• Genomics & Informatics
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• 제18권4호
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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.

• 한국동물위생학회지
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• 제40권3호
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• pp.187-192
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• 2017

In this report, fifteen monoclonal antibodies (MAbs) against an avian influenza virus (H9N2 subtype) were newly produced and characterized. These MAbs proved to react to the epitopes of nucleocapsid protein (NP), hemagglutinin (HA), neuraminidase (NA) and non-structural protein 1 (NS1) of Korean H9N2 strain, respectively. Two HA-specific MAbs showed the ability to inhibit the hemagglutination activity of H9N2 subtype avian influenza virus when tested by hemagglutination inhibition (HI) assay. All MAbs did not cross-react with other avian-origin viruses (Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus and avian rotavirus) by immunofluorescence test or enzyme-linked immunosorbent assay. The MAbs produced in this study could be useful as the materials for diagnostics and therapeutics against Korean-lineage H9N2 virus infections.

• 한국가금학회지
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• 제46권3호
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• pp.173-183
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• 2019

조류인플루엔자 바이러스 H7 subtype에 속하는 바이러스 중 일부는 가금류에 감염할 경우 고병원성이 발휘된다. 또 H7 아형 AIV중 일부는 사람에 감염하여 사망 등을 유발할 수도 있다. 본 연구는 야생조류로부터 분리된 H7 아형 조류인플루엔자 바이러스 6주(H7N7 아형 4주, H7N1 아형 2주)를 대상으로 8개 유전자 분절 전체의 염기서열을 분석하여 병원성, 사람 감염 가능성 등 그 특성을 조사하였다. 계통유전학적 분석결과, 국내에서 분리된 H7 아형 분리주들은 8개 유전자(HA, NA, PB2, PB1, PA, NP, M, NS) 모두 Eurasian lineage로 분류되었으나, Eurasian lineage 내에서도 각기 다른 sublineage로 분류되어 유전적 다양성이 있는 것으로 분석되었다. 한국 분리주 6주는 HA 단백질 분절부위 아미노산은 두 종류(PEIPKGR 및 PELPKGR)의 motif를 가지고 있었으나, 모두 저병원성 바이러스 특성을 가지고 있었다. 숙주세포 결합 특이성과 관련 있는 HA 단백질 receptor-binding site를 분석한 결과, 한국 분리주 모두는 사람 세포 수용체 결합특이성보다는 조류 세포 수용체 결합 특이성을 가지는 것으로 나타났다. 사람 감염 가능성을 높게 하는 부위에서의 아미노산 치환(PB2 단백질의 E627K 및 PB1단백질의 I368V)도 나타나지 않았고, 또한 NA stalk region에서의 결손도 관찰되지 않았다. 이상의 결과를 미루어 볼 때 한국 야생조류에서 분리된 H7 아형 6주 모두는 저병원성 바이러스로 최근 중국에서 사람 감염이 나타나고 있는 H7N9 바이러스와는 유전적으로 다른 계열의 바이러스인 것으로 판단된다.

• 생명과학회지
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• 제28권5호
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• pp.555-562
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• 2018

Swine influenza virus (SIV)는 돼지 개체군에서 가장 흔한 질병을 일으키는 바이러스 중 하나이며 그 subtype은 hemagglutinin (HA)와 neuraminidase (NA)에 의해 결정됩니다. 최근 SIV subtype 진단 방법이 개발되고 있으나 SIV의 리보뉴클레오타이드 서열의 많은 변이로 인해 PCR 보다는 항원-항체 반응을 이용하는 방법이 주로 사용되고 있다. 본 연구에서는 SIV 하위 유형의 신속한 결정을 위하여 2008년 이후 국내에서 발생한 SIV의 다중염기서열 정렬을 통하여 10개의 subtype 진단 프라이머 세트를 개발하고 이를 이용한 one-step RT-PCR 반응을 최적화하였다. 또한 감염력이 높고 독성이 있는 인플루엔자 H1N1 (pH1N1)의 아형에서 확인된 독특한 M 유전자서열을 검출함으로써 pandemic SIV를 조기에 결정하도록 특이적 프라이머를 설계하였다. 2008년부터 2014년까지 한국에서 발생한 9종의 SIV RNA를 활용하여 SIV의 아형 및 pandemic 가능성을 결정하기 위해 시험 분석한 결과 모든 진단 프라이머 세트는 SIV 아형을 정확하게 결정하였으며 pandemic SIV를 검출할 수 있는 것으로 확인되었다. 결과적으로 이들 프라이머 세트를 이용한 최적화된 one-step RT-PCR 분석이 SIV 아형의 신속한 진단에 유용하다는 것이 확인하였다. 이러한 결과는 SIV 하위 유형 및 pandemic SIV가 확산되기 전에 조기 발견을 위한 키트로 개발될 수 있음을 시사한다.

