• Toxicological Research
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• v.17
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• pp.157-166
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• 2001

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared to the well known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.10a
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• pp.31-50
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• 2000

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene e(pression. However, we have now developed a series of reroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors the intron and exon sequences from heterologous cellular or viral genes are present, When compared to the well blown MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.75-79
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• 1991

The yeast L-A virus (4.6 kb dsRNA genome) encodes the major coat protein and a "gag-pol" fusion minor coat protein that separately encapsidate itself and $M_{1}$, a 1.8 kb dsRNA satellite virus encoding a secreted protein toxin (the killer toxin). The teast chromosomal SKI genes prevent viral cytopathology by lowering the virus copy number. Thus, $ski^{-}$ mutants are ts and cs for growth. We transformed a ski2-2 virus-infested mutant with a yeast bank in a high copy cloning vector and selected the rare healthy transformants for analysis. One type of transformant segregated M-O L-A-O cells with high frequency. Elimination of the DNA clone from the ski2-2 strain eliminated this phinotype and introduction of the DNA clone recovered from such transformants into the parent ski2-2 strain, or into ski3 or ski6 mutants gave the same phenotype. This killer-curing phenotype was due to the curing of the helper L-A dsRNA virus. The 6.5 kb insert only had this activity when carried on a high copy vector and in $ski^{-}$ cells (not in $SKI^{+}$ cells). This 6.5 kb insert acts as a mutagen on L-A dsRNA producing a high rate of deletion mutations.mutations.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.11b
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• pp.199-200
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• 2002

GX-12 is a naked DNA vaccine developed by research team of Dong-A Pharmaceutical Company, Green Cross Company and Genexine for the treatment of HIV infection. It consists of four separate plasmids (pGX10-GE HX, pGX10-dpol JR, pGX10-VN/TV JR, pGX10-hIL-12m), which were constructed by inserting the HIV-1 gag-env, pol, regulatory genes and a human IL-12 mutant gene into pGX10 plasmid vectors.(omitted)

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.61-70
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• 1983

Synchronized cat kidney cells chronically infected with feline leukemia virus (FeLV) were used to study virus production, the synthesis of group specific antigen (gag) and envelope (env) proteins, the expression of env protein on the cell surface during the cell cycle, and the stability of viral RNA. As detecting method, we developed the radioimmunoassay (RIA) system using beta-emission of $^{131}I$ and demonstrated the validity of this system by comparison with routine RIA system using gamma-emission of $^{125}I$. The produced virus was analysed by developed RIA interval was determined by measuring reverse transcriptase activity. The results show that infected cells produce the complete virus particle containing products of gag, env and pol genes of FeLV, and maximum virus production occurs during mitosis of synchronized cells. Labeling of the cell surface of synchronized cells with $^{131}I$ shows that the amount of $gp70^{env}$ on the cell surface parallels cellular gorwth. Therefore, the cell cycle-dependent release of virus is not petition RIA of synchronized cells with $^{131}I$ labeled viral proteins synthesis during the cell cycle. The rate of synthesis of gag protein shows three peaks, corresponding to the $G_1,\;late\;S\;and\;late\;G_2$ phases of cell cycle. But the rate of synthesis of env protein dose not change, suggesting that in these cells the synthesis of these two gene products in controlled seperately. In Actionomycin D treated cells, the synthesis of viral proteins decreased sharply from 8 hours after treatment, and the late S and $G_2$ peaks of gag protein synthesis were disappeared. This shows the stability of viral RNA for about 6 hours in the absence of continuing viral RNA synthesis.

• Korean Journal of Agricultural Science
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• v.38 no.1
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• pp.45-50
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• 2011

This study was conducted to investigate the farm situation about bovine leukemia virus(BLV) infection that greatly influence productivity in dairy cattle and compare the accuracy of diagnosis for BLV infection between PCR and ELISA techniques. Blood samples of 193 heads from 5 herds in Chungnam and Chungbuk area were used to analyze BLV gene and serum, and the results were obtained as follows. The amplified BLV gene in dairy cattle by PCR technique resulted in 226 bp, 596 bp and 434 bp, respectively, for gag, pol and env, which were well amplified. The infection rates of BLV virus diagnosed by PCR and ELISA techniques ranged from 80.55 to 100% and from 22.22 to 86.95%, respectively, and the infection rates among 5 herds were significantly different in both methods (P<0.05). Further, the average infection rates of 5 herds were 87.05 and 63.21%, respectively, for PCR and ELISA techniques. Kappa statistics for examining consistency of diagnosis by PCR and ELISA techniques showed 0.246, which represents low consistency. Consequently, PCR based BLV technique was considered as a corrective measure for diagnosis of BLV infection in Holstein dairy cattle.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.181-187
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• 2006

In recent years the number of patients waiting for organ transplantation has greatly outpaced the supply of human organs available, which leads to a renewed interest in pig-to-human xenotransplantation as an alternative. However, one of the biggest barriers in the xenotransplantation is presence of porcine endogenous retroviruses(PERV) that can infect human cells. In this study, to present a possible solution for this problem we tried to inhibit expression of PERVs using shRNAs(short hairpin RNA) at the level of RNA synthesis and virus release. The shRNA targeting the sequence of PERV A, B type was cloned into pSIREN-RetroQ vector under the control of polymerase-III U6-RNA gene promoter. Quantitative real-time PCR was performed to detect my alterations in mRNA production of PERV A, B targeted by the shRNA in each done. Depending on the target sequence of the shRNA, the transcription of PERV was decreased to as much as 4% and the number of progeny viruses was reduced to less than 1/200,000. Transgenic pigs producing such shRNAs may result in a highly reduced PERV expression in cells and organs, which is a prerequisite for safe xenotransplantations.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.107-118
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• 1999

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over $500/{\mu}l$ from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9. A series of 9 overlapping PCR products were amplified from cultured PBMC and cloned About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between $NF-{\kappa}B$ I and Sp1 III, and duplication of $TCF-1{\alpha}$ in LTR. We could not find any deletion of amino acid in Nef, Gag, Pol and Env gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

• Biomedical Science Letters
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• v.22 no.3
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.