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

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

• Journal of Microbiology and Biotechnology
• /
• v.20 no.1
• /
• pp.63-70
• /
• 2010

Novel influenza A (H1N1) is a newly emerged flu virus that was first detected in April 2009. Unlike the avian influenza (H5N1), this virus has been known to be able to spread from human to human directly. Although it is uncertain how severe this novel H1N1 virus will be in terms of human illness, the illness may be more widespread because most people will not have immunity to it. In this study, we compared the codon usage bias between the novel H1N1 influenza A viruses and other viruses such as H1N1 and H5N1 subtypes to investigate the genomic patterns of novel influenza A (H1N1). Totally, 1,675 nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A virus, including H1N1 and H5N1 subtypes occurring from 2004 to 2009, were used. As a result, we found that the novel H1N1 influenza A viruses showed the most close correlations with the swine-origin H1N1 subtypes than other H1N1 viruses, in the result from not only the analysis of nucleotide compositions, but also the phylogenetic analysis. Although the genetic sequences of novel H1N1 subtypes were not exactly the same as the other H1N1 subtypes, the HA and NA genes of novel H1N1s showed very similar codon usage patterns with other H1N1 subtypes, especially with the swine-origin H1N1 influenza A viruses. Our findings strongly suggested that those novel H1N1 viruses seemed to be originated from the swine-host H1N1 viruses in terms of the codon usage patterns.

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

• Journal of Life Science
• /
• v.28 no.5
• /
• pp.555-562
• /
• 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 Poultry Science
• /
• v.46 no.3
• /
• pp.173-183
• /
• 2019

Based on their virulence, the avian influenza viruses (AIVs) are classified into two pathotypes: low pathogenic avian influenza (LPAI) virus and highly pathogenic avian influenza (HPAI) virus. Among the 16 HA subtypes of AIV, only the H5 and H7 subtypes are classified as HPAI. Some AIVs, including H5 and H7 viruses, can infect humans directly. Six H7 subtype isolates from wild birds of the H7N7 (n=4) and H7N1 (n=2) subtypes were characterized in this study. Phylogenetic analysis showed that eight viral genes (HA, NA, PB2, PB1, PA, NP, M, and NS) of the H7 isolates clustered in the Eurasian lineage, the genetic diversity of which is indicated by its division into several sublineages. The Korean H7 isolates had two motifs, PEIPKGR and PELPKGR, at the HA cleavage site, which have been associated with LPAI viruses. Six H7 isolates encoded glutamine (Q) and glycine (G) at positions 226 (H3 numbering) and 228 of HA, suggesting avian-type receptor-binding specificity. None of the Korean H7 isolates had the amino acid substitutions E627K in PB2 and I368V in PB1, which are critical for efficient replication in human cells. The Korean H7 isolates showed no deletions in the NA stalk region and in NS. These results suggest that the Korean H7 isolates from wild birds are different from the H7N9 influenza viruses isolated in China in 2013, which are capable of infecting humans.

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

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

• Journal of Microbiology and Biotechnology
• /
• v.19 no.11
• /
• pp.1470-1474
• /
• 2009

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.4
• /
• pp.405-411
• /
• 2011

Avian influenza virus vaccines produced in oil-emulsified inactivated form with antigen content of at least 160 hemagglutinin units (HAU) induced immunity in birds. However, in addition to enhancing the effect of the adjuvant(s), other additional supplemented biological compounds included in inactivated vaccines could produce higher levels of antibody. We examined in chickens, Vietnamese ducks, and muscovy ducks the adjuvant effect of Sophy ${\beta}$-glucan (SBG), a ${\beta}$-1,3-1,6 glucan produced by the black yeast Aureobasidium pollulans strain AF0-202, when administered with an avian influenza H5 subtype vaccine. In Experiment 1, 40 chickens (ISA Brown hybrid), allocated to four groups of ten each, were immunized with Oil-H5N1(VN), Oil-H5N1(CN), Oil-H5N2(CN), and saline (control group), respectively. In Experiment 2, chickens (ISA Brown hybrid), muscovy ducks (French hybrid), and Vietnamese ducks (indigenous Vietnamese) were used to further assess the effect of SBG on immunogenicity of the Oil-H5N1(VN) Vietnamese vaccine. ELISA and hemagglutination inhibition (HI) assays were used to assess the antibody response. The H5 subtype vaccines initiated significantly higher immune responses in the animals dosed with SBG, with 1.0-1.5 $log_2$ higher HI titers and 10-20% ELISA seroconversion, compared with those not dosed with ${\beta}$-glucan. Notably, some of the animals dosed with SBG induced HI titers higher than 9.0 $log_2$ following boosting immunization. Taken together, our serial studies indicated that SBG is a potential effector, such as enhancing the immune response to the H5 vaccines tested.