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Effective Antitumor Activity of a Recombinant Vaccinia Virus Expressing Murine Interleukin 4.

Keejung YOON ; Sunyoung KIM ; Ning Yi JIN

Journal of the Korean Society of Virology.1998;28(1):71-78.

Vaccinia virus is the prototype orthopoxvirus that has been used as a vaccine strain for small pox. This virus has been used to express a variety of cellular and viral genes in mammalian cells at high levels. Interleukin-4 (IL-4) has been found to stimulate the proliferation of T cells and enhance the cytolytic activity of cytotoxic T lymphocytes. To test the immunotherapeutic potential of IL-4 delivered in vivo by poxvirus, a recombinant vaccinia virus expressing the murine IL-4 gene (RVVmIL-4) was constructed. A high level of IL-4 production was confirmed by infecting HeLa cells and measuring IL-4 in cell culture supernatant by ELISA. As a tumor model, two cell lines were used: the murine T leukemic line P388 and the murine breast cancer line TS/A. CDF1 mice were intraperitoneally inoculated with 1 x 105 cells of P388. Mice were injected at the same site with 5 x 105 PFU of recombinant vaccinia virus; first, 3 days after the injection of tumor cells and thereafter once every week for 3 weeks. Intraperitoneal injections of RVVmIL-4 significantly prolonged the survival time of mice inoculated with tumor cells. All mice injected with RVVmIL-4 remained alive for 30 days after the postinoculation of tumor cells, while 100% and 70% of the animals injected with saline or wild type vaccinia virus died, respectively. In another tumor model using TS/A, tumor was established by subcutaneously inoculating 2 x 105 tumor cells to BALB/c mice. After tumor formation was confirmed on day 4 in all mice, 5 x 106 PFU of RVVmIL-4 was inoculated subcutaneously three times, once every week for 3 weeks. The TS/A tumor was eradicated in two of the nine mice. Seven of the nine mice treated with RVVmIL-4 developed a tumor, but tumor growth was significantly delayed compared to those treated with saline or wild type vaccinia virus. These results indicate that recombinant vaccinia viruses may be used as a convenient tool for delivering immunomodulator genes to a variety of tumors.
Animals ; Breast Neoplasms ; Cell Culture Techniques ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Genes, Viral ; HeLa Cells ; Humans ; Injections, Intraperitoneal ; Interleukin-4* ; Interleukins* ; Mice ; Orthopoxvirus ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic ; Vaccinia virus* ; Vaccinia*

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Evaluation of Anti-Herpes Simplex Virus Type 1 Activity of Acyclovir by Using Mouse Intracerebral Infection Model.

Chong Kyo LEE ; Hae Soo KIM

Journal of the Korean Society of Virology.1998;28(1):63-69.

To establish in vivo antiviral evaluation system by using murine herpesvirus intracerebral infection model, 5-6 female BALB/c mice per group aged 5 weeks were inoculated i.c. into cerebrum with different inocular HSV-1 F. Signs of clinical disease noted everyday for one month. Observed were body weight decrease, neurological signs and death caused by encephalitis. Mice discontinued body weight decrease were recovered from the disease, and keratitis was often observed during recovery. The groups inoculated with higher than 1,000 PFU showed 100% mortaltiy and LD50 was <100 PFU/mouse. To study the effect of virus inoculum sizes on antiviral effect of acyclovir (ACV), mice inoculated with different inocula were administered i.p. with different doses of ACV immediately after infection, and twice a day for 5 days. The higher inculum size, the less protective. ED50 of ACV was >25, >25, 18.4 and 8.0mg/kg b.i.d. in the group infected with 1,000,000, 100,000, 10,000 and 1,000 PFU/mouse, respectively. LD50 of ACV was 62.5 mg/kg b.i.d. Therapeutic index of ACV was <2.5, <2.5, 3.0 and 7.0 in the groups with inocula 1,000,000, 100,000, 10,000 and 1,000 PFU/mouse, respectively. Inoculum size 1,000 PFU/mouse showing 100% mortaltiy and 5-6 days mean time to death, 5 days drug administration and 14 days observation will be future exeperimental conditions.
Acyclovir* ; Animals ; Body Weight ; Cerebrum ; Encephalitis ; Female ; Herpesvirus 1, Human ; Humans ; Keratitis ; Lethal Dose 50 ; Mice*

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Expression of Human Papillomavirus Type 16, Prototype and Natural Variant E7 Proteins using Baculovirus Expression System.

