바이러스 불활화 공정에 대한 Hepatitis A Virus와 Murine Encephalomyocarditis Virus의 민감도 비교

김인섭,Kim, In-Seop 한국미생물학회 2003 미생물학회지 Vol.39 No.4

Murine encephalomyocarditis virus (EMCV)는 혈장유래의약품의 바이러스 안전성 검증을 위해 hepatitis A virus (HAV)의 모델 바이러스로 사용되어왔다. 근래에 혈액응고인자제제에 의한 HAV 감염사례가 보고되면서 혈장유래의약품의 HAV 안전성 검증에 대한 국제적인 규제가 강화되어가고 있다. 본 연구에서는 HAV와 EMCV의 바이러스 불활화 공정에 대한 민감도를 평가하여, 혈장유래의약품 제조공정에서 HAV 불활화 공정의 검증법을 표준화하고자 하였다. HAV와 EMCV의 바이러스 불활화 공정에 대한 민감도를 평가한 결과 HAV가 60$^{\circ}C$ 열처리, low pH 처리(pH 3.9), 0.1 M NaOH 처리, 동결건조 공정 모두에서 EMCV보다 더 저항성이 큰 것을 확인할 수 있었다. EMCV는 특히 열처리와 0.1 NaOH 처리에 민감하게 불활화 되었지만, HAV는 큰 저항성을 나타내었다. 열처리의 경우 2시간 안에 EMCV는 검출한계 이하로 감소하였지만, HAV는 5시간 후에 검출한계 이하로 감소하였다. 0.1 M NaOH 처리시 EMCV는 15분 안에 검출한계 이하로 감소하였지만, HAV는 120분 정도의 처리에도 감염성 바이러스가 검출되었다. pH 3.9에서 25$^{\circ}C$로 14일 동안 항온하였을 때 HAV와 EMCV의 log 감소인수는 각각 1.63, 3.84이었다. 또한 혈액응고 8인자 제조공정의 동결건조 과정에서 HAV와 EMCV의 log 감소인수는 각각 1.21, 4.57이었다. 이와 같은 결과는 혈장유래의약품 제조공정의 HAV 불활화 또는 제거 검증시 모델 바이러스로 사용된 EMCV의 검증 결과를 해석함에 있어 보다 신중함을 가져야 한다는 것을 보여준다. 또한 보다 정확한 HAV검증 결과를 얻고자 한다면 모델 바이러스인 EMCV 보다 HAV를 사용하는 것이 보다 더 타당하다고 사료된다. Murine encephalomyocarditis virus (EMCV) has been used as a surrogate for hepatitis A virus (HAV) for the validation of virus removal and/or inactivation during the manufacturing process of biopharmaceuticals. Recently international regulation for the validation of HAV safety has been reinforced because of the reported cases of HAV transmission to hemophiliac patients who had received ntihemophilic factors prepared from human plasma. The purpose of the present study was to compare the resistance of HAV and EMCV to various viral inactivation processes and then to standardize the HAV validation method. HAV was more resistant than EMCV to pasteurization (60oC heat treatment for 10 hr), low pH incubation (pH 3.9 at 25oC for 14 days), 0.1 M NaOH treatment, and lyophilization. EMCV was completely inactivated to undetectable levels within 2 hr of pasteurization, however, HAV was completely inactivated to undetectable levels after 5 hr treatment. EMCV was completely inactivated to undetectable levels within 15 min of 0.1 M NaOH treatment, however, residual infectivity of HAV still remained even after 120 min of treatment. The log reduction factors achieved during low pH incubation were 1.63 for HAV and 3.84 for EMCV. Also the log reduction factors achieved during a lyophilization process of antihemophilic factor VIII were 1.21 for HAV and 4.57 for EMCV. These results indicate that HAV rather than EMCV should be used for the virus validation study and the validation results obtained using EMCV should be precisely reviewed.

