Human cytomegalovirus (HCMV) replication and $Ca^{2+}$ response in human cell lines of neuronal origin were investigated. SK-N-SH (neuroblastoma cells) and A172 cells (glioblastoma cells) were used. SK-N-SH cells were permissive for HCMV multiplication with a delay of one day compared to virus multiplication in human embryo lung (HEL) cells. The delay of HCMV multiplication in SK-N-SH cells appeared to be correlated with a delay in the $Ca^{2+}$ response. The cytoplasmic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) began to increase at 12 h p.i. in HCMV-infected SK-N-SH cells, while $[Ca^{2+}]_i$ increase in HCMV-infected HEL cells was observed as early as 3 h p.i. On the whole, the level of the increase in $[Ca^{2+}]_i$ in SK-N-SH cells was about 30% of that in HEL cells. On the other hand, in A172 cells infected with HCMV, neither production of infectious virus nor detectable increase in $[Ca^{2+}]_i$ was observed. Treatment with TPA of HCMV-infected SK-N-SH cells resulted in $[Ca^{2+}]_i$ increase at 6 h p.i. The stimulatory effect of TPA on HCMV- induced $[Ca^{2+}]_i$ increase continued until 12 h p.i., but TPA failed to stimulate the $Ca^{2+}$ response in SK-N-SH cells at 24 h p.i., suggesting that the effect of TPA had disappeared in SK-N-SH cells at that time point. In conclusion, SK-N-SH cells are permissive for HCMV replication and the delay in $Ca^{2+}$ response may be a consequence of the lower responsiveness of SK-N-SH cells than HEL cells to HCMV infection.

Respiratory Syncytial virus (RSV) is an important cause of acute lower respiratory tract infections in human, with infants and young children being particularly susceptible. In the temperate zones, sharp annual outbreaks of RSV occur during the colder months, in both the northern and the southern hemisphere. RSV is unusual in that it can repeatedly reinfect individuals throughout life and infect babies in the presence of maternal antibody. RSV isolates can be divided into two subgroups, A and B, on the basis of their reactions with monoclonal antibodies, and the two subgroups are also distinct at the nucleotide sequence level. The specific diagnosis of RSV infection was best made by isolation of virus in tissue culture, identification of viral antigen, or by specific serologic procedures. Recently, rapid detection of RSV and analysis of RSV strain variation became possible by development of methods of reverse transcription and polymerase chain reaction amplification. In this study, to determine the genetic diversity of RSV found in Korea, 173 bp and 164 bp spanning selected regions of the RSV F and SH genes were enzymatically amplified and sequenced, respectively. Eight for F gene and three for SH gene were detected in 66 nasopharyngeal swap samples tested. Two major antigenic subgroups, A and B were confirmed from Korean samples (seven for subgroup A and one for subgroup B). At the nucleotide level of the F gene region, Korean subgroup A strains showed 95-99% homologies compared to the prototype A2 strain of subgroup A and 93-100% homologies among Korean subgroup A themselves. For the SH gene region, Korean subgroup A strain showed 97.5% homology compared to the prototype A2 strain of subgroup A, and Korean subgroup B strain showed 97% homology compared to the prototype 18537 strain of subgroup B. Most of base changes were transition and occured in codon position 3, which resulted in amino acid conservation. Using the maximum parsimony method, phylogenetic analysis indicated that Korean RSV strains formed a group with other RSV strains isolated from the United States, Canada, the Great Britain and Australia.

