 63.5% homology with the American HCV prototype. It implicated that the Korean isolates of HCV were more closely related to the Japanese HCV. Viral serine-like proteinase DNA was isolated and expressed in E. coli KM-392 strain and two expressed proteins, 15-22KDa and 25-32 Kda, were reactive to monoclonal anti- body against HCV and to sera of hepatitis C patient by immunoblotting analysis. This result indicates that 15-22KDa and 25-32 Kda serine-like proteinase of HCV expressed in E. coli could be used as an serodiagnostic antigen for anti-HCV and immunogens for HCV vaccine.

1993년 한국에서 발생한 Aseptic meningitis 환자로부터 장내바이러스 RNA 검출

김문보,김기순,김창민,서순덕,윤재득,신학균 대한바이러스학회 1994 Journal of Bacteriology and Virology Vol.24 No.1

Aseptic meningitis is known that the sign of neurological diseases are present except paralysis even though the main sign and symptoms are fever and headache with nuchal rigidity of which is stiffness of the neck muscles due to meningeal irritation but the damage is minor. Outbreakes are very common in young aged group during the warm season through the infection is spread mainly by the fecal-oral route from virus excretors to contacts and virus in pharyngeal secretion is also source of infection. Since sporadic clinical cases of aseptic meningitis has reported in 1990, one of epidemic during the month of May, 1993 due to let enterovirus teams in NIH, Korea carried out seroepidemiological investigation against cases of aseptic meningitis which is a first time in Korea. Stool and CSF specimens from 3S patients with aseptic meningitis were tested by neutralization test and PCR derived from the highly conserved 5-noncoding enterovirus' genomic region. We found a higher infection rate in the 1-9 years old group(68.5 %) and in the male sex(68.5 / ). ECHO type 9 and 30 were isolated in fecal specimens and coxsackie type B was isolated in CSF. The PCR assay yielded positive results in 7 cases where enterovirus diagnosis were obtained by serological test.

한국인에서 분리된 C형 간염 바이러스의 항원성 단백질 발현

김문보 中央大學校 遺傳工學硏究所 1993 遺傳工學硏究論集 Vol.6 No.1

우리나라 C형 간염 보균자에서 HCV DNA를 분리하여 noncoding 부위와 core부위의 DNA염기서열을 밝히고, E. coli에서 항원성 단백질을 발현시킨 결과 다음과 같은 결과를 얻었다. 1. Noncoding 부위와 핵 부위에서 HCV DNA의 염기서열을 분석한 결과 분리된 HCV의 염기서열은 미국형과 63.5%, 일본형과 67.5%의 동질성을 보여 미국형보다 일본형에서 유사하였다. 2. HCV core DNA로 발현시킨 핵 단백질의 항원성을 관찰한 결과 분자량 20-22 KDa와 47KDa 부위에서 항원성을 보였다. 3. HCV의 유전자 내에 serine proteinase의 DNA가 존재함을 확인하였으며, 이들 유전자에 의해 발현된 단백질은 serine-like proteinase였고, 이들 효소는 C형 간염 양성자 혈청 및 단클론 항체와 특이적으로 면역반응 하므로서 그 항원성이 증명되었다. 4. 이들 효소 단백질의 분자량은 SDS-PAGE상에서 15-22 KDa과 25-32 KDa이었다. Hepatitis C virus (HCV) is the major causative agent of post transfusion non-A, non-B hepatitis which is often liver disease and developed into the malignant disease such as liver cirrhosis and hepato cellular carcinoma. An experiment to express antigenic protein of HCV was carried out by PCR using isolated DNA from sera of Korean HCV carriers. And the nucleotide and deduced amino acid sequence of noncoding and core region of HCV were also analyzed by DNAstar software. It showed sequence homology at the nucleotide level of about 67.5% with the Japanese HCV prototype, and about 63.5% homology with the American HCV prototype. Core protein of structural region of HCV was expressd in Escherichia coli(E.coli) XL-1 blue strain. Two induced protein, 22 KDa and 47 KDa, were reactive to HCV monoclonal antibody and sera of patients with chronic HCV by immunoblotting analysis. Viral serine-like proteinase DNA was isolated and expressed in E.coli KM-392 strain and two expressed proteins, 15-22 KDa and 25-32 KDa, also were reactive to monoclonal antibody and patients sera by immunoblotting anaysis. These result indicates that 22 and 47 KDa core protein, and 15-22 and 25-32 KDa serine-like proteinase of HCV expressed in E.coli could be used as an serodiagnostic antigen for anti-HCV and immunogens for HCV vaccine.