Hee Sung HAN ; Sun Ho KEE ; Hyung Jun KIM ; Kyung A CHO ; Yoon Won KIM ; Min Kee CHO ; Woo Hyun CHANG ; Soon Bong HWANG

Journal of the Korean Society of Virology.1998;28(1):53-62.

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Asparagine ; Baculoviridae* ; Blotting, Western ; Chromatin ; Cytoplasm ; Humans* ; Immunoprecipitation ; Insects ; Open Reading Frames ; Phosphoproteins ; Serine ; Sf9 Cells

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Expression and Characterization of the Human Immunodeficiency Virus Type 1 Mutant Envelope Glycoproteins in Mammalian Cells.

Ji Yoon RYU ; Jin Seu PARK

Journal of the Korean Society of Virology.1999;29(3):183-193.

Human immunodeficiency virus type 1(HIV-1) envelope glycoprotein is synthesized as a 160KDa precursor, gp160, that is cleaved by a cellular protease to form the gp120 and gp41 subunits. Mammalian expression vectors were designed that are capabae of efficient expression of various mutant envelope glycoproteins derived from a molecular clone of HIV-1. To construct these vectors, one type of mutation was made at the gp120-gp41 cleavage site by oligonucleotide directed mutagenesis. And another mutation was made to change amino acids in the membrane spanning region of HIV-1 gp41 important for membrane anchorage. Next, these two mutations were combined to generate a vector to have double mutations in cleavage site and membrane spanning region. These mutants were transiently expressed in mammalian cells. The effect of these mutations on envelope glycoprotein synthesis, proteolytic processing and secretion was determined. In addition, cell surface expression and ability of the glycoprotein to induce syncytium formation were examined. This study provides a mammalian expression system that is capable of efficient expression and secretion of soluble gp160.
Amino Acids ; Clone Cells ; Giant Cells ; Glycoproteins* ; HIV* ; HIV-1* ; Humans* ; Membranes ; Mutagenesis, Site-Directed

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Expression and Purification of Herpes Simplex Virus Type 1 Protease.

Pan Kee BAE ; Jin Wook PAENG ; Jee Hyun KIM ; Hae Soo KIM ; Sang Gi PAIK ; In Kwon CHUNG

Journal of the Korean Society of Virology.1999;29(3):175-182.

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. HSV-1 protease is essential for DNA packaging and virus maturation. To perform high throughput for potent inhibitors, the efficient production of larger amounts of highly purified enzyme and protease activity assay method must be established. In this report, expression in E. coli and purification of the protease gene of HSV-1 strain F was investigated. The protease gene was cloned pET28, and the nucleotide sequence of protease catalytic domain of HSV-1 compared strain F with other strains (KOS and CL101). In these results the F strain was different in base sequence. However, the amino acid sequence was identifical. The HSV-1 protease was purified with His-tagged affinity column. The analysis of HSV-1 protease activity Was performed by high performance liquid chromatography.
Amino Acid Sequence ; Base Sequence ; Catalytic Domain ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Clone Cells ; DNA Packaging ; Drug Therapy ; Herpes Simplex* ; Herpesvirus 1, Human* ; Simplexvirus*

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Efficacy of Acyclovir on Replication in Infected Tissues and Virus Reactivation from Explanted Tissues in Mouse Encephalitis Model of Herpes Simplex Virus Type 1.

Chong Kyo LEE ; Jee Hyun KIM ; Pan Kee BAE ; Mi Kyung PI ; Hae Soo KIM

Journal of the Korean Society of Virology.1999;29(3):165-174.

To investigate viral pathogenesis and in vivo efficacy of acyclovir (ACV) in mouse HSV-1 encephalitis models, female BALB/c mice aged 5 weeks were inoculated with strain F either intranasally (IN) or intracerebrally (IC). ACV-treatment by intraperitorneal injection with 0, 5, 10 and 25 mg/kg b.i.d. for 6days was commenced 1 h after infection. Body weight and signs of clinical disease were noted daily up to 2 weeks. ED50 of ACV in IN infection was 5mg/kg and 14.1 mg/kg in IC infection. Tissues of cental nervous system were collected from 2 mice per group everyday up to 5 days p.i. and the virus titers were measured. In IN infection model, high titers in eyes and trigeminal nerves were observed. ACV-treatment showed significant reduction of the titers in all the isolated. In IC infection model, cerebrum, cerebellum and brain stem showed high virus titers. ACV-treatment showed less significant reduction of virus titers than that in IN infection model. Reactivation of explanted trigeminal nerves from mice 30 day p.i. was monitored. In all of ACV treated mice reactivation was observed, i.e. even the highest dose of ACV did not inhibit the establishment of viral latency.
Acyclovir* ; Animals ; Body Weight ; Brain Stem ; Cerebellum ; Cerebrum ; Encephalitis* ; Female ; Herpes Simplex* ; Herpesvirus 1, Human* ; Humans ; Mice* ; Nervous System ; Simplexvirus* ; Trigeminal Nerve ; Virus Latency

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Molecular Characterization of the L Segment of Hantann Virus, Strain Howang.

