• The Plant Pathology Journal
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• 제22권2호
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• pp.155-160
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• 2006

A strain of Pepper mottle virus (PepMov) was isolated from chili pepper plants in Korea. In host range study, this virus, designated PepMoV-SNU1, shared most characteristics with PepMoV isolates reported previously. Thermal inactivation point ($45^{\circ}C\;to\;75^{\circ}C$) and dilution end point ($10^{-1}\;to\;10^{-4}$) of PepMoV-SNU1 showed differences depending on the propagation hosts. Cylindrical and pinwheel-shaped inclusions were always observed in pepper leaf tissues infected with the virus alone. Unexpectedly, a special structure of pinwheel shaped inclusion surrounded with unknown small spots was also observed in the leaf section when co-infected with a strain of pepper mild mottle virus. The partial sequence of coat protein gene and 3' untranslated region of PepMoV-SNU1 showed 98% identity with those of other PepMoV isolates. A primer pair derived from 3' end of the coat protein gene and poly A tail regions were designed. Optimal detection condition of PepMoV-SNU1 by RT-PCR was tested to determine appropriate annealing temperature and additional volumes of oligo-dT (18-mer), dNTP, and Taq polymerase. Under the optimized condition, an expected 500 Up PCR-product was detected in pepper leaves infected with PepMoV-SNU1 but not in healthy plants.

• 한국미생물·생명공학회지
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• 제43권1호
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• pp.79-83
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• 2015

박과는 스쿼시, 호박, 애호박, 조롱박, 오이 및 수박 등 100여 속이 넘는 속이 포함된 식물과이며, 종자의 수입량이 매우 많은 작물이다. 이들이 우리나라로 수입될 때, 규제바이러스인 Squash mosaic virus (SqMV), Cucumber green mottle mosaic virus (CGMMV) 및 Kyuri green mottle mosaic virus (KGMMV)에 대하여 검역을 수행한다. 본 연구에서는 SqMV, CGMMV 및 KGMMV를 신속·높은 감도로 검출할 수 있는 특이적 RT-PCR 및 nested PCR 프라이머 조합과 유전자변형-양성대조구 플라스미드를 개발하였다. 또한 본 연구에서 개발한 진단시스템을 2010년부터 2014년 상반기까지 현장적용하여, SqMV 47건, CGMMV 67건 및 KGMMV 17건을 검출하였다.

• The Plant Pathology Journal
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• 제40권2호
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• pp.125-138
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• 2024

Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Journal of Veterinary Science
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• 제21권2호
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• pp.24.1-24.11
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• 2020

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.

• Journal of Veterinary Science
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• 제23권5호
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• pp.32.1-32.11
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• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• 대한임상검사과학회지
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• 제51권3호
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• pp.329-335
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• 2019

매년 호흡기 바이러스 감염으로 인해 수 백만명의 소아들이 사망한다. 호흡기 바이러스 감염의 원인 병원체 중 Human rhinovirus (HRV)는 코감기의 주요 원인 균으로 면역력이 약한 영, 유아, 노인 그리고 천식 환자에게 심각한 호흡기 감염의 원인으로 작용하는 병원체이다. 2012년 1월부터 2018년 12월까지 천안 단국대학교 병원 진단검사의학과에 호흡기 바이러스 검사가 의뢰된 호흡기 검체 9,010개의 검체를 real time reverse transcription PCR (real time RT-PCR) 방법으로 검사했다. 총 12종의 호흡기 바이러스를 real-time RT-PCR로 검출했다. 연구기간 중 평균 검출률은 21.3%이었고, HRV 양성 환자의 평균 연령은 6.5세였다. 7월의 검출률이 32.4%로 가장 높게 나타났고 2월이 8.3%로 가장 낮았다. 연령대별로 검출률을 분석해봤을 때 10세 미만의 검출률이 가장 높았다. HRV의 중복 감염률은 35.3%이고, 가장 흔한 조합은 Adenovirus와의 조합이었다. 호흡기 바이러스 감염증은 비슷한 임상 증상을 가지고 있어 빠른 진단이 이루어 져야 적절한 시기에 적절한 치료를 할 수 있다. 호흡기 바이러스 감염은 보통 면역력이 약한 어린아이와 노인에서 주로 발생하는 것으로 알려져 있다. 하지만 본 연구에서는 10세 미만에 이어 10대 환자들의 검출률이 높았다. 그리고 1,2월을 제외하고 15% 이상의 detection rate를 보였다. HRV의 감염 양상에 대한 꾸준한 연구가 필요할 것으로 사료된다.