한탄바이러스 게놈 RNA의 packaging mechanism에 관한 연구

박찬,유정희,이연승,이호동,박근용,이평우 국립보건연구원 1999 국립보건원보 Vol.36 No.-

한국산 조류와 박쥐의 Hantavirus 감염에 대한 혈청역학적 조사

이호왕,백락주,이연태 대한바이러스학회 1991 Journal of Bacteriology and Virology Vol.21 No.2

Hantaan virus, the etiologic agent of Korean hemorrhagic fever (KHF) was discovered from the lungs of Apodemus agrarius caught in Songnae-dong, Dongduchun city, Kyunggi province in 1976 in Korea and it was designated Hantaan virus after Hantaan river in 1980. Seoul virus, Puumala virus, Prospect Hill virus and Leaky virus that are rnembers of genus Hantavirus were isolated from lungs of house rats, Clethrionomys glareolus, Microtus pennsylvanicus and Mus musculus, respectively. New we know that natural reservoirs of hantaviruses are not only field mice but also house rats and other animals in many parts of the world. This experiment was designed to find the distribution of Hantavirus infection among the wild birds and bats in Korea from 1989 to 1990. The results were as follows ; 1. Among the 166 wild brids of 15 species, 14 Paradoxomis vebbiana and 1 Emberiza elegans were immunofluorescent (IF) antibody positive against Hantaan virus. IF antibody titers against Hantaan virus of seropositive sera from wild birds ranged between 1:16 to 1:256. 2. Among the 143 wild bats of 2 species, 3 Rinolophus ferram-equinam and 1 Vespertilo abramus were IF antibody positive against Hantaan virus, and 2 Rhinolophus ferrum-equinum were IF antibody positive agsinst Seoul virus. IF antibody titers against Hantaan and Seoul virus of seropositive sera from wild bats ranged 1:16 to 1:256. The above results are the first evidences of hantavirus infection among wild birds and bats in Korea.

Maaji Virus의 Humster 계대 및 적응

김윤철,백우현,이평우 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.1

The methods that make Hantavirus grow consist of inoculation into the experimental animals and cultured cells. The cultured cells, such as Vero-E6 and A549 cells, have been usually used for isolation of the virus and the animals, such as mice and rats, are used for large scale preparation of the virus so far. Furthermore, the cell can be used to maintain the virus and assay the infectivity and the animals can be used for the experiment of viral pathogenicity and challenge for assessment of vaccine. Apodemus mice, the own natural host of the virus, has been used for challenge test of Hantaan virus. However it has been pointed out to difficult handling and breeding the animal in laboratory. Therefore, we attempted to establish a new animal model for challenge test at the time of isolation of Maaji virus which is a new hantavirus similar but distinct to Hantaan virus. In suckling hamster, the titer of Maaji virus and the lethality to mice of the virus were increased gradually in the titer and lethality through passage by intracerebral (IC) inoculation. We tried to re-adapt this brain virus to lung of weanling hamster. The brain passaged virus was inoculated into weanling hamster intramuscularly. Again, the titer of the virus in lung was also increased by continuous passage of this virus. This facts could regarded as adaptation to new environment in which the virus proliferates. To identity the virus passaged in hamster with Maaji virus, both of the virus passaged in hamster brain and lung were compared with Maaji virus (MAA-I) and Hantaan virus (HTN 76-118) by means of restriction fragment length polymorphism (RFLP) and slingle strand conformation polymophism (SSCP). As a result, we conclude that Maaji virus could be adapted successfully to weanling hamster through this passage strategy. Utilizing this adapted Maaji virus strain, hamster model is able to be used for challenge test in hantaviral vaccinology and further experiments utilizing hamster system as a rather available and convenient lab animal are expected.

