• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2037-2043
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• 2017

The surface protein hemagglutinin (HA) mediates the attachment of influenza virus to host cells containing sialic acid and thus facilitates viral infection. Therefore, HA is considered as a good target for the development of diagnostic tools for influenza virus. Previously, we reported the isolation of single-stranded aptamers that can distinguish influenza subtype H1 from H5. In this study, we describe a method for the selective electrical detection of H1 using the isolated aptamer as a molecular probe. After immobilization of the aptamer on Si wafer, enzyme-linked immunosorbent assay (ELISA) and field emission scanning electron microscopy (FE-SEM) showed that the immobilized aptamer bound specifically to the H1 subtype but not to the H5 subtype. Assessment by cyclic voltammetry (CV) also demonstrated that the immobilized aptamer on the indium thin oxide-coated surface was specifically bound to the H1 subtype only, which was consistent with the ELISA and FE-SEM results. Further measurement of CV using various amounts of H1 subtype provided the detection limit of the immobilized aptamer, which showed that a nanomolar scale of target protein was sufficient to produce the signal. These results indicated that the selected aptamer can be an effective probe for distinguishing the subtypes of influenza viruses by monitoring current changes.

• Proceedings of the Korea Contents Association Conference
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• 2014.11a
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• pp.57-58
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• 2014

인플루엔자 A 바이러스의 아형인 H5N1은 고병원성으로 조류 독감을 일으킨다. H5N1 바이러스는 원래 조류끼리만 감염되는 독감이고, 사람에게는 전염되지 않는다고 알려져 있었으나, 2003년에 베트남과 중국을 시작으로 현재까지 168명의 사망자가 기록되고 있다. 그러나 현재 시판되고 있는 진단키트(Rapid diagnostic kits)들은 H5N1 에 특이적인 것이 아니라 influenza A virus 모두를 진단한다. 따라서 influenza 감염여부는 확인 할 수 있지만, 이것이 H5N1 인지는 확인 할 수가 없다. H5N1은 전염성이 강하기 때문에 빠르게 진단하여 감염조류를 살 처분 하여야 더 많은 경제적 손실을 줄일 수 있다. 따라서 H5N1 에만 특이적인 epitope를 네트워크 기반으로 예측하여 진단제에 응용할 수 있도록 하고자 한다. 각 서열 정보는 Openflu (http://openflu. vital-it.ch/browse.php)에서 얻었다. H5N1은 H1N1에서 유래되었기 때문에 두 subtype의 차이점을 알아보고자 TCOFFEE에서 multiple sequence alignment를 수행한 결과 N-terminal 부분이 상이하였다. 상이한 H5N1의 N-terminal 부분이 H5N1 virus에 감염된 모든 host에서 존재하는지 알아보기 위해 host가 사람인 경우와 조류인 경우를 TCOFFEE에서 alignment 하였다. 그 결과 H5N1의 N-terminal 부분은 사람과 조류에서 보존적이었다. 따라서 H5N1의 N-terminal이 다른 subtype과 유사하지 않고 H5에만 특이적이기 때문에 진단키트 제작을 위한 epitope로 사용할 수 있을 것으로 기대된다.

• Genomics & Informatics
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• v.18 no.4
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• pp.40.1-40.8
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• 2020

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse-transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.

• Journal of Life Science
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• v.28 no.5
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• pp.555-562
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• 2018

Swine influenza virus (SIV) causes one of the most common diseases of the pig population, and its subtypes are determined by hemagglutinin (HA) and neuraminidase (NA). Recently, the SIV subtype diagnosis has been developed. The method using antigen-antibody reaction rather than PCR was mainly used because of the large change in the ribonucleotide sequences of SIV. Here, we have developed 10 diagnostic primer sets through multi-nucleotide sequences alignment of spreaded SIV since 2008 in Korea and then optimized the reaction of the one-step RT-PCR for rapid determination of SIV subtype. In addition, specific primers were designed to early determine the pandemic SIV by detecting unique M sequences proven in highly infectious and virulent subtypes of the influenza H1N1 (pH1N1). Here, some of the SIVs spread in Korea from 2008 to 2014 have been tested to determine the subtypes and pandemic potential of SIV. All diagnostic primer sets were found to be able to accurately determine the SIV subtype and to detect the pandemic SIV. In conclusion, it was confirmed that the optimized one-step RT-PCR analysis using these primer sets is useful for rapid diagnosis of SIV subtypes. These results can be used for development of SIV subtype diagnostic kit to early detect before virulent SIV spreads do.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.23-30
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• 2007

The most rapid Real-time PCR based detection method for Avian influenza A virus (AIV) subtype H5N1 was developed. The target DNA sequence in this study was deduced from H5N1 subtype-specific 387 bp partial gene of hemagglutinin, and was synthesized by using PCR-based gene synthesis on the ground of safety. Real-Time PCR was performed by $GenSpector^{TM}$ using microchip-based, total $1{\mu}l$ of reaction mixture with extremely short time in each steps in PCR. The detection including PCR-amplication and analysis of melting temperature was totally completed within 13 min. The H5N1-specific 189 bp PCR product was correctly amplified until 2.4 molecules of hemagglutinin gene as minimum of templates. This kind of PCR was designated as Quick Real-Time PCR in this study and it could be applied to detect not only AIV H5N1, but also other pathogens using PCR-based detection.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.30-37
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• 2011

