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Isolation of porcine epidemic diarrhea virus (PEDV) in Korea
권창희,권병준,정태성,기영진,허동호,황의경,이재진,안수환,Kweon, Chang-hee,Kwon, Byung-joon,Jung, Tae-sung,Kee, Young-jin,Hur, Dong-ho,Hwang, Eui-kyung,Rhee, Jae-chin,An, Soo-hwan The Korean Society of Veterinary Science 1993 大韓獸醫學會誌 Vol.33 No.2
설사로 폐사한 자돈의 장 절편을 이용하여 돼지 유행성 설사바이러스(PEDV), 돼지 전염성 위장염 바이러스(TGEV), 돼지 로타 바이러스(PRV)에 대한 병인학적 검사를 형광항체반응을 통하여 조사하였던 바 돼지 유행성 설사 바이러스의 감염에 의한 자돈의 폐사를 확인하였다. 간접형광항체검사시 돼지 유행성 설사 바이러스에 대한 양성반응을 보인 가검재료를 이용 Vero세포에 연속 계대한 후 plaque assay를 통하여 크로닝된 돼지 유행성 설사 바이러스 KPEDV-9주를 작성하였다. 돼지 유행성 설사 바이러스에 대한 면역혈청과 바이러스가 분리된 농장에서 채취된 돼지 혈청을 이용 돼지 유행성 설사 바이러스에 대한 구조단백성분을 분석하였던 바 88K(M.W.), 74K, 70K, 58~54K, 54~46K, 44~40K 및 33~32K에 상당하는 단백성분을 검출할 수 있었다. The etiological survey on porcine epidemic diarrhea virus(PEDV) by immunofluorescence antibody test(IFA) showed the positive rusult from the intestines of piglet died from acute diarrhea. The viral agent of PED was also isolated from intestine, which showed positive reaction by immunofluorescence test. After passage in Vero cell, the viral agent was further cloned by plaque purification and designated as KPEDV-9. The immunoblotting analysis using hyperimmune sera and porcine sera revealed the presence of several polypetide bands with molecular weight(M.W.) of 88K, 74K, 70K, 58~54, 54~46K, 44~40K and 33~32K, respectively.
보행실조증 자돈의 뇌조직에서 돼지 호흡기 코로나바이러스의 분리
권창희,한명국,이재길,황의경,강영배,이광원,Kweon, Chang-hee,Han, Myung-guk,Lee, Jae-gil,Hwang, Eui-kyung,Kang, Yung-bai,Lee, Kwang-won 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.2
A cytopathogenic virus was isolated from the brain tissues of pig showing ataxia. The biophysical, morphological and serological assay showed that the isolate belongs to a coronavirus. The differential identification of the isolate with monoclonal antibodies against A and X sites of transmissible gastroenteritis virus indicated that the virus has a characteristics of porcine respiratory coronavirus. The RT-PCR on nucleocapsid region of TGEV also showed that the isolate has the same conserved sequence. The diverse pathogenesis of PRCV and its implication in field were discussed.
돼지 폐포탐식세포로부터 adenovirus의 국내분리주 작성
권창희,조재진,권병준,송재영,채찬희,Kweon, Chang-hee,Cho, Jae-jin,Kwon, Byung-joon,Song, Jae-young,Chae, Chanhee 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.1
One cytopathogenic virus was isolated in alveolar macrophages from pig without any apparent respiratory clinical signs. Biophysical properties and electron microscopy of the isolate showed the characteristics of adenovirus. Intracytoplasmic inclusion bodies were seen in virus-inoculated cells. The genetic analysis indicated the presence of DNA with the size of >20Kb. In a serological survey of 40 serum samples collected from two different farms in slaughter house, 9 sera were positive for neutralizing antibody against the isolate. The potential implications of the isolate as the causative agent in respiratory disorder were discussed.