• The Plant Pathology Journal
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• 제21권3호
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• pp.258-261
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• 2005

We conducted a survey on pepper virus diseases in 31 regions in Korea from November 2001 to December 2004. Using electron microscopy, test plant reaction, rapid immuno-filter paper assay (RIPA), reverse transcription-polymerase chain reaction (RT-PCR) and/or analysis of viral nucleotide sequences, we found a number of viruses from 1,056 samples that we collected. These included Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), Pepper mild mottle virus (PMMoV), Broad bean wilt virus 2 (BBWV2), Tobacco mild green mosaic virus (TMGMV), and Tomato spotted wilt virus (TSWV). Of the samples analyzed, $343(32.5\%)$ were infected with CMV, $209(19.8\%)$ with PepMoV, $141(13.4\%)$ with PMMoV, $12(1.1\%)$ with BBWV2, $40(3.8\%)$ with TMGMV, $5(0.5\%)$ with TSWV, $153(14.5\%)$ with CMV and PepMoV, $54 (5.1\%)$ with CMV and PMMoV, $31(2.9\%)$ with PepMoV and PMMoV, $3(0.3\%)$ with CMV and BBWV2, $1(0.1\%)$ with CMV, PepMoV and BBWV2, $8(0.8\%)$ with CMV, PepMoV and PMMoV, and $30 (2.8\%)$ samples were infected with viruses which were not identified. CMV was the most predominant virus in all inspected fields and the number of the samples infected with PMMoV was relatively low as compared PepMoV infection level in pepper. TMGMV was only found in the southern part of Korea, while TSWV was isolated in Anyang and Yesan. However, we did not encounter in this survey the Alfalfa mosaic virus (AMV), Potato virus Y (PVY), Tobacco mosaic virus (TMV), and Pepper vein chlorosis virus (PVCV).

• 대한의생명과학회지
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• 제17권4호
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• pp.297-304
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• 2011

Influenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen that continues to evolve and burden in the human public health. It is able to spread efficiently from human to human and have the potential to cause pandemics with significant morbidity and mortality. It has been estimated that every year about 500 million people are infected with this virus, causing about approximately 0.25 to 0.5 million people deaths worldwide. Influenza A viruses are classified into different subtypes by antigenicity based on their hemagglutinin (HA) and neuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes and access for epidemiological analysis of this subtypes demanded a rapid development of specific diagnostic tools. Also, rapid identification of the subtypes can help to determine the antiviral treatment, because the different subtypes have a different antiviral drug resistance patterns. In this study, our aim is to detect influenza A virus subtypes by using real-time nucleic acid sequence based amplification (NASBA) which has high sensitivity and specificity through molecular beacon. Real-time NASBA is a method that able to shorten the time compare to other molecular diagnostic tools and is performed by isothermal condition. We selected major pandemic influenza A virus subtypes, H3N2 and H5N1. Three influenza A virus gene fragments such as HA, NA and matrix protein (M) gene were targeted. M gene is distinguished influenza A virus from other influenza virus. We designed specific primers and molecular beacons for HA, NA and M gene, respectively. In brief, the results showed that the specificity of the real-time NASBA was higher than reverse transcription polymerase chain reaction (RT-PCR). In addition, time to positivity (TTP) of this method was shorter than real-time PCR. This study suggests that the rapid detection of neo-appearance pandemic influenza A virus using real-time NASBA has the potential to determine the subtypes.

• 식물병연구
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• 제19권4호
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• pp.288-293
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• 2013

국내외적으로 ACLSV, ASGV, ApMV, ASSVd와 같은 바이러스 및 바이로이드 병의 발생으로 사과 과실의 생산량 감소와 기형적인 외형 등 많은 문제점들이 보고되었다. 하지만 사과 바이러스의 감염에 대한 방제 대책은 거의 알려진 바가 없는 실정이다. 따라서 본 논문에서는 사과 신품종인 '단홍', '홍안', '새나라', '썸머드림'을 분양하기에 앞서 바이러스 무병묘를 생산하는 시스템을 확립하고자 하였다. $37^{\circ}C$가 유지되는 항온 항습장치에서 4주간 열처리를 하였으며 기내에서 경정 배양을 하였다. 열처리된 각각의 사과 신품종들은 바이러스 진단 프라이머를 통해 RT-PCR을 수행하여 바이러스 진단을 수행하였다. 결과적으로 '단홍'은 28%의 바이러스 무병묘를 확보할 수 있었으며 '홍안'은 16%, '새나라'와 '썸머드림'은 12%의 확률로 바이러스 무병 사과를 확보할 수 있었다. 본 연구결과는 열처리 및 경정배양을 통해 사과 신품종에서 바이러스 무병묘 생산 시스템 구축이 가능함을 보여주었다.