This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

다음 데이터의 수집에 사용 된 모든 돼지는 오하이오 주립 대학 기관 동물 관리 및 사용위원회에 의해 보호 된 (동물 사용 프로토콜 번호 2009A0134-R1). 1. 바이러스 성 전송 매체의 준비 및 시료 채취 튜브 <ol><li> 정제수 900 ml의 뇌 심장 주입 37 g을 넣고 가열 70 ° C가 완전히 분말을 용해하는 동안 교반 막대와 자석 교반기로 충분히 혼합한다. </li><li> 15 분 동안 121 ℃에서 오토 클레이브 국물. 국물까지 벤치 위에 차가운 실내 온도에 도달한다. 4 ° C에서 냉장 하룻밤 필요한 경우. </li><li> 자기 교반 막대, 페니실린 G 나트륨 염 6.02 g의 실온에서 순수한 물 50 ㎖에 황산 스트렙토 마이신 10 g과 혼합하여 용해. 100 ㎖에 총 볼륨을 가지고 추가 정제수를 추가합니다. 멸균 병에 0.22 μm의 폴리 에테르 설폰 멤브레인을 통해 솔루션을 필터. </li><li> 무균는 냉각 뇌 심장 주입 국물로 필터링 된 페니실린과 스트렙토 마이신 솔루션을 추가하고 바이러스 수송 배지를 만들기 위해 철저하게 (교반 막대 또는 손으로 소용돌이 플라스크)를 섞는다. </li><li> 바이러스는 무균 전송 매질의 pH를 테스트; 필요한 경우, 염산 또는 수산화 나트륨을 사용하여 7.4 ± 0.2로 pH를 조정합니다. </li><li> 장과 하몬 기재된 바와 같이, RRT-PCR에 의해 인플루엔자 바이러스의 매트릭스 단백질을 시험함으로써 바이러스 전송 매체의 품질 보증 테스트를 수행한다. (22) </li><li> 8 ml의 용량 멸균 고밀도 폴리에틸렌 병에 바이러스 수송 배지 5 ㎖를 분배. 현장에서 사용하기위한 튜브에 라벨. </li><li> 샘플 수집 할 때까지 -20 ℃에서 표준 냉동 박스에서 바로 사용할 수있는 튜브를 저장합니다. </li></ol> 돼지의 코 닦음 2. 컬렉션 <ol><li> 컬렉션 샘플 직전에 튜브의 해당 번호를 해동; 한 바이알 샘플링 돼지 당 필요합니다. 룸에서 해동온도는 약 30 ~ 45 분 소요됩니다. 해동 유리 병 사용까지 얼음 팩에 냉정을 유지. </li><li> 돈 적절한 개인 보호 장비를 상황에 의해 결정 (등 예 : 작업복, 부트 커버, 마스크, 귀마개,)로. </li><li> 동물의 영역을 입력합니다. 필요한 경우, 작은 영역으로 돼지를 한정하지 않지만 억제 더 않습니다. 동물을 쉬고 환기하지 마십시오. </li><li> 일회용 시험 장갑 한 켤레를 착용 할 것. 터치 돼지, 사람, 또는 무생물에 의해 장갑이 오염되지 않도록주의 할 것. </li><li> (2를.에서 × 2.) 5.08 cm × 멸균 5.08 CM을 제거하기 위해 장갑을 낀 손을 사용하여면 거즈 패드의 래퍼에서. 멸균 거즈 패드는 가능하면 사용되어야한다. 개별 포장 거즈 패드는 패키지 당 두 거즈 패드로 감싸 그보다 사용하기 더 편리합니다. </li><li> 한 손의 손가락이 샘플링 가능한 패드의 많은 노출과면 거즈 패드를 잡으십시오. </li><li> 복용, 돼지의 주둥이에 걸쳐 거즈 패드를 닦아가능하면 별도의 관리는 외부 콧 구멍을 입력합니다. 같은 거즈 패드로 볼 수 비강 분비물 (약 1 ml)에 수집합니다. </li><li> 같은 손을 사용하여, 바이러스 전송 매체를 포함하는 바이알에 배치를 용이하게하도록 자체적으로 거즈 패드를 접어. </li><li> 다른 손을 사용하여 바이알로부터 캡을 제거하고, 바이알 내로 접혀 거즈 패드를 배치했다. 바이알을 다시 정리해 보면하고 거즈 패드와 바이러스 수송 배지의 혼합을 보장하기 위해 유리 병을 흔들어. </li><li> 해당 콘센트에 장갑과​​ 장소를 제거합니다. 각 돼지 사이에 장갑을 변경합니다. </li><li> 유리 병 식별자를 확인합니다. 기록 돼지 식별 번호, 나이, 성별, 임상 증상 및 연구자 필요하다고 인정하는 다른 음. </li><li> 즉시 수집 후 샘플을 진정. 여러 동결 - 해동 사이클을 방지하기 위해 동결 전에 샘플의 분취 량을 고려한다. 즉시 수집 후 가능한 한 실험실 교통을위한 드라이 아이스에 샘플을 배치합니다. 유리 병에 보관 -80테스트가 시작 될 때까지 C를 °. </li></ol> 3. 탐지 인플루엔자의 바이러스 핵산 <ol><li> 5 분 동안 37 ° C 건조 비드 용기에 샘플을 해동 한 후 20 ~ 30 분 동안 실온에서 벤치 위에 샘플을 해동 완료. 구슬에 여러 병의 동시 첨가는 목욕 가열하게됩니다; 주의 튜브를 과열하지으로 행사되어야한다. </li><li> RNA 추출을위한 바이러스 수송 배지 100 μl를 제거합니다. <ol><li> 많은 수의 샘플로부터 RNA를 추출하기 위해 높은 처리량 (96 웰 플레이트 포맷) 자성 비드 플랫폼을 사용한다. (31) </li></ol></li><li> 빠른 검색 인플루엔자 바이러스 실시간 역전사의 PCR 분석을 사용합니다. (31) </li></ol> 인플루엔자 4. 분리 코 닦음에서 바이러스 <ol><li> 섹션 3.1에서 상술 한 바와 같이 샘플을 해동 </li><li> (1,000 겐타 마이신 황산염과 해동 샘플을 치료μg의 / ㎖), 암포 테리 신 B (22.5 μg의 / ㎖), 카나마이신 설페이트 (325 μg의 / ㎖). </li><li> Madin-다비 개 신장에 접종 (MDCK) 전술 한 바와 같이 상피 세포. (32) </li></ol>

