우리는 시약으로 우리를 제공 요헤이 야마우치와 로버타 만치니 감사합니다. 아 연구소는 마리 퀴리 초기 교육 네트워크 (ITN)에 의해 지원되었다, 스위스 국립 과학 재단 (National Science Foundation)에 의해 유럽 연구위원회 (ERC) (SINERGIA).

버퍼 및 재고 솔루션 1. 준비 <ol><li> 80 ML의 DDH 2 O.에 470 mg의 트리스 염산염 390 mg을 MES 수화물 580 mg의 NaCl을 용해하여 (20 MM의 MES, 30 mM 트리스, 100 mM의 염화나트륨)을 MNT 버퍼를 준비 pH를 7.4로 버퍼를 조정하고 DDH 2 O. 100 ml의 최종 부피로 가져 </li><li> 80 ml의 DH 2 O 각각에 9.76 g의 MES 수화물을 용해하여 500 mM의 MES 버퍼의 3 개의 동일한 솔루션을 준비합니다. 농축 된 NaOH로 적정하여 각각의 pH 5.8, 5.4, 5.0,에 버퍼를 조정합니다. DH 2 O. 100 ml의 최종 부피로 각 버퍼를 가지고 </li><li> DH 2 O 80 ㎖에 각각 7.88 g 트리스 염산에 용해시켜 500 mM 트리스 버퍼의 두 개의 동일한 솔루션을 준비하고 진한 HCl로 적정하여, 각각 7.4와 6.4로 pH를 조정합니다. DH 2 O. 100 ml의 최종 부피로 버퍼를 가지고 </li><li> DH 2 O 50 % 글리세롤 솔루션을 준비하고 glyc 때까지 잘 저어EROL는 균일하게 혼합이다. 유리 병으로 Steritop 필터 부 (0.22 ㎛의 공극 크기)를 통해 용액을 필터. </li><li> 10 % NP-40 용액 DH 2 O. 1 M NaCl을 준비 </li><li> 4 ML의 DDH 2 O. 두 개의 정제를 용해시켜 25 배 농축 된 단백질 분해 효소 억제제를 준비 </li></ol> 글리세롤 그라디언트 2. 준비 <ol><li> 5 ml의 세제 버퍼 마스터 믹스 준비 (PH 7.4, 6.4, 5.8, 5.4, 5.0) 시험을 각각의 pH 조건에 대한 용액 (300 mM의 NaCl을 2 % NP-40을 2 배는 단백질 분해 효소 억제제를 집중). 이를 위해, 1 M의 NaCl 9 ㎖, 10 % NP-40 6 ml 일 25X 프로테아제 저해제 스톡 2.4 ㎖, 9 ML의 DDH 2 0 섞는다. </li><li> 각각의 pH 조건 피펫 대한 50 ㎖ 원뿔형 튜브에 마스터 믹스의 4.4 ml의. </li><li> pH 값의 경우 5.8 위의 튜브에 각각의 pH를 조정 500 mM 트리스 주식 솔루션의 0.6 ml를 추가합니다. pH가 5.8와 하부를 들어 각각의 pH를 조정 500 mM의 MES 주식 솔루션의 0.6 ml를 추가합니다. </li><li> 5 확인300 mM의 NaCl을 함유하는 완충액 ml의 2 배 프로테아제 억제제는, 60 mM 트리스 pH를 7.4 DDH 2 O. 조정 이 세제없이 제어 그라데이션 버퍼로서 기능한다. </li><li> 필요한 경우, 각각 농축 HCI 또는 수산화 나트륨 용액을 첨가하여 pH를 7.4, 6.4, 5.8, 5.4 및 5.0의 용액 pH를 미세 조정한다. </li><li> 여섯 가지의 25 % 글리세롤 용액의 결과로 세제 함유 세제 프리 버퍼의 혼합물을 5 ml의 50 % 글리세롤 5 mL를 추가한다. 산도 표시기 스트립을 사용하여 pH 값을 확인한다. 