• The Plant Pathology Journal
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• v.37 no.2
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• pp.162-172
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• 2021

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.54-61
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• 2009

An infectious full-length clone of Soybean mosaic virus (SMV) strain G5H was constructed under the control of the cauliflower mosaic virus 35S promoter. The cloned SMV G5H established infections upon simple rub-inoculation of soybean leaves with intact plasmid DNA. We demonstrated that this SMV G5H infectious DNA clone caused typical characteristic symptoms and virulence of SMV strain G5H in twelve tested soybean cultivars. Soybean cultivars Lee74, Somyungkong and Sowonkong developed systemic mosaic symptom while Kwanggyo, Taekwangkong, Hwangkeumkong and Geumjeongkong-l showed systemic necrosis. In contrast, Geumjeongkong-2, Jinpumkong-2, L29, V94-5152 and Ogden showed resistant response against SMV-G5H infection. We also determined full-length sequence of cloned SMV-G5H. The phyogenetic analyses reveal that SMV-G5H is most closely related to SMV-G5, and support that SMV-G5H might be derived from SMV-G5 by recombination rather than mutation.

• Research in Plant Disease
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• v.20 no.3
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• pp.219-222
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• 2014

Several tomato production regions in Korea were surveyed for tomato yellow leaf curl disease (TYLCD). Tomato leaf samples showing TYLCD-like symptoms were collected from Tongyeong (To), Geoje (Gi), and Gimhae (Gh) cities of the southern part of Korea. Tomato yellow leaf curl virus (TYLCV) was detected and the full-length genomes of the isolates were sequenced. The TYLCV isolates found in Korea shared high sequence identity (> 99%) with TYLCV-IL [JR:Omu:Ng] (AB110217). Phylogenetic relationship analysis revealed that they formed two groups (with little genetic variability), and the To, Gj, and Gh isolates belonged to the TYLCV-IL group. An infectious clone of TYLCV-To (JQ013089) was constructed and agroinoculated into Nicotiana benthamiana, Nicotiana tabacum var. Xanthi, Petunia hybrida, Capsicum annuum, and Lycopersicon esculentum cv. Hausumomotaro. Agroinfection with a dimeric infectious clone of TYLCV-To induced severe leaf curling and stunting symptoms in these plants, excluding C. annuum. Tomato plants then developed typical yellow leaf curl symptoms.

• The Plant Pathology Journal
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• v.32 no.1
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• pp.70-76
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• 2016

The infectious full-length cDNA clone of zucchini yellow mosaic virus (ZYMV) isolate PA (pZYMV-PA), which was isolated from pumpkin, was constructed by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Simple rub-inoculation of plasmid DNAs of pZYMV-PA was successful to cause infection of zucchini plants (Cucurbita pepo L.). We further engineered this infectious cDNA clone of ZYMV as a viral vector for systemic expression of heterologous proteins in cucurbits. We successfully expressed two reporter genes including gfp and bar in zucchini plants by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs. Our method of the ZYMV-based viral vector in association with the simple rub-inoculation provides an easy and rapid approach for introduction and evaluation of heterologous genes in cucurbits.

• The Plant Pathology Journal
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• v.30 no.3
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• pp.310-315
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• 2014

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is responsible for one of the most devastating viral diseases in tomato-growing countries and is becoming a serious problem in many subtropical and tropical countries. The climate in Korea is getting warmer and developing subtropical features in response to global warming. These changes are being accompanied by TYLCV, which is now becoming a large problem in the Korean tomato industry. The most effective way to reduce damage caused by TYLCV is to breed resistant varieties of tomatoes. To accomplish this, it is necessary to establish a simple inoculation technique for the efficient evaluation of resistance to TYLCV. Here, we present the rolling circle amplification (RCA) method, which employs a bacteriophage using phi-29 DNA polymerase for construction of infectious TYLCV clones. The RCA method is simple, does not require sequence information for cloning, and is less expensive and time consuming than conventional PCR based-methods. Furthermore, RCA-based construction of an infectious clone can be very useful to other emerging and unknown geminiviruses in Korea.

• Proceedings of the Microbiological Society of Korea Conference
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• 2003.05a
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• pp.83-86
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• 2003

Major advances in positive-sense RNA virus research have been facilitated by the development of reverse genetics systems. These systems consist of an infectious cDNA clone that encompasses the genome of the virus in question. This clone is then used as a template for the subsequent synthesis of infectious RNA for the generation of synthetic viruses. However, the construction of infectious cDNA for the Japanese encephalitis virus (JEV) has been repeatedly thwarted by the instability of its cDNA. As JEV is an important human pathogen that causes permanent neuropsychiatric sequelae and even fatal disease, a reliable reverse genetics system for this virus is highly desirable. The availability of this tool would greatly and the development of effective vaccines as well as facilitate studies into the basic biology of the virus, including the molecular mechanisms of viral replication, neurovirulence, and pathogenesis. We have successfully constructed a genetically stable infectious JEV cDNA containing full-length viral RNA genome. Synthetic RNA transcripts generated in vitro from the cDNA were highly infectious upon transfection into susceptible cells, and the cDNA remained stable after it had been propagated in E. coli for 180 generations. Using this infectious JEV cDNA, we have successfully expressed a variety of reporter genes from the full-length genomic and various subgenomic RNAs in vitro transcribed from functional JEV cDNAS. In summary, we have developed a reverse genetics system for JEV that will greatly facilitate the research on this virus in a variety of different fields. It will also be useful as a heterologous gene expression vector and aid the development of a vaccine against JEV.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1735-1740
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• 2008

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.361-368
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• 2005

A Korean isolate of Pepper mild mottle virus (PMMoV-Kr) was isolated from a diseased hot pepper plant and its biological and molecular properties were compared to that of PMMoV-J and PMMo V -So The genomic RNA of PMMoV-Kr consists of 6,356 nucleotides. The nucleotide and amino acid sequences identities of four viral proteins and two noncoding regions among PMMoV-Kr, PMMoV-S and PMMoV-J were $96.9\%\;to\;100.0\%\;and\;97.5\%\;to\;98.6\%$, respectively. Full-length cDNA amplicon of PMMoV-Kr was directly amplified by RT-PCR with a set of 5'-end primer anchoring T7 RNA promoter sequence and 3'-end virus-specific primer. Capped transcript RNAs from the full-length cDNA clone were highly infectious and caused characteristic symptoms of wild type PMMoV when mechanically inoculated to systemic host plants such as Nicotiana benthamiana and pepper plants.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.1-8
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• 2015

In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7^th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.