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M., Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+23,+RNA+extraction+from+swine+samples+and+detection+of+influenza+A+virus+in+swine+by+real-time+RT-PCR.">Ch. 23, RNA extraction from swine samples and detection of influenza A virus in swine by real-time RT-PCR.</a> Animal Influenza Virus. , 277-293 (2014).</li><li>Zhang, J., Gauger, P. C., Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+22,+Isolation+of+swine+influenza+virus+in+cell+cultures+and+embryonated+chicken+eggs.">Ch. 22, Isolation of swine influenza virus in cell cultures and embryonated chicken eggs.</a> Animal Influenza Virus. , 265-276 (2014).</li><li>Bowman, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inability+to+Screen+Exhibition+Swine+for+Influenza+A+Virus+Using+Body+Temperature.">The Inability to Screen Exhibition Swine for Influenza A Virus Using Body Temperature.</a> Zoonoses Public Health. , (2015).</li><li>Prickett, J. R., Kim, W., Simer, R., Yoon, K. J., Zimmerman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral-fluid+samples+for+surveillance+of+commercial+growing+pigs+for+porcine+reproductive+and+respiratory+syndrome+virus+and+porcine+circovirus+type+2+infections.">Oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections.</a> J. Swine Health Prod. 16 (2), 86-91 (2008).</li><li>Prickett, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Porcine+reproductive+and+respiratory+syndrome+virus+infection+in+porcine+oral+fluid+samples:+a+longitudinal+study+under+experimental+conditions.">Detection of Porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions.</a> J. Vet. Diagn. Invest. 20 (2), 156-163 (2008).</li><li>Pepin, B., Liu, F. F., Main, R., Ramirez, A., Zimmerman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+oral+fluid+from+individually+housed+sows.">Collection of oral fluid from individually housed sows.</a> J. Swine Health Prod. 23 (1), 35-37 (2015).</li></ol>

The authors have nothing to disclose.