주 : 측정 된 pH가 목적하는 값에서 크게 다를 경우, 각각의 용액을 다시 제조한다. </li><li> 7 비율 : 3에서 DH 2 O 50 % 글리세롤 주식을 혼합하여 15 % 글리세롤 솔루션을 준비합니다. </li></ol> IAV의 3 초 원심 분리 <ol><li> 5 ML의 주사기와 옛 성은을 사용하여 매우 명확한 원심 분리 튜브에 (13.2 ml를, 14mm X 89mm)을 3 ml의 15 % 글리세롤 솔루션을 추가DLE (9cm 길이 21 G). 이 그라데이션의 무결성을 방해 하듯 튜브의 내부 벽에 방울을 두지 마십시오. 여섯 원심 분리 튜브, 시험 법에 다섯 pH 조건 각각에 대해 하나 제어 샘플에 대한 하나의 총에 대해이 작업을 반복합니다. </li><li> 조심스럽게 5 ㎖ 주사기 긴 바늘을 사용하여 15 %의 글리세롤 층 아래에​​ 3.4 ml의 25 % 글리세롤 용액을 놓는다. 두 개의 레이어를 혼합하지 않도록주의하십시오. 각각의 pH를 조정 글리세롤 솔루션을 테스트하는 여섯 조건에 대해이 작업을 반복합니다. 참고 : 다음 단계 클래스 II 바이오 안전성 캐비닛에서 작업 할 수 있습니다. </li><li> 부드럽게 1 ml의 MNT 버퍼에 희석 IAV (X31, H3N2)를 포함 뇨 유체를 정화 30 μL와 글리세롤 그라디언트 오버레이 각 그라데이션 (약 20 ~ 30 μg의 총 바이러스 성 단백질에 해당). </li><li> 균형 튜브를 반대하고 SW41의 초 원심 분리 회 전자에 배치합니다. 55,000 XG에 150 분, 12 ° C에 대한 원심 분리기. </li><li> 원심 분리 후, 신중하게깨끗한 파스퇴르 피펫 및 흡입기를 사용하여 상층 액, 즉, 두 글리세롤 레이어를 제거합니다. 40 μL (1X) LDS 샘플 완충액 비 환원성의 펠렛을 재현 탁. 완전히 펠렛을 용해 다운 수회 피펫을하고 중요하다. 1.5 ml의 microcentrifuge 관에 샘플을 전송합니다. </li></ol> 펠렛 분획 및 쿠마시 염색 4. SDS-PAGE <ol><li> 10 분 동안 95 ° C에서 모든 샘플을 가열. 이들은 SDS-PAGE에 의해 분석 될 때까지이 지점에서 샘플을 -20 ℃에서 저장 될 수있다. </li><li> 로드 프리 캐스트 구배 (4~12%) 상에 용해 된 펠릿 20 μl를 비스 - 트리스 미니 젤 버퍼를 실행 1 배 MOPS SDS 200 V에서 1 시간 동안 실행합니다. </li><li> DDH 2 O. 40 % 메탄올 및 10 % 빙초산 고정 용액을 </li><li> 1 시간 동안 고정 용액 내에서 겔을 쿠마 부화 콜로이드 용액의 충분한 부피를 가진 15cm 세포 배양 접시에서 하룻밤 동안 부드럽게 얼룩실온에서 진탕. 주 : 염색 용액의 증발을 방지하기 위해 접시를 가까이하는 것이 중요하다. </li><li> DDH 2 O.에서 젤 Destain 겔 배경이 명확해질 때까지 DDH 2 O마다 15 ~ 20 분 교체합니다. 이 밴드 정량 검사 될 때까지 4 ° C에서 DDH 2 O에서 젤을 저장합니다. </li><li> 높은 해상도에서 젤을 스캔 단백질 밴드 강도의 정량화를위한​​ 시판 또는 사용자 정의 만든 소프트웨어를 사용합니다. 각 밴드에 근접한 영역에서 바탕 신호를 빼고 (PH 7.4) 세제없는 대조 시료에 이들 값을 정규화. </li></ol>

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The authors have nothing to disclose.