2006년과 2007년 상주와 구례에서 발생한 오이 바이러스병의 병징 특성

조점덕,김정수,이중환,고숙주,최홍수,이수헌,최국선 한국식물병리학회 2011 식물병연구 Vol.17 No.2

Virus diseases occurring on cucumber was surveyed at main plantation areas of ‘Sangju’ and ‘Gurye’ in 2006and 2007. Viral infection rate on cucumber was ranged from 14% to 90% in fields and the average infection rate was 46% at Sangju area. Cucumber cultivated at Gurye area had viral incidence ranged 9−100% and averaged 48%. The majorly infected viruses were Cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, Papaya ring spot virus and Watermelon mosaic virus-2, and their infection rates were 23.5%,13.0%, 9.0% and 2.0%, respectively in 2006 and 2007. Mixed infection rate of duplex, triplex and tetraplex was 31.5%, 7.5% and 2.5%, respectively. The rate of infection type was 47.7% by single infection, 31.5% by double infection and 88.7% by both infection types. Important viruses involved in mixed infection types were Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, Papaya ring spot virus and Broad been wilt virus2, orderly. Symptom in single infection was almost same on ‘Dadagi’ line and ‘Chicheong’ line of cucumber cultivars. Cucumber green mottle mosaic virus produced various symptoms of chlorotic spot, vein chlorosis and vein wrinkle and so on. Main symptoms of vein chlorosis, and severe mosaic and malformation were induced by Zucchini yellow mosaic virus. Mild symptoms were occurred relatively by Papaya ring spot virus and Watermelon mosaic virus2. 오이 주요재배지인 상주와 구례에서 오이에 발생하는 바이러스병을 2006년과 2007년에 조사하였다. 경상북도 상주 지역 오이의 병징 발생률은 농가 포장에 따라서 14%에서 90%이었으며 평균 발생률은 46%이었다. 전라남도 구례 지역의 경우 농가 포장에 따라서 9%에서 100%이었으며 평균 발병률은 48%이었다. 주요 발생 바이러스는 CGMMV, ZYMV, PRSV와 WMV2의 4종류였다. 이러한 바이러스는 단독감염과 복합감염 형태로 발생하였다. 2006년과 2007년의 평균 감염률을 보면 단독감염의 경우 ZYMV가 23.5%로 가장 많이 발생하고 있었으며, PRSV가 13.0%, CGMMV가 9.0%, WMV2가 2.0% 순서이었다. 복합감염의 경우에는 2종 바이러스 복합감염이 31.5%, 3종 복합감염이 7.5%, 4종 복합감염이 2.0% 발생하였다. 오이에 발생하고 있는 바이러스 감염형태는 단독감염과 2종 복합감염이 각각 47.7%와 31.5%로 총 88.7%로 대부분을 차지하였다. 오이에 발생한 바이러스의 복합감염으로 많이 발생되는 바이러스로는 ZYMV, CGMMV, PRSV, BBWV2의 순서이었다. 단독감염된 오이의 병징은 다다기 계통이나 취청 계통 오이 모두 비슷한 병징이 나타났으나, CGMMV의 경우 퇴록 반점, 엽맥 퇴록, 엽맥 쭈그러짐 등 매우 다양한 병징이 나타났다. ZYMV는 엽맥 퇴록 병징을 위주로 심한 모자이크 및 기형 병징이 나타났다. 그 이외의 PRSV, WMV-2의 병징은 CGMMV와 ZYMV에 의한 병징에 속하는 비교적 약한 병징이었다.

Managing Virus Diseases Through an Understanding of Virus Vector Interactions and Virus Complexes

Robert R. Martin 한국응용곤충학회 2016 한국응용곤충학회 학술대회논문집 Vol.2016 No.04

In perennial crops virus diseases are usually caused by mixed infections rather than by individual viruses. Understanding the contribution of each virus in disease development, the interactions between viruses and how each virus spreads in the field allows for development of control measures that are targeted for disease control rather than controlling all viruses in a complex. There are multiple types of virus-vector interactions and this information can be used to inform vector control strategies to manage virus diseases. Information on virus-vector interactions and insect biology for controlling a disease caused by a virus complex in raspberry will be presented. Understanding the biology of multiple vectors as well as multiple types of virus-vector interactions for a vector of multiple viruses will be presented as a model for managing virus disease in strawberry in different environments. The goal is to describe a systems approach for controlling virus diseases in vegetatively propagated crops from developing clean plants through to fruit production.