폴리 에스테르로 만들어진 코 면봉을 사용하여 돼지에서 시료를 수집하는 것은 IAV 감시를 수행에 도움이 입증되었습니다; 그러나, 비강 면봉 절차의 사용은 억제 용 스네어의 사용으로 인해 필요한 감시 활동을 방해한다. 비강 와이프는 시료 채취시에 사람과 돼지의 스트레스를 최소화하기 위해 현재 돼지 샘플링 기법의 정제를 나타낸다. 이 방법은 개발 및 전시 돼지 설정에서 확인되었지만 돼?...

<html> 인플루엔자 A 바이러스 (IAV)는 국내 조류, 돼지, 인간을 포함하여 많은 종의 호흡기 질환을 유발한다. 인해 발생할 수있는 분할 IAV 게놈 빠른 바이러스 진화의 reassortment에 새로운 IAV 균주에 자주 등장. 돼지는 일반적으로 북미 신종 (H1N1, H1N2, H3N2) 사이에서 순환 IAV의 세 가지 주요 서브 타입은있다. (1) 여러 호스트 종 IAVs의 유전자 재 배열을위한 혼합 용기 역할을 할 수 종 있지만, 인간의 여러 IAV 소개는이 그 하위 유형 내에서 광범위한 IAV 다양성되었다. 돼지를 감염 IAVs의 2 급속한 진화는 인간, 조류 및 돼지 바이러스로부터 유전자 세그먼트를 포함하는 트리플 된 재조합 IAV 3는 미국에서 돼지들 사이에서 확산 된 1998 년부터 분명했다. 4 내부 유전자 그 트리플 재조합 체의 IAV에서 세그먼트는 현재 돼지를 감염 IAVs 가운데 매우 널리 남아있다. (5) &quot;&gt; 전세계, IAV는 전형적인 임상 증상은 발열, 식욕 부진, 무기력, 기침을 포함하는 돼지의 호흡기 질환의 중요한 원인은, 콧물과 가난한 체중 증가 재채기, 호흡 고심한 흔적. IAV 어디 생식 농장을 뿌리는 것이 특히 비용이 많이 드는 될 수 있습니다 때문에 IAV 유도 발열과 약한 태어난 새끼 돼지에 오류가 기록되어있다. 6, 7을 미국 내에서 IAV는 일반적으로 상업 돼지 떼와 IAV 감염 돼지들 사이의 광범위한 항원 및 유전자 다양성과 계속 진화 검출이의 제어를 방해하고있다 바이러스. 8-11 트리플 된 재조합 북미 돼지의 혈통과 유라시아 조류 같은 돼지 혈통에서 유전자 세그먼트를 포함하는 돼지 혈통 IAV는 전 세계적으로 대유행 발생 때 2009 년에 실현 된 돼지의 유전자 재 배열로 인한 전염병 IAV 균주의 출현에 대한 공공 건강 문제 인간. 12 유행성 바이러스 (A (H1N1) pdm09)는 이후이발병 돼지와 재조합 된 IAV는 13, 14 균주 및이 새롭게 재조합 된 균주 중 일부는 인간에게 다시 전송하고있다. (15) 유전자 재 배열 이벤트 및 대유행 가능성이있는 새로운 IAV 균주의 출현 돼지 필수적으로 IAV 바이러스 순환의 적극적인 감시를 만드는의 주파수를, 특히 돼지 - 인간 인터페이스에서. 돼지 - 인간 인터페이스 IAV 양방향 종간 송신을 위해 중요하다. 인간 간 돼지 상업용 돼지의 생산에서 발생하는 송신 돼지 인구에 현재 존재 IAV 다이버 다량 담당한다. 농업 박람회는 미국에있는 사람과 돼지의 comingling 가장 큰 설정하고 변종 H3N2 IAV의 발발시, IAV의 인수 공통 전송을위한 사이트. 2012 년 15-21 알려진, 경우의 93 %는 참석을보고 일 농업 박람회 질환 발병하기 전에. 15 게놈 분석인수 공통 전송을 확인 인간 균주에 비해 전시 돼지에서 바이러스 균주의. IAV 감염 (21) 전시 돼지 종종 직접 진단 테스트의 필요성을 나타내는 질병, 21-23의 임상 증상을 표시하지 않습니다. 눈에 띄게 아픈 돼지의 샘플링은 혼자 성공적으로 돼지에 IAV의 유병률을 파악하지 않으며 돼지들 사이에서 신흥 IAV의 새로운 변종을 식별하는 데에 의존 할 수 없습니다. 활동 감시 IAV의 새로운 변종 돼지 및 돼지 및 공중 보건 모두 그들의 위협을 평가하기의 검출을위한 절대적으로 필요하다. 대부분의 IAV 감시 활동은 자발적 때문에 최소한의 파괴적인 방법이 필요하다. IAV가 돼지를 감염에 대한 세 가지 주요 도축 샘플 수집 절차는 다음과 같습니다 비강 면봉, 구강 유체 및 코 와이프. IAV 목록을 합성 섬유의 삽입을 감지하는 개별 돼지를 샘플링 현재 권장되는 방법으로 콧 구멍에 면봉을 밀고비강 분비물과 상피 세포를 수집 할 수 있습니다. (24, 25)를 돼지가 수동으로 또는 올무는 동물의 크기에 따라와 돼지를 억제해야이 절차, 훈련받은 사람의 팀을 피하려고 할 수 있기 때문에. (26)를 구속 과정에 대한 힘드는입니다 직원 및 돼지에 대한 스트레스. 경쟁 동물에 추가 스트레스의 인식이 감시 활동에 소유자가 저항 할 수 있도록 또한, 전시 돼지는 종종 박람회에서 여러 대회에 참여하고 있습니다. 80-100% IAV에 감염된 가축에 이르기까지 IAV 검출의 확률로, 구강 유체는 돼지의 인구 IAV의 분자 검출을위한 면봉을 비강에 인기있는 대안이되고있다. 