• 한국발생생물학회지:발생과생식
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• 제24권3호
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• pp.177-185
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• 2020

Although many aquaporin (AQP) transcripts have been demonstrated to express in the female reproductive tract, the defined localizations and functions of AQP subtype proteins remain unclear. In this study, we investigated the expression of AQP1, AQP3, AQP5, AQP6, and AQP9 proteins in female reproductive tract of mouse and characterized their precise localizations at the cellular and subcellular levels. Immunofluorescence analyses for AQP1, AQP3, AQP6, and AQP9 showed that these proteins were abundantly expressed in female reproductive tract and that intense immunoreactivities were observed in mucosa epithelial cells with a subtype-specific pattern. The most abundant aquaporin in both vagina and uterine cervix was AQP3. Each of AQP1, AQP3, AQP6, and AQP9 exhibited its distinct distribution in stratified squamous or columnar epithelial cells. AQP9 expression was predominant in oviduct and ovary. AQP1, AQP3, AQP6, and AQP9 proteins were mostly seen in apical membrane of ciliated epithelial cells of the oviduct as well as in both granulosa and theca cells of ovarian follicles. Most of AQP subtypes were also expressed in surface epithelial cells and glandular cells of endometrium in the uterus, but their expression levels were relatively lower than those observed in the vagina, uterine cervix, oviduct and ovary. This is the first study to investigate the expression and localization of 5 AQP subtype proteins simultaneously in female reproductive tract of mouse. Our results suggest that AQP subtypes work together to transport water and glycerol efficiently across the mucosa epithelia for lubrication, proliferation, energy metabolism and pH regulation in female reproductive tract.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• 센서학회지
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• 제23권4호
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• pp.278-283
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• 2014

In this study, we propose an immunosensor using zinc oxide nanorods (NRs) inside PDMS channel for detecting the influenza A virus subtype H7N9. ZnO with high isoelectric point (IEP, ~9.5) makes it suitable for immobilizing proteins with low IEP. In this proposed H7N9 immunosensor structure ZnO NRs were grown on the PDMS channel inner surface to immobilize H7N9 capture antibody. A sandwich enzyme-linked immunosorbent assay (ELISA) method with was used 3,3',5,5' tetramethylbenzidine (TMB) for detecting H7N9 influenza virus. The immunosensor was evaluated by amperometry at various H7N9 influenza antigen concentrations (1 pg/ml - 1 ng/ml). The redox peak voltage and current were measured by amperometry with ZnO NWs and without ZnO NWs inside PDMS channel. The measurement results of the H7N9 immunosensor showed that oxidation peak current of TMB at 0.25 V logarithmically increased from 2.3 to 3.8 uA as the H7N9 influenza antigen concentration changed from 1 pg/ml to 1 ng/ml. And then we demonstrated that ZnO NRs inside PDMS channel can improve the sensitivity of immunosensor to compare non-ZnO NRs inside PDMS channel.