바이러스 캡시드는 준 안정 거대 분자 복합체이다. 비리의 어셈블리가 바이러스 게놈의 이입 및 응축을 필요로하지만, 감염의 다음 라운드의 시작이 소형 캡시드 구조의 해체에 따라 달라집니다. 바이러스는 세포 수용체, 보호자, 단백질 분해 효소, 모터 단백질 또는 헬리뿐만 아니라 pH와 이온 스위치 (26, 27)에 의해 제공되는 물리적 인 힘을 포함하는 코팅 - uncoating주기를 제어하는 다양?...

인플루엔자 A 바이러스 (IAV)는 포락선 바이러스와 오르 쏘 믹소 바이러스과 (orthomixoviridae)의 가족에 속한다. 그 유전자는 세그먼트, 음의 감지 및 단일 가닥 RNA 게놈에 인코딩됩니다. 인간, IAV 계절 전염병의 발생에서 발생하는 호흡기 감염을 원인과 글로벌 전염병 일 가능성을 맺는다. 숙주 세포의 표면에이 시알 산 잔기에 결합하면, IAV는 클라 의존적 세포 내 이입 및 클라 독립 경로 3-8에 의해 내재화된다. 세포 내 이입 공포의 산성 환경 (PH &lt;5.5) 바이러스의 융합과 후반 엔도 좀 막 9 초래 IAV 스파이크 당 단백질 헤 마글 루티 닌 (HA)의 주요 구조적 변화를 트리거합니다. 사이트 오 - IAV 캡시드되면 말 엔도 좀 (레),이 핵에 바이러스 리보 (vRNPs)의 전송 다음 세포질에 코팅되지 않은됩니다에서 탈출했다 (여기 또한 바이러스의 &quot;핵심&quot;이라한다)F 바이러스 복제 및 전사 10-13. HA 산의 활성화에 앞서 바이러스는 연속 14-16 분해 용 코어를 준비하게 이입 시스템에서 pH의 점차적 감소를 경험한다. 이 &quot;프라이밍&quot;양성자와 K +10,14,16의 유입을 매개 바이러스 막 중의 M2 이온 채널 단계. 바이러스 내부에 이온 농도의 변화는 상호 작용이 바이러스의 매트릭스 단백질 (M1)과 여덟 vRNP 번들로 구축 방해 및 막 융합 10,14-17 다음 세포질 IAV의 uncoating을 용이하게한다. 프라이밍 공정 직접 정량 분석​​은 엔도 좀 실험적 액세스 어렵다는 사실에 의해 저해되었다. 진입 과정은 또한 매우 비동기이다. 또, 릴리스 바이러스 RNA 또는 감염성 측정 QRT-PCR 같은 종점 분석법은 바이러스 capsi의 생화학 적 상태에 대한 상세한 그림을 제공하지항목의 특정 단계 라. siRNA를하거나 약물 치료에 의해 ​​엔도 좀의 교란이 크게 IAV 항목 18 ~ 20의 이해에 기여하고 있지만, 미세 조정은 엄격하게 통제 엔도 좀 성숙 프로그램에서 불특정 부작용에 어렵고 쉽다. 이러한 문제를 방지하기 위해 속도 구배 원심 분리 (17)의 사용에 기초하여 이전에 개발 된 시험 관내 프로토콜을 채택하고있다. 대부분의 단백질 분해 절단 및 EM 분석 하였다 세제 처리의 조합을 기반으로 한 다른 시도가 21, 22, 23, 24, 대조적으로,이 방법은 쉽게 정량화 결과 수 있었다. 다른 구배 층을 통해 원심 분리는 침강 입자에 노출 제어 된 방식으로 상황 변화에 반응 할 수있다. 제시된 프로토콜, IAV 정화 막액 또는 정제 된 바이러스 입자로부터 유도는 2 층 글리세롤로 침강구배하는 바닥층은 비이 온성 세제 NP-40 (도 1)을 포함한다. 바이러스 제 세제 함유층 들어갈 때, 바이러스 외피 지질 및 엔벨로프 당 단백질 부드럽게 가용화 남아있다. 코어 8 개의 vRNP 다발로 구성되며, 매트릭스 층에 의해 둘러싸인 펠렛 분획으로 안정한 구조와 퇴적물. 이러한 M1과 vRNP 관련 핵 단백질 (NP)과 같은 바이러스 코어 단백질은, SDS-PAGE 및 쿠마시 염색으로 펠렛으로 식별 될 수있다. 특히, 시판 구배 겔을 활용하고 고감도 콜로이드 쿠마시 염색 25, 고정밀 바이러스 코어 - 관련 단백질에도 소량의 검출을 가능하게한다. 이는 pH를, 염 농도, 및 추정 uncoating 요인 핵심 구성 요소 및 핵심 STABIL의 침강 거동에 영향을 상이한 조건 여부를 시험하기위한 기준을 설정<html> 성만. 이를 위해, 글리세롤 구배 만 이하, 세제 함유층 인자 또는 관심 조건을 도입함으로써 변경된다. 이 기술은 IAV 코어 (16)의 무결성에 대한 pH 값 염화칼슘 농도 가지의 효과를 조사하고 특히 가치있다. IAV의 uncoating의 중간 단계의 pH 5.5와 낮은 16, 17 (그림 1)에서 vRNP 해리 뒤에 약간 산성 pH (&lt;6.5)에서 매트릭스 층을 포함하는 해리를 모니터링 할 수 있습니다. 후자 단계는 상기 늦은 이입 환경 (16)을 반영 글리세롤 층이 높은 K + 농도의 존재에 의해 개선되었다. 바이러스 코어는 그라데이션을 침전으로서 따라서, 그들은 변화 환경 엔도 좀에서 모방 조건을 경험 하였다. 결과는 세포 생물학 분석에서 도출 된 결과를 보완, 체외에서 바이러스 코어의 단계적 해체했다. t &quot;&gt; 여기에 제시된 방법은 빠른 활성화 및 IAV의 높은 재현성 분석했다 (X31 및 A / WSN / 33) 및 IBV (B가 리 / 40 /) 산성 pH에 의해 트리거 및 분해뿐만 아니라 K +16,17 증가 uncoating 그것은 생각할 알칼리 pH를 17 노광시의 코어 paramyxovirus. 