27, 28는 또한, 경구 용 유체의 넓은 창을 제공 할 수있다 초기 감염 다음과 같은 비강 면봉보다 IAV 감지. 그러나, IAV 경구 유체로부터 분리 바이러스 분리 시도의 50 % IA 결과로 문제가 있었다V 복구. (29) 돼지에 IAV 감시 동안 비강 면봉 대신에 코 와이프를 사용하면 상기의 한계를 극복한다. 비강 와이프는 구속 스네어의 사용을 필요로하지 않고, 과정의 동물 또는 증인을 강조하지 않고 수행 될 수있다. 최소한의 기술 교육은 감시 샘플을 수집, 돼지 소유자를 포함한 비 수의학 전문가, 수 비강 잎사귀를 수집하기 위해 필요합니다. 코 와이프는 이전에 바이러스 (30)와 샘플링이 비 침습적 방법에 대한 자세한 프로토콜은 아래에 설명되어 검출 및 인플루엔자의 분리를위한 비강 면봉에 비교 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="1051">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">211059</td>
			<td style="width:356px;"></td>
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			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
			<td style="width:119px;">0382</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Gentamicin Solution</td>
			<td style="width:360px;">Mediatech, Inc.</td>
			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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			<td height="21" style="height:21px;width:216px;">Amphotericin B Solution</td>
			<td style="width:360px;">Fisher</td>
			<td style="width:119px;">S 24 25</td>
			<td>250 ug/ml</td>
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			<td height="21" style="height:21px;width:216px;">Kanamycin Sulfate</td>
			<td style="width:360px;">Teknova</td>
			<td style="width:119px;">K2105</td>
			<td>5000 ug/ml</td>
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			<td style="width:360px;">TPP Techno Plastic Products AG&#160;</td>
			<td style="width:119px;">99150</td>
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			<td height="21" style="height:21px;width:216px;">Nalgen Diagnostic Bottles</td>
			<td style="width:360px;">Thermo Scientific</td>
			<td style="width:119px;">342002-9025</td>
			<td>HDPE with white PP closure</td>
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			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
			<td style="width:360px;">Covidien</td>
			<td style="width:119px;">441211</td>
			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Nitrile Exam Gloves</td>
			<td style="width:360px;">Saftey Choice</td>
			<td style="width:119px;">19-170-010 (A-D)</td>
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</table>