• 대한수의학회지
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• 제52권4호
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• pp.223-230
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• 2012

The worldwide distribution and continuing genetic mutation of avian influenza virus (AIV) has been posed a great threat to human and animal health. A comparison of 3 isolates of AIV H9N2, A/chicken/Korea/KBNP-0028/00 (H9N2) (KBNP-0028), A/chicken/Korea/SNU8011/08 (H9N2) (SNU 8011) and an inactivated oil vaccine strain A/chicken/Korea/01310/01 (H9N2) (01310), was performed. The former 2 AIVs were isolated from field cases before and after the application of an inactivated H9N2 vaccine in 2007, respectively. The antigenic relationship, viral shedding, tissue tropism and genetic analysis were examined. The comparison of virus shedding from the cloaca and the oropharynx revealed that both isolates were more frequently isolated from the upper respiratory tract (90~100%) 1 day post inoculation (DPI) compared with isolation 5 DPI from gastrointestinal tracts (10~60%). Moreover, the isolate KBNP-0028 were recovered from all organs including bone marrow, brain and kidneys, indicating higher ability for broad tissue dissemination than that of SNU 8011. KBNP-0028 replicated earlier than other strains and with a higher titer than SNU 8011. In full-length nucleotide sequences of the NA gene and a partial sequence of the HA gene of SNU 8011, we found that there might be significant changes in tissue tropism, virus replication and genetic mutation in AIV H9N2 isolates.

• 약학회지
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• 제34권4호
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• pp.224-237
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• 1990

A single uniform population of specific, saturable, high affinity binding site of $[^3H]QNB$ guinuclidinyl benzilate(QNB) was identified in the rat cerebral microsomes. The Kd value(37.2 pM) for $[^3H]QNB$ calculated from the kinetically derived rate constants was in agreement with the Kd value(48.9 pM) determined by analysis of saturation isotherms at various receptor concentrations. Dimenhydrinate(DMH), histamine $H_1-blocker$, increased Kd value for $[^3H]QNB$ QNB without affecting the binding site concentrations and this effect resulted from the ability of DMH to slow $[^3H]QNB-receptor$ association. Pirenzepine inhibition curve of $[^3H]QNB$ binding was shallow(nH = 0.52) indicating the presence of two receptor subtypes with high ($M_1-site$) and low($M_2-site$) affinity for pirenzepine. Analysis of these inhibition curves yielded that 68% of the total receptor populations were of the $M_1-subtype$ and the remaining 32% of the $M_2-subtype$. Ki values for the $M_1-$ and $M_2-subtypes$ were 2.42 nM and 629.3 nM, respectively. Ki values for $H_1-blockers$ that inhibited $[^3H]QNB$ binding varied with a wide range ($0.02-2.5\;{\mu}M$). The Pseudo-Hill coefficients for inhibition of $[^3H]QNB$ binding by most of $H_1-blockers$ examined except for oxomemazine inhibition of $[^3H]QNB$ binding were close to one. The inhibition curve for oxomemazine in competition with $[^3H]QNB$ was shallow(nH = 0.74) indicating the presence of two receptor populations with different affinities for this drug. The proportion of high and low affinity was 33:67. The Ki values for oxomemazine were $0.045{\pm}0.016\;{\mu}M$ for high affinity and $1.145{\pm}0.232\;{\mu}M$ for low affinity sites. These data indicate that muscarinic receptor blocking potency of $H_1-blockers$ varies widely between different drugs and that most of $H_1-blockers$ examined are nonselective antagonist for the muscarinic receptor subtypes, whereas oxomemazine might be capable of distinguishing between subclasses of muscarinic receptor.

• 생명과학회지
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• 제17권3호통권83호
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• pp.340-344
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• 2007

조류인플루엔자바이러스는 사람 및 동물에 상당한 위협을 가하고 있다. 이 연구는 알칼리성 소독용액이 H5N1, H3N2, H6N1, H9N2형 조류인플루엔자바이러스를 불활성화시킬 수 있는지를 조사했다. 알칼리성 소독용액을 생리식염수에 100배 희석한 경우 조류인플루엔자가 MDCK 세포에서 증식하는 것을 완전히 억압하였다. 형광항체법을 이용한 실험에서도 알칼리성소독액을 처리한 H9N2 조류인플루엔자바이러스는 MDCK세포에서 증식이 억제되었고, 알칼리성 소독액을 처리하지 않은 H9N2조류인플루엔자바이러스는 증식이 억제되지 않았다. 이 결과는 알칼리성 소독액은 고병원성 H5N1을 포함을 조류인플루엔자바이러스를 방역하는데 도움이 될 수 있음을 시사한다.