Isolation of porcine reproductive and respiratory syndrome virus(PRRSV) in Korea
권창희,권병준,이한정,조재진,황의경,신진호,윤용덕,강영배,안수환,김용희,허원,전무형,Kweon, Chang-hae,Kwon, Byung-joon,Lee, Han-jung,Cho, Jae-jin,Kang, Yung-bai,An, Soo-hwan,Kim, Yong-hee,Huh, Won,Jun, Moo-hyung,Wensvoort, G.,Yoon, Yong-d The Korean Society of Veterinary Science 1994 大韓獸醫學會誌 Vol.34 No.1
유사산 태아의 폐, 청색증을 나타내는 자돈으로부터 돼지생식기 및 호흡기증후군(PRRS)의 원인체로 추정되는 바이러스주(KPRRSV) 들을 분리하였다. 분리된 바이러스주는 돼지콜레라, 돼지오제스키병, 돼지뇌심근염바이러스에 대한 형광항체반응에서는 음성이었으며 기니픽혈구에 대한 혈구응집 능력을 나타내지 않았다. 그리고 포유 마우스의 뇌내 접종시 이상을 나타내지 않았으나 돼지생식기 및 호흡기증후군에 대한 형광항체검사시 양성반응을 나타내었다. 분리된 바이러스는 돼지폐포탐식세포(porcine alveola macrophages)에서 세포변성효과(cytopathic effect)를 나타내었으며 세포변성효과를 나타내었던 바이러스주중 일주(KPRRSV-1)를 돼지폐포탐식세포에서 7대 연속 계대하여 돼지에 접종한 후 혈청을 분리하여 미국 및 유럽지역에서 분리된 돼지유행성 유사산 및 호흡기증후군의 바이러스를 탐식세포에 감염시켜 효소면역방법 (immunoperoxidase monolayer assay)으로 분석한 결과 분리된 바이러스는 미국형 돼지호흡기 및 유사산증후군에 가까운 항원형으로서 판명되었다. Three viral strains, causing CPE in porcine alveolar macrophage cell, were isolated from aborted fetus, serum from young pig showing blue-ear sign and lung of suspected pig, respectively. The differential diagnostic results showed no characteristics of Aujeszky's disease virus(ADV), hog cholera virus (HCV), Japanese encephalitis virus(JEV), porcine parvovirus(PPV) and encephalomyocarditis virus (EMCV). However, positive reactions were demonstrated by IFA using monospecific porcine antibodies against Lelystad virus. When the paired sera of experimentally inoculated swine with one of isolate, KPRRSV-l were tested by IPMA, the result indicated that the isolate was related to United States isolate than European LV.
Biophysical characteristics of a noncytopathic bovine viral diarrhea virus
권창희,Castro E Anthony,우희종,Kweon, Chang-hee,Anthony, Castro E,Woo, Hee-jong The Korean Society of Veterinary Science 1992 大韓獸醫學會誌 Vol.32 No.1
A noncytopathic bovine viral diarrhea virus(NC BVDV) strain isolated and purified from persistently infected primary bovine fetal lung(BFL) cells was studied by biophysical methods. The buoyant density of particles of the NC BVDV strain was shown to be between 1,090 and $1,114g/cm^3$ and the maximum virus infectivity occured at $1,098g/cm^3$. Immunoelectron microscopic examination by using the partially purified virus revealed regular spherical particles 30~80nm in diameter. Differences in the genomic size of cytopathic and noncytopathic BVDV from infected cells were not found. A comparison of viral proteins of a noncytopathic and cytopathic strain(NADL) by immunoprecipitation using monoclonal antibody indicated that NC BVDV, compared to cytopathic NADL, was cell associated. 세포 비병원성 소 설사병 바이러스가 감염된 우태아 폐세포를 이용 바이러스를 순수분리하여 이화학적 성상을 검사하였던 바 다음의 결과를 얻었다. 바이러스의 비중은 $1.090{\sim}1,114gm/cm^3$로 검출되었으며 $1,098gm/cm^3$에서 최대 감염가를 나타내었다. 면역 전자현미경을 이용한 형태학적 분석결과 30~80nm의 바이러스성 입자를 관찰할 수 있었으며 유전인자의 추출 분석결과 병원성 바이러스와의 전기영동상 유전자 규모에 있어서 차이는 인정되지 않았다. 그러나 비세포 병원성 소 설사병 바이러스는 병원성 바이러스에 비하여 세포외 배출이 적었으며 세포내에서 보다 많이 존재하는 것으로 관찰되었다.