대략 36,000 base pairs (bp)의 두 가닥짜리 DNA를 지놈으로 가진 사람 아데노바이러스 (Ad)는 DNA 상동성(相同性) 및 생물학적/생화학적 성격이 특이한 49개의 혈청형이 알려져 있는데, 이들 대부분의 Ad가 영유아군 및 면역능이 저하된 성인에서 치사적 결과를 초래할 수 있다. Ad의 세포향성(向性)(tropism)은 매우 다양하여 종류에 따라 상기도 감염, 각결막염, 영유아 장염등을 유발하는데 최근 Ad의 다양한 병원성에 대한 원인을 분자생물학적 수준에서 규명하려는 노력의 일환으로 지역에 따라 주되게 출현하는 Ad형 규명이 활발히 이루어지고 있다. Ad 동정/확인은 표면을 이루고 있는 group 공통항원인 hexon 단백질을 탐지하는 효소면역 측정법 (EIA)에 의하며, Ad형별은 Ad fiber의 세포독성 중화시험에의 한다. 그러나, 세포독성 중화시험이 엄청난 노동력 및 시간을 요구하면서도 민감도/특이도가 만족스럽지 못하여 이를 개선하기 위하여 검체 또는 세포배양에서 Ad DNA를 추출하여 제한효소 절단형태를 비교하는 방법이 개발되었는데 이는 세포배양에 잘 자라지 않는 바이러스주의 형별뿐만 아니라 지역 분리주들의 지놈 변형주를 관찰하는 분자생물학적/분자역학적 연구에도 도움이 되고 있다. 국내에는 Ad와 관련된 소아장염의 빈도가 rotavirus에 의한 것 다음으로 빈번한데도 Ad40/41외에 주되게 출현하는 장내 Ad형들이 전혀 규명된 바 없고, 한국형 Ad들의 지놈형태가 전혀 보고된 바가 없다. 또한 세계적으로 Ad형별 조사지역이 늘어감에 따라 유아장염과 연관된 Ad 역시 Ad40, 41이 외의 형들이 Ad40, 41을 능가하는 것으로 보고되고 있는 지역도 있으나 국내에서는 Ad40, 41이외의 형들은 그 역학적 중요도가 전혀 알려져 있지 않다. 이로서 본 연구의 목적은 Ad주들에 특이 중화항체를 이용한 세포독성 중화시험과 Ad DNA 절단법을 적용하여 한국형 장내 Ad주들의 형별을 처음으로 시도함과 동시에 1989-1991사이 출현한 Ad들의 유전적 변형을 관찰하려는 것이었다. 두 방법 모두 사용하였을 때 주되게 출현하는 장내 Ad형들은 Ad4l, Ad2, Ad7, Ad5, 및 Ad40이었다. Ad40/41-양성 검체를 제외한 Ad hexon-EIA양성들의 77.5%를 형별 할 수 있었던 Ad DNA의 제한효소 절단방법은 형들간의 교차중화로 특이성이 낮았던 중화방법 (47.5%)보다 매우 효율적이어서 두 가지 방법을 함께 적응하였을 때는 40주중의 81.5%인 35주를 형별 할 수 있었다. 또한Ad DNA 제한 효소 절단방법은 Ad7 변이주 (Ad7b)도 탐지 할 수 있었다.

C형 간염바이러스 (HCV)는 각 개체간에 뉴클레오티드 서열상의 다양성을 나타내고, 이러한 유전적 다양성이 임상병리적 증상과 밀접한 연관이 있을 것으로 고려되어 왔다. 본 연구에서는 HCV E1과 NS5B 부위의 염기서열 분석을 통해 한국의 C형 간염바이러스의 분포와 다양성에 관해 분석하고, 발생계통도를 그려 HCV간의 진화적 거리를 확인하였다. 염기서열분석은 서울대학교 병원과 충남대학교 병원으로부터 얻은 56개의 HCV-양성 혈청을 대상으로 RT-PCR과 PCR 과정을 통해 얻은 유전자 산물을 클로닝하여 수행되었다. 56개의 혈청중 53개의 샘플에서 HCV RNA가 검출되었다. 이들 53개 샘플에 대한 분석 곁과, 유전형 1a, 1b, 2a, 2b, 7a가 각각 5.7, 45.3, 45.3, 1.9, 1.9%로 분포하고 있고, 1b형과 2a형이 한국에서의 주요한 HCV 유전형으로 밝혀졌다. 본 연구는 염기서열 분석을 통해 한국에서 1b형과 마찬가지로 2a형도 높은 빈도로 분포하고 있고, 비록 분포 빈도는 낮지만 1a 형과 7a 형도 존재하고 있음을 밝힌 최초의 보고이다.