사람호흡기바이러스검출에 대한 다중PCR과 단일PCR의 성능 비교

김솔잎,엄기원,조종래,엄태현 대한진단검사의학회 2016 Laboratory Medicine Online Vol.6 No.4

Background: The use of the multiplex polymerase chain reaction (PCR) technique for respiratory viruses has become popular in Korea owing to its convenience and sensitivity. However, concerns remain with regard to possible interference due to multiplexing. Methods: We compared the analytical sensitivity and virus interference of a commercially available, multiplex PCR kit (AdvanSure Respiratory virus real-time PCR kit, LG Life Sciences, Korea) with that of singleplex PCR to detect 11 viruses including coronavirus 229E and OC43; parainfluenza virus 1 (PIV 1), parainfluenza virus 2 (PIV 2), and parainfluenza virus 3 (PIV 3); influenza virus A (INF A) and influenza virus B (INF B); respiratory syncytial virus A (RSV A) and respiratory syncytial virus B (RSV B); adenovirus; and rhinovirus A, B, and C. Results: The lowest detected viral concentrations of coronavirus 229E and OC43, INF A and B, RSV A and B, adenovirus, and rhinovirus A, B, and C were the same for both, multiplex and singleplex systems. However, the lowest detected viral concentrations of PIV1, 2, and 3 differed by 1 dilution factor between the two systems. Threshold cycle (Ct) values for mixed viruses within the same well were not significantly influenced by each other, where the difference between Ct values ranged from 0.24 to 1.99. Conclusions: Analytical sensitivity of multiplex PCR was comparable to that of singleplex PCR for respiratory viruses. No significant interference was observed with mixed virus samples using multiplexed PCR. 배경: 다중 polymerase chain reaction (PCR)은 사용이 편리하고 민감도가 높아 국내 에서 호흡기바이러스 검출의 방법으로 흔히 사용되고 있다. 그러나 하나의 반응웰에서 여러 PCR을 동시에 진행하기 때문에 간섭의 우려가 있다. 방법: 저자들은 상용화된 바이러스를 이용하여 11종의 바이러스(coronavirus 229E, OC43; parainuenza virus 1 (PIV1), parainuenza virus 2 (PIV2), and parainuenza virus 3 (PIV3); inuenza virus A (INF A), inuenza virus B (INF B); respiratory syncytial vivirus (RSV) A), respiratory syncytial virus B (RSV B); adenovirus; rhinovirus A, B, and C)를 검출할 수 있는 다중PCR 키트(AdvanSure Respiratory virus real-time PCR kit, LG Life Sciences, Korea)의 분석민감도와 간섭 정도를 단일PCR키트와 비교하였다. 결과: 코로나바이러스 229E 및 OC43, 인플루엔자 A 및 B, RSV A 및 B, 아데노바이러스, 리노바이러스 A, B, C의 측정가능한 최소농도는 다중PCR과 단일PCR이 같았다. 그러나 PIV1, 2, 3의 최소농도는 두 검사법 간에 1 희석배수의 차이가 있었다. 하나의 반응웰에 혼합된 바이러스들의 역치주기(threshold cycle, Ct) 변화는 0.24-1.99 범위로서, 서로 영향을 주지 않는 것으로 판단되었다. 결론: 호흡기바이러스 측정을 위한 다중PCR의 분석민감도는 단일PCR과 비슷하였다. 다중PCR에서 혼합된 바이러스들 간에 의미있는 간섭은 관찰되지 않았다.