韓國人에서 分離된 淋菌의 藥劑耐性에 關한 硏究

宋哲鏞,金汶普 중앙대학교 기술과학연구소 1984 기술과학연구소 논문집 Vol.14 No.-

The measurement of the sensitivity of gonococci to antibiotics is great importance in the rational uses of chemotheraphy, evolution of new agents and epidemiological studies. Especially, penicillinase-producing Neisseria gonorrhoeae (PPNG) must be regarded as a serious threat in public health. In connection with recent increasing discoveries of PPNG strains, this study was proposed to investigate the isolation rates of N. gonorrhoeae to the high risk populations and antimicrobial susceptibility of current strains. The strains of N. gonorrhoeae tested Seoul, Incheon, Kyungki, Gangwon, Chung Buk, Chung Nam, Kyung Buk and Busan were collected April to September in 1982. Antibiotics used for the susceptibility test by the agar dilution method (Wilkinson 1980) were penicillin-G, ampicillin, tetracycline, chlorampenicol, cefuroxime, spectinomycin, kanamycin, sulfamethoxazole and trimethoprim. The result was summarized as follows: The isolation rates of N. gonorrhoeae of the prostitute girls were about 7.2% and determinated PPNG strains were about 20.2%. However, according to the areas, there were many differences about the determination rate of PPNG from 0% to 41.7%. PPNG strains were shown higher resistant to penicillin-G and ampicillin than non-PPNG strains. The minimal inhibitory concentration (MIC) range of penicillin-G was 0.25 ㎍/ml∼64 ㎍/ml and 19 Cultures (20.21%) were resistant (MIC≥4.0 ㎍/ml). MIC range of ampicillin was 0.25 ㎍/ml ∼ 32 ㎍/ml and 76 cultures (80.8%) had MIC≥4.0 ㎍/ml. Spectinomycin was susceptible to all strains of PPNG and non-PPNG which were in MIC≤16 ㎍/ml. MIC range of sulfamethoxazole was 16 ㎍/ml ∼ 512 ㎍/ml and MIC range of sulfamethoxazole plus trimethoprim was 8 ㎍/ml ∼ 64 ㎍/ml. MIC range of cefuroxime was 0.125 ㎍/ml ∼ 0.5 ㎍/ml and cefuroxime was susceptible to all strains of PPNG and non-PPNG which were in MIC≤0.5 ㎍/ml.