Histopathological vascular changes in hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus include increased vascular permeability, disseminated intravascular coagulation, thrombocytopenia and changes in coagulation activity. Although vascular endothelial cells of main target organs such as kidney infected with Hantaan virus are not damaged but swelling of endothelial cells, perivascular exudates and infiltration of mononuclear cells and fresh interstitial hemorrhages are common. However, the pathogenesis of cell infiltration and hemorrhages around vascular endothelial cells are not well understood. Some endothelial cell molecules or vascular adhesins that acts as adhesion moleulces for leukocyte are expressed on endothelial cells close to site of inflammation. However, whether the expression of endothelial adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and endothelial leukocyte adhesion molecule (ELAM) on vascular endothelial cells are increased by infection with Hantaan virus has not been studied. In this study, the relationship between the expression of VCAM-1, ICAM-1 and ELAM and adhesion of mononuclear cells on endothelial cells of human blood vessels infected with Hantaan virus was investigated. The endothelial cells of umbilical vein was passaged three times in culture medium and the monolayered cells were infected with $10^5\;pfu/ml$ of Hantaan virus grown in Vera E6 cell cultures. The multiplication of virus in cultured endothelial cells was monitored by immunohistochemistry and the expression of adhesion molecules was demonstrated by immunohistochemistry using monoclonal antibodies against VCAM-1, ICAM-1 and ELAM. And in situ hybriditation against ICAM-1 was also performed. The endothelial adhesion molecules, VCAM and ICAM, were expressed after 6 hours postinfection, respectively, and their expressions lasted for 72 hours. Similar expression of VCAM and ICAM appeared on endothelial cells by infection with virus, but the expression of ELAM was not recognized up to 72 hours postinfection. Microscopically, it was noted that many monocuclear cells adhered on endothelial cells infected with viruses. In an electronmicroscopic study, the transendothelial migration of mononuclear cells was observed on monolayered endothelial cells infected with virus. This results suggested that the endothelial adhesion molecules, particulary VCAM and ICAM, might be expressed on endothelial cells by infection with Hantaan virus and these molecules play a key role in the adhesion and extravasation of inflammatory cells around blood vessels.

For easy and rapid screening of hemorrhagic fever with renal syndrome (HFRS) without any laboratory equipment, dot blot enzyme immunoassay was developed and tried to detect anti-hantavirus antibodies. The nucleocapsid protein of Hantaan virus was isolated by affinity chromatography and used for making the dot strip. 28 of 29 Hantaan virus infected sera showed positive signals and 21 of 22 HFRS negative sera showed no positive signals. Anti-Seoul virus monoclonal antibody also exibited positive signal but the intensity of colorization was approximately 5 fold less than that of anti-Hantaan monoclonal antibody. The sensitivity of dot blot assay was equal or superior to indirect immunofluorescent assay (IFA) or ELISA test. Overall, the screening results with dot blot assay showed 92.2 % of concordance with IFA or ELISA test. This results suggests that dot blot assay could be applied a tool for easy and rapid screening of HFRS.

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.

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

To understand whether the bats inhabiting in Korea play role as hosts harboring Hantavirus that cause acute febrile diseases, a total number of 802 bats of 9 species were collected from seven provinces in Korea from 1989 to 1995 and tested for the presence of antibodies to Hantavirus by means of immunofluorescent antibody (IFA) technique. The results are summarized as follow. 1. Total 802 captured bats were classified into 9 different species with the following distribution. They were Rhinolophus ferrumequinum, Eptesicus serotinus, Miniopterus sehreibersii, Vespertilio superans, Myotis mystatinus, Murina leucogaster, Myotis formosus, Myotis macrodactylus and Plecotus auritus with numbers and rates of 423 (52.74%), 291 (36.28%), 47 (5.86%), 28 (3.49%), 8 (1.00%), 1 (0.12%) and 1 (0.12%), respectively. The predominant species of the bats was Rhinolophus ferrumequinum with 52.74% of the captured. 2. Among 9 species of bats, species of Rhinolophus ferrumequinum and Eptesicus serotinus were positive with Hantavirus antibody of strain numbers 76-118. The rate of antibody positive was 3.78%. 3. The seasonal differences of Hantavirus antibody in 802 bats tested were 5.83%, 4.17%, 3.67% and 0.64% in winter, spring, summer and autumn, respectively. Again the highest viral antibody prevalence was detected in winter. It could be concluded through the study that certain species of bats inhabiting in Korea play a definite role as the host animals of certain species of Hantavirus.

Hantavirus is the etiologic agent of hemorrhagic fever with renal syndrome (HFRS). It has been known that the natural reservoirs of Hantavirus are not only field mice but also other animals in parts of the world. In this study, to research on the host range of Hantavirus, immunofluorescent antibody against Hantavirus was investigated in wild birds from 1991 to 1992, duck from 1991 to 1992 and squirrels (Tamias sibiricus) in 1990 in Korea. The results were as follows: 1. Of total 179 wild birds of 14 species, Emberiza elegans elegans and Passer montanus dy-bowsky were antibody positive. The positive rates were 3.92% (2 out of 51) and 1.64% (1 out of 61), respectively. 2. The antibody titers of wild birds were 1:16 and 1:64 in Emberiza elegans elegans, 1:16 in Passer montanus dybowsky. 3. The positive rate of antibody in ducks was 2.3% (3 out of 129). 4. The positive rate of antibody in squrrels was 48.10% (38 out of 79). According these results, we newly showed that passer montanus dybowsky, domestic ducks and Tamias sibiricus possessed the antibody against Hantavirus.