This ultra real-time PCR (UPCR) based diagnosis system for avian influenza A virus (AIV) subtype was designed. The target primer in this study was derived from H5N1 subtype-specific 133 bp partial gene of hemagglutinin (HA), and was synthesized by using PCR-based gene synthesis on the ground of safety. UPCR was operated by Mini-Opticon Q-PCR Quantitative Thermal Cycler using aptamer-based molecular beacon, total 10 ${\mu}l$ of reaction mixture with extraordinarily short time in each steps in PCR. The detection including UPCR and analysis of melting temperature was totally operated within 15 min. The AIV-specific 133 bp PCR product was correctly amplified until 5 molecules of HA gene as minimum of templates. This kind of PCR was drafted as UPCR in this study and it could be used to detect not only AIV subtype, but also other pathogens using UPCR-based diagnosis.

• Korean Journal of Veterinary Research
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• v.47 no.4
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• pp.399-407
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• 2007

Cause of high lethality and dissemination to human being, new development of rapid method for the detection of highly pathogenic Avian Influenza Virus (AIV) is still necessary. For the detection of AIV subtype H5N1, typical pathogenic AIV, new method to confirm sub-typing of this virus is also needed. For the purpose of ultra-rapid detection and sub-typing of hemagglutinin and neuraminidase of AIV, this study was planned. As the results we could demonstrate an ultra-rapid multiplex real-time PCR (URMRT PCR) for the detection of AIV In this study, the URMRT PCR were optimized with synthesized AIV H5- and AIV Nl-specific DNA templates and GenSpector TMC, which is a semiconductor process technology based real-time PCR system with high frequencies of temperature monitoring. Under eight minutes, the amplifications of two AIV subtype-specific PCR products were successfully and independently detected by 30 cycled ultra-rapid PCR, including melting point analysis, from $1{\times}10^3$ copies of mixed template DNA. The URMRT PCR for the detection of AIV H5N 1 developed in this study could be expected to apply not only detections of different AIVs, but also various pathogens. It was also discussed that this kind of the fastest PCR based detection method could be improved by advance of related technology in near future.

• Genomics & Informatics
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• v.18 no.1
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• pp.5.1-5.5
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• 2020

Highly pathogenic avian influenza (HPAI) viruses have caused severe respiratory disease and death in poultry and human beings. Although most of the avian influenza viruses (AIVs) are of low pathogenicity and cause mild infections in birds, some subtypes including hemagglutinin H5 and H7 subtype cause HPAI. Therefore, sensitive and accurate subtyping of AIV is important to prepare and prevent for the spread of HPAI. Next-generation sequencing (NGS) can analyze the full-length sequence information of entire AIV genome at once, so this technology is becoming a more common in detecting AIVs and predicting subtypes. However, an analysis pipeline of NGS-based AIV sequencing data, including AIV subtyping, has not yet been established. Here, in order to support the pre-processing of NGS data and its interpretation, we developed a user-friendly tool, named prediction of avian influenza virus subtype (PAIVS). PAIVS has multiple functions that support the pre-processing of NGS data, reference-guided AIV subtyping, de novo assembly, variant calling and identifying the closest full-length sequences by BLAST, and provide the graphical summary to the end users.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Journal of Preventive Medicine and Public Health
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• v.52 no.5
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• pp.308-315
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• 2019

Objectives: Estimating influenza-associated mortality is important since seasonal influenza affects persons of all ages, causing severe illness or death. This study aimed to estimate influenza-associated mortality, considering both periodic changes and age-specific mortality by influenza subtypes. Methods: Using the Microdata Integrated Service from Statistics Korea, we collected weekly mortality data including cause of death. Laboratory surveillance data of respiratory viruses from 2009 to 2016 were obtained from the Korea Centers for Disease Control and Prevention. After adjusting for the annual age-specific population size, we used a negative binomial regression model by age group and influenza subtype. Results: Overall, 1 859 890 deaths were observed and the average rate of influenza virus positivity was 14.7% (standard deviation [SD], 5.8), with the following subtype distribution: A(H1N1), 5.0% (SD, 5.8); A(H3N2), 4.4% (SD, 3.4); and B, 5.3% (SD, 3.7). As a result, among individuals under 65 years old, 6774 (0.51%) all-cause deaths, 2521 (3.05%) respiratory or circulatory deaths, and 1048 (18.23%) influenza or pneumonia deaths were estimated. Among those 65 years of age or older, 30 414 (2.27%) all-cause deaths, 16 411 (3.42%) respiratory or circulatory deaths, and 4906 (6.87%) influenza or pneumonia deaths were estimated. Influenza A(H3N2) virus was the major contributor to influenza-associated all-cause and respiratory or circulatory deaths in both age groups. However, influenza A(H1N1) virus-associated influenza or pneumonia deaths were more common in those under 65 years old. Conclusions: Influenza-associated mortality was substantial during this period, especially in the elderly. By subtype, influenza A(H3N2) virus made the largest contribution to influenza-associated mortality.