C형 간염 바이러스의 검출 및 유병률 조사 : 우리나라 C형간염의 의학적 특성

윤재득,김문보,김기순,서순덕,안광수,신학균,정상인 국립보건연구원 1994 국립보건원보 Vol.31 No.1

1994년도 무균성 뇌막염 환아에서 원인바리러스 동정

김종현,김문보,윤재득,김기순,서순덕,박재현,김동수 대한감염학회 1995 감염 Vol.27 No.1

목 적 : 뇌막염은 뇌막의 염증성 반응으로 여러가지 병원균에 의해서 올 수 있다. 특히 소아에서는 무균성 뇌막염이 많은 것으로 알려져 있고 이중 대다수가 바이러스에 의한 것이다. 특히 장바이러스가 무균성 뇌막염의 약 85%를 차지하고 있다. 1993년도 우리나라에서는 echovirus 9, 30에 의한 뇌막염의 전국적인 대유행이 있었다. 이러한 원인 바이러스는 매년 달라질 수 있으므로 1994년도 봄철에 뇌막염으로 입원하는 환자들의 원인 바이러스를 동정하여 보고하는 바이다. 방 법 : 1994년도 5월과 6월에 걸쳐 연세대학교 의과대학 세브란스 소아과에 입원하여 무균성 뇌막염으로 진단된 환아 11예를 대상으로 임상적 관찰 및 바이러스 동정을 시행하였다. 가검물은 환아의 뇌척수액과 대변을 동시에 채취하여 배양된 RD세포와 HEP-2 세포에 접종하여 세포병변을 관찰한 후 enterovirus serum pool 에 의한 중화 시험을 하였다. Piore와 Nicoletta의 방법에 의하여 세포배양액에서 RNA를 추출하고 PCR에 의하여 oligonucleotide를 합성하였다. 그 후 noncoding RNA PCR에 의하여 DNA를 합성하고 전기영동을 거쳐 확인하였다. 결 과 : 1) 임상양상 및 경과는 전형적인 무균성뇌막염의 양상을 보였다. 2) 총 11예 중 4예에서 echovirus가 자랐으며, 1예에서 뇌척수액에서만 배양된 것을 제외하고는 3예에서 뇌척수액과 대변에서 모두 echovirus 3가 배양되었다. 1예에서는 뇌척수액에서 echovirus 3이외에 coxsackie B virus가 검출되었다. 3) PCR product도 전형적인 장바이러스에 합당한 소견을 보였다. 결 론 : 1994년도 봄철에 연세대학교 의과대학 세브란스병원 소아과에 무균성뇌막염으로 입원한 환자의 원인 바이러스는 echovirus 3에 의한 감염이었다. Background : Aseptic meningitis, an acute inflammation of the meninges, is a common illness during childhood. Although the etiologic agent is not always identified, viruses are the usual responsible agents. Enterovirus especially causes approximately 85% of all cases of aseptic meningitis. In 1993, there was a nationwide epidemic of aseptic meningitis by echovirus 9 and 30. This study was done to detect the causative virus of aseptic meningitis in 1994. Methods : To isolate the causative viruses, stool and cerebrospinal fluid specimens from the patients with aseptic meningitis, who were admitted to Severance Hospital in 1994, were collected. Cultured RD cell and HEP-2 cell were inoculated with specimens to see the cytopathic effects. Neutralizing antibody tests using enterovirus serum pool were done on the specimens with the cytopathic effects. RNA was isolated from the cultured supernatants of the infected cells by Piore and Nicoletta method. Oligonucleotide was synthesized by PCR, which was run on polyacrylamide gel after purification with HPLC. After running the DNA produced by using Geneamp RNA PCR Kit, electrophoresis was done. Results : Echovirus was isolated from 4 out of 11 patients. Among these four, echovirus 3 was isolated from both CSF and stool in three patients. In one of these three, Coxsackie B virus was isolated as well from CSF. In the remaining one patient, echovirus 3 was present in CSF only. PCR product from this virus showed a 114 bp band on gel electrophoresis, which was the same PCR product from enterovirus isolated from stool specimens of the patients with aseptic meningitis in 1993. Conclusion : The causative virus of aseptic meningitis in patients who were admitted to Severance Hospital during the spring season of 1994 was Echovirus 3.

유방암에서의 3차원 조직배양 항암제 감수성 검사(HDRA)의 유용성 검토

윤호성,안세현,곽희숙,김문보 대한외과학회 2001 대한외과학회 학술대회 초록집 Vol.2001 No.5

한국인에서 E형 간염 바이러스의 항체 유병률에 관한 조사

신학균,윤재득,정연호,김문보,서순덕,김정서 대한바이러스학회 1992 Journal of Bacteriology and Virology Vol.22 No.2

Serosurvey of hepatitis E virus (HEV) infection, known as enterically transmitted non-A, non-B hepatitis (ET-NANBH), was done in Korea between May through August, 1991 using anti HEV EIA method developed by Genelabs Inc. in 1990. The coated polypeptide antigens were expressed in Baculovirus vector from the specific HEV cDNA clones derived from two patients infected with HEV, one from Burma (antigen A) and the other from Mexico (antigen B). The 1,079 examinees was arbitrarily grouped into risk (406) and non-risk (673) group according to the serologic markers of hepatitis B virus (HBsAg, HBsAb, and HBcAb), anti-hepatitis C virus (HCV), and alanine transarninase (ALT) value but not to the risk of HEV. The overall seroprevalences of HEV in non-risk and risk group were 10.4 %, and 8.9%, respectively. Among the non-risk subgroups, the prevalence of anti-HEV was about 10 % regard- less of the result of HBsAb or HBcAb. There was no sexual and regional difference. The peak prevalence was shown at the age between 30 and 39. In the risk groups, the highest prevalence was 15% shown in the anti-HCV positive group and the lowest was the 5.9% of HBsAg positive group. There was a little higher prevalence rate in male group but there was no significant statistical difference between male and female. Anti-HEV posit,ive rates of normal group were relatively higher than the expected risk group and these results strongly suggested the possibility of existence of HEV infection in Korea, particularly in rural areas where the tap water supply facilities are not enough.

쯔쯔가무시病에 關한 血淸疫學的 硏究

申鶴均,金喜衍,張永植,金汶普,鄭潤鎬,徐順德 국립보건연구원 1989 국립보건원보 Vol.26 No.