본 연구는 인간면역결핍바이러스의 입자를 비이온성 계면활성제로 처리할 때 바이러스 입자구조에서 분리되어 방출되는 바이러스 구조단백질들의 분포를 sucrose gradient로 분석하여, 바이러스 입자를 구성하는 바이러스 구조단백질과 바이러스입자의 생물리학적 특성을 연구하였다. 바이러스입자들을 0.16% NP40 (Nonidet P-40)으로 처리할 때, 바이러스 capsid 단백질과 바이러스 막 단백질 (membrance protein)들은 다른 바이러스 구성성분들과 잘 분리되었다. 계면활성제처리에서 방출되지 않은 구성 성분들은 matrix 단백질, nucleocapsid 단백질, reverse transcriptase, integrase 및 바이러스 RNA genome로써, 이들은 subviral 구조를 형성한다. 이러한 결과는 상대적으로 다른 바이러스들의 capsid 단백질과 면역 결핍 바이러스의 capsid 단백질 (p24)를 비교할 때, 면역결핍바이러스의 capsid 단백질은 바이러스핵을 형성할 때, capsid 단백질 사이의 결합력이 매우 약한 것으로 추정된다. 또한 바이러스 조절단백질의 하나인 vpr 단백질을 함유하는 바이러스입자를 NP40 처리하여 분석하였을 때, vpr 단백질은 subviral 구조에 존재하는 것으로 나타났다.

Human papillomavirus (HPV), especially type 16 and 18, has been closely associated with carcinomas and uterine cevical cancer, recently. From in vitro assays, E6 and E7 genes of HPV16 are closely linked with transformation of cell lines of rodent fibroplasts. However, the transforming activity of E6 and E7 genes of HPV type 16 in vivo has not been fully elucidated. For explaining this mechanism, we prepared a expression system with the promoter of mouse mammary tumorvirus long terminal repeat and E6E7's open reading frames. This expression system was introduced in rodent cell lines, No. 7, 3Y1 and shown normal transforming abilities. And, we produced transgenic mice with E6, E7 expression system. These transgenic mice were confirmed from Southern blot analysis. One male of them was observed enlargement of the testis after 5 months postdelivery.

Herpes simplex virus type-1의 vero 세포에서의 증식기작을 규명하기 위해 전자현미경으로 관찰하였고, 유전학적 특성을 규명하기 위해 유전자도서관을 작성하였고, thymidine kinase (TK) 유전자를 클로닝을 하였다. 감염 48시간 후 많은 수의 바이러스 nucleocapsid가 핵뿐만 아니라 세포질에서 관찰되었다. 바이러스는 세포에 감염된 후 핵내에서 복제증식한 후 세포질내로 이동하였으며, 이때 핵막을 통과하면서 외투막을 갖고 세포질로 이동하여 세포밖으로 나가는 것을 관찰할 수 있었으며, 또한 일부 nucleocapsid는 세포막을 출아하여 비리온으로 출아되었다. HSV-1의 DNA를 BamHI과 BglII 제한효소로 각각 절단하여 DNA의 절단 양상을 조사하였다. BamHI에 의해 절단된 단편은 27개 이었고, 그들 분자량의 범위는 1.1 - 14 kb이었으며, BglII에 의해 절단된 단편은 16개이었고, 분자량의 범위는 4.5 - 20.1 kb이었다. Southern blot 방법으로 TK 유전자를 포함하고 있는 단편을 확인하였는데, pHLA-12와 pHLB-14클론에 포함되어 있었고 각 단편의 분자량은 3.74와 6.41 kb이었다.

Hyphantria cunea nuclear polyhedrosis virus p10유전자와 프로모터의 염기서열을 결정하였고, p10단백질의 아미노산 서열을 유도했다. pBP10재조합클론 (Cha et. al., 1991)에 삽입이 되어있는 p10유전자의 염기서열을 결정한 결과 p10유전자의 ORF는 285 bp였고, p10단백질은 95개의 아미노산으로 구성 되었으며, 분자량은 10.26 kDa이었다. 프로모터내에는 TATA box와 전사개시부위인 TAAG 염기가 발견되었다. poly (A) signal부위인 AATAAA염기서열은 3'-말단상류의 65염기부위에 위치했다. p10단백질의 N-말단은 소수성이었으며, C-말단은 고도로 친수성이었다. p10단백질에는 cysteine, histidine, tryptophane, tyrosine, glutamine, asparagine잔기가 없었다.