한탄바이러스 및 서울바이러스에 대한 단세포군 항체생산과 그 특성분석

반상자,이호자,박순희,조해월 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.1

To study the antigenic characteristics and differentiation serotypes of Hantavirus, the produced hybridoma secreting monoclonal antibodies(Mabs) against Hantaan virus, 76/118 strain and Seoul virus, 80/39 strain were produced by fusion of mouse myeloma cell, Sp2/0-Ag14 with spleen cells isolated from Balb/c mice immunized with Hantaan virus or Seoul virus. From the result in this study, the Mabs against Hantaan virus were screened by indirect immunofluorescent antibody techinque(IFA). 587 hybridoma cells were produced out of 768 wells and the fusion frequency was 76%. Seven among 587 hybridoma cells produced Mabs against Hantaan virus continuously. Isotype analysis of Mabs against Hantaan virus revealed that all these Mabs belong to the IgG2a subclass. All 7 Mabs reacted to nucleocapsid (N) proteins (MW.49-50Kd) of Hantaan virus by immunoblot assay. Mabs against Seoul virus were screened, 985 hybridoma cells were produced out of 1152 and the fusion frequency was 86%. Eight arnong 985 hybridoma cells produced Mabs against Seoul virus continuously. Isotype analysis of these Mabs against Seoul virus revealed the IgM and IgG2a subclass. These 8 Mabs reacted to nucleocapsid (N) proteins (MW.49-50Kd) of Seoul virus as demonstrated by immunoblot. Interestingly, these Mabs had neutralizing activity determined by PRNT analysis. Therefore it is suspected that the antigenic sites on N protein may be involved in the virus neutralization. The serological reactivity of 7 Mabs against Hantaan virus with Hantaan virus were strong, although Seoul, Puumala and prospect hill virus were not reacted to these Mabs. 8 Mabs against Seoul virus, reacted to Seoul virus strongly. Hantaan and Puumala virus reacted slightly to these Mabs but Prospect hill virus did not. To comparative study of sensitivity test between IFA and double sandwich ELISA method for human sera using ELISA of which these Mabs coated were tested, the ELISA method was more sensitive than IFA. These Mabs react.ed specifically to Hantaan and Seoul virus. There are antigenic relationships among Hantaviruses could be analysed by using these Mabs.

바이러스성 호흡기 감염의 역학 조사 및 각종 검체의 바이러스 동정률 비교

김순선,조영걸,김유겸,홍수종,김은순,우영대,주용규 대한감염학회 1999 감염 Vol.31 No.4

종자전염검역바이러스 3종(CRLV, SpLV 및 WClMV)을 검정하기 위한 RT-PCR 및 Nested PCR 프라이머 개발

이시원,차미정,김상목,허노열,신용길,이수헌 경상대학교 농업생명과학연구원 2014 농업생명과학연구 Vol.48 No.3

종자의 수입 시, 검역관련 종자전염바이러스는 가장 문제가 되는 식물병이다. 본 연구에서 PCR 검역체계가 보고되지 않은 3종의 종자전염바이러스, Cherry rasp leaf virus (CRLV), Spinach latent virus(SpLV) 및 White clover mosaic virus (WClMV)를 검출하기 위하여 reverse transcriptionpolymerase chain reaction (RT-PCR)과 nested polymerase chain reaction (nested PCR) 방법을 도입하였다. 각각의 바이러스별로 2 세트의 RT-PCR primer가 선발되었으며, 증폭산물에서 더욱 높은 감도로 검출 할 수 있는 nested PCR primer set를 개발하였다. 본 연구에서 사용한 RT-PCR과 nestedPCR 방법은 종자로부터 CRLV, SpLV 및 WClMV를 검역하는 고효율적 진단시스템으로 제공될 것이다. Seed-transmitted viruses regarding quarantine are the most problematic plant diseases in cropseed industry. In this study, three seed-transmitted viruses [Cherry rasp leaf virus (CRLV),Spinach latent virus (SpLV) and White clover mosaic virus (WClMV)] that have not previouslybeen studied for PCR diagnostic system in Korean quarantine were targeted for the detection. For successful virus detection, we employed a diagnostic technique based on reverse transcriptionpolymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nested PCR) methods. Two RT-PCR primer sets for each virus were finally selected for the diagnosis. Nested primersets developed in the present study were shown to be highly sensitive in detection andverification of the target viruses. Overall, RT-PCR and nested PCR were proven to be a usefuldiagnostic technique for the detection of CRLV, SpLV and WClMV in quarantine.