Yong Kyu CHU ; Dae Yong SONG ; Hyung Mo KOO ; Ho Wang LEE

Journal of the Korean Society of Virology.1999;29(3):155-163.

Hantaan virus (HTNV), the etiologic agent of hemorrhagic fever with renal syndrome (HFRS), belongs to the genus Hantavirus, and has three single negative straded RNA genome segments. HTNV strain Howang isolated from the blood of severe case of Korean HFRS is more virulent than HTNV 76/118 and the M and S genome segments' nucleotide sequence of Howang strain showed 93.5% and 94% homology to each segment of HTNV 76/118. We have obtained 6533 nucleotides long sequence of the L genome segment of Howang strain using reverse transcriptase in conjunction with PCR amplification and compared to other hantaviruses. The messenger sense of the L segment contains one long single long open reading frame of 2151 amino acids, which encodes a deduced RNA dependent RNA polymerase of 246.4 kDa caculated molecular weight protein. The nucleotide sequence of the Lsegment of Howang strain shows 93%, 74%, 66%,65% homology to HTNV 76/118, Seoul virus 80/39, Puumala virus Hallnas B1 and Sin Nombre virus, respectively. The amino acid sequence of the L segment of Howang strain shows 99%, 85%, 68%, 68% homology to HTNV 76/118, Seoul virus 80/39, Puumala virus Hallnas B1 and Sin Nombre virus, respectively.
Amino Acid Sequence ; Amino Acids ; Base Sequence ; Genome ; Hantaan virus ; Hantavirus ; Hemorrhagic Fever with Renal Syndrome ; Molecular Weight ; Nucleotides ; Open Reading Frames ; Polymerase Chain Reaction ; Puumala virus ; RNA ; RNA Replicase ; RNA-Directed DNA Polymerase ; Seoul virus ; Sin Nombre virus

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Construction of a Novel Recombinant Baculovirus Producing Polyhedra with Bacillus thuringiensis Cry1Ac Crystal Protein.

Yeon Ho JE ; Byung Rae JIN ; Jong Yul ROH ; Jin Hee CHAN ; Seok Kwon KANG

Journal of the Korean Society of Virology.1999;29(3):145-153.

We have now construted a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) Cry1Ac crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Suprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus paticles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantrea cunea, was strikingly improved in comparison with the wild-type AcNPV.
Bacillus thuringiensis* ; Bacillus* ; Baculoviridae* ; Insects ; Nucleopolyhedrovirus

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Pseudotype HIV-1 Particles Carrying CD4.

Seung Won PARK ; Tai Gyu KIM ; Ji Chang YOU ; Manfred SCHUBERT ; Soon Young PAIK

Journal of the Korean Society of Virology.2000;30(1):83-99.

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.
Adsorption ; Animals ; Antibodies ; COS Cells ; DNA ; Helper Viruses ; HIV-1* ; Membranes ; Plasmids ; Product Packaging ; RNA ; Virion

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Cloning and Sequence Analysis of the Full-length cDNA of Coxsackievirus B3 Isolated in Korea.

Yoon Seok CHUNG ; Ki Soon KIM ; Jeong Koo PARK ; Yoon Sung LEE ; Soo Youn SHIN ; Doo Seong CHEON ; Youngmee JEE ; Moon Bo KIM ; Byoung Kuk NA ; Jae Deuk YOON ; Kwang Ho LEE ; Chul Yong SONG

Journal of the Korean Society of Virology.2000;30(1):71-81.

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 VR1, which closely associated with cardiovirulence, was replaced with Arg155 by single nucleoptide substitution from A2916 to T2916. Moreover, additional amino acid substitutions were observed in the flanking region of Asp155. Taken together, aminoacid(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.
Amino Acid Substitution ; Amino Acids ; Cardiomyopathies ; Clone Cells* ; Cloning, Organism* ; DNA, Complementary* ; Genome ; Humans ; Korea* ; Meningitis, Aseptic ; Nucleotides ; Open Reading Frames ; RNA Replicase ; RNA, Messenger ; Sequence Analysis* ; Sequence Homology ; Uracil

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