Monoclonal antibodies against structural proteins of bovine viral diarrhea virus
권창희,우희종,Kweon, Chang-hee,Zee, Yuan Chun,Woo, Hee-jong The Korean Society of Veterinary Science 1992 大韓獸醫學會誌 Vol.32 No.1
소 설사병 바이러스 구조단백에 대한 단크론항체를 작성하여 혈청중화시험, 전기영동, 면역침전반응을 이용하여 분석하였던 바 다음의 결과를 얻었다. 중화능력이 있는 항체의 경우 56K내지 54K의 구조단백에 대응하였다. 그외 중화력을 나타내지 않는 항체는 45K와 36K의 바이러스 항원과 대응하였다. 순수정제된 바이러스의 전기영동 분석결과 12종 이상의 바이러스 단백성분이 구조단백질로서 검출되었으며 중화능력을 나타내는 항체를 이용한 면역침전 결과는 이들의 존재를 뒷받침하였다. 중화단백성분의 세포내 전구물질의 검출은 불가능하였으나 방사선동위원소 부착즉시 세포배지에서 바이러스의 존재를 확인할 수 있었다. Staphylococcus aureus $V_8$효소를 이용한 항원의 부분소화 분석결과 45K와 36K의 바이러스 항원은 서로 상관이 있는 것으로서 입증되었다. Monoclonal antibodies against structural proteins of bovine viral diarrhea virus(BVDV) were derived by classical hybridoma techniques. These antibodies were characterized by serum neutralization, immunoblotting and immunoprecipitation. The neutralizing monoclonal antibody reacted with the 56kd to 54kd(M.W.) viral protein in western blotting and immunoprecipitation analysis. Although there was no neutralizing activity, another monoclanal antibody reacted with the 45kd protein by immunoprecipitation and with both the 45kd and 36kd proteins in immunoblotting analysis. respectively. Densitometer scanning of purified BVDV and the immunopreipitation of whole virus particles with neutralizing monoclonal antibody revealed the presence of more than twelve viral polypeptides. Although no possible precursor form of protein was identified with the neutralizing monoclonal antibody. the presence of intact virion was detected in the infected cell supernatant immediatelty after pulse labeling, indicating rapid translational processing as well as packaging of the virus. The partial peptide mapping of 45kd and 36kd proteins with Staphylococcus aureus V 8 protease showed that these two proteins are related.
권창희,권병준,강영배,안수환,Kweon, Chang-hee,Kwon, Byung-joon,Kang, Yung-bai,An, Soo-hwan The Korean Society of Veterinary Science 1994 大韓獸醫學會誌 Vol.34 No.2
돼지 유행성 설사 바이러스(KPEDV-9)주를 이용하여 세포내 증식성을 비롯한 혈청학적 역학조사를 수행하였던 바 다음의 결과를 얻었다. 돼지 유행성 설사바이러스 국내 분리주는 Vero 세포에 연속계대시 증식성이 증가되었으며 90대 계대시 $10^{5.5}TCID_{50}/ml$의 역가를 나타내었다. 조직배양 순화주를 이용하여 간염세포내에서 20Kb 이상의 RNA가 존재함을 확인할수 있었으며 전자현미경 검사시 5~10nm의 외피항원 및 80~300nm크기의 coronavirus특징을 나타내었다. 설사증상을 나타내는 돼지의 장가검재료를 이용하여 유행성 설사 바이러스의 감염실태를 조사하였던 바 18%에 상당하는 감염 양성율을 확인하였으며 ELISA법에 의한 항체검사결과 전국적으로 약 45%의 항체 양성율을 나타내었다. Korean isolate, porcine epidemic diarrhea virus (KPEDV-9) was adapted through serial passages in vero cell. The viral yield reached up to $10^{5-6}$ $TCID_{50}/ml$ at the passage level of 90th. The cell adapted virus was characterized through genetic and morphological examinations. The RNA extracted from virus infected cell revealed the presence of RNA band with molecular size of >20Kb. The electron microscopic examination on purified virus showed the pleomorphic appearance of enveloped particles with 5-10nm surface projections, which fit with the shape of coronavirus. The etiological survey on swine diarrhea by immunofluorescence test(FA) indicated 17.5% positive rate on the PEDV infection. In addition, the incidence were detected both in piglets within two weeks old as well as fattening pigs. Serological survey by ELISA revealed the overall 45% positive result, thus, indicating the PEDV infection are widespread throughout this country.
Pseudorabies virus의 gp50과 gp63 유전자 클로닝에 관한 연구
권창희,송재영,김병한,이중복,이재진,안수환,이영순,Kweon, Chang-hee,Song, Jae-young,Kim, Byoung-han,Lee, Jung-bok,Lee, jae-chin,An, Soo-hwan,Lee, Yong-soon,Susumu, Maeda 대한수의학회 1991 大韓獸醫學會誌 Vol.31 No.3
The DNA fragment representing for Pseudorabies gp50 and gp60(Shope) was cloned by recombinant techniques. The viral DNA was extracted from the infected cells and digested with Bam HI. The 6.8 Kb of Bam HI fragment was isolated from agarose gel and further digested with Nde I followed by Klenow treatment. The blunt ended 4.9Kb fragment was cloned into pTZ18R plasmid vector. The upstream region of gp50 was further manipulated to remove its 5' promoter region and create EcoRl site for possible eukaryotic expression system. The result of partial sequencing of cloned DNA indicated that Shope strain showed 95% homology with gp50 of Rice strain.