nasal wipes for influenza a virus detection and isolation from swine

인플루엔자 바이러스가 빠르게 돼지 인구의 진화와 새로운 변종이 지속적으로 등장하고 있기 때문에 돼지에서 인플루엔자 A 바이러스에 대한 감시는 인간과 동물의 건강에 중요하다. 돼지는 그들에게 출현과 새로운 인플루엔자의 유지 보수 바이러스 균주에 대한 중요한 호스트를 만드는 인플루엔자 A 바이러스의 다양한 계통에 감염 될 수 있습니다. 상업 돼지 농장, 농업 박람회, 라이브 동물 시장 등 다양한 설정에서 돼지를 샘플링 현재 IAV 균주 순환의 포괄적 인 뷰를 제공하는 것이 중요하다. 이 돼지의 물리적 구속을 필요로하기 때문에 현재의 금 표준 도축 샘플링 기술 (비강 면봉 즉 컬렉션) 노동 집약적이다. 코 와이프 ​​동물없이 구속을 최소한으로 돼지의 주둥이에 걸쳐 직물의 조각을 문질러 포함한다. 코는 절차를 수행 할 간단하고 전문 수의사 또는 동물 취급 교육 인력을 필요로하지 않습니다 닦습니다. W비강 면봉보다 약간 덜 민감 hile은 바이러스 탐지 및 차단 속도는 코가 낮은 스트레스 샘플링 방법이 필요한 경우 개별 돼지를 샘플링하기위한 실행 가능한 대안을 쳐하게하기에 충분하다. 진행 프로토콜 가능한 코를 수집하기 위해 필요한 단계가 개인 돼지 닦아 윤곽선.

이 방법의 성공적인 사용은 RRT-PCR은 어떤 환경 파편 샘플링 중에 픽업에서 RNA 추출 및 RRT-PCR 동안 내부 통제의 사용과 함께, 쇼 샘플은 PCR 억제제를 포함하지 않은 것으로 결과를 얻을 수 있습니다. 샘플 접종 후 바이러스 분리 우물은 샘플에서 볼 환경 찌꺼기가 없도록해야한다. PCR은 바이러스 핵산, 반드시 가능한 바이러스를 감지하기 때문에 PCR은 종종 바이러스 분리보다 높은 IAV 긍정적 인 비?...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

돼지에서 인플루엔자 바이러스 탐지 및 격리를위한 코 닦음

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K 조회수.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

비디오: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

플라스미드 DNA의 재조합 독감 바이러스의 생성

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

인플루엔자에의 DNA Microarray에 ampliPHOX Colorimetric 검색

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

감염된 세포의 염색질에서 인플루엔자 바이러스 Ribonucleoprotein 컴플렉스의 동질 정제

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

인간 백신 닭 계란에 인플루엔자 바이러스 전파

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

그라데이션 원심 분리에 의해 인플루엔자 A 바이러스 캡시드의 생체 외 분해에

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

감염 마우스 모델에서 인플루엔자 A 바이러스 연구

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

인플루엔자 바이러스 단일 클론 항 체를 중화의 탈출 변종의 생성

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...