접근법은 셀 입력시 바이러스 캡시드 구조 캡시드 분해의 생화학 적 특성에 대한 통찰력을 얻기 위해 다른 외피 바이러스에 적용 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1103">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">cOmplete&#8482;, EDTA-free protease inhibitor tablets</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:116px;">11873580001</td>
			<td style="width:473px;">The stock solution can be stored at 2 to 8 &#176;C for 1 to 2 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Merck Millipore</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol anhydrous BioChemica</td>
			<td>AppliChem</td>
			<td>A1123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Merck Millipore</td>
			<td>100317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long injection needle (21 GA, 9 cm, bevel or blunt-end)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>Now commercially available as IGEPAL&#174; CA-630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; 4-12% Bis-Tris mini gels, 10 wells, 1.0 mm</td>
			<td>Life Technologies</td>
			<td>NP0321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; LDS sample buffer (4X)</td>
			<td>Life Technologies</td>
			<td>NP0008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; MOPS SDS running buffer (20X)</td>
			<td>Life Technologies</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH indicator strips, pH 4.0 - 7.0</td>
			<td>Merck Millipore</td>
			<td>109542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QC Colloidal Coomassie Stain</td>
			<td>BIO RAD</td>
			<td>1610803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>106406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodiumhydroxide</td>
			<td>Merck Millipore</td>
			<td>106498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steritop&#8482; filter unit</td>
			<td>Merck Millipore</td>
			<td>SCGPT05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti, ultracentrifuge rotor set</td>
			<td>Beckman Coulter</td>
			<td>331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris hydrochloride&#160;</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td>
			<td>Virapur</td>
			<td></td>
			<td>Freshly thawed on 4&#176;C</td>
		</tr>
	</tbody>
</table>

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.

이미 설명한 바와 같이, 엔도 좀에서 프라이밍 능력 uncoating IAV 코어를 렌더링하는 데 필요합니다. 코어의 양성자는 M1 및 vRNPs의 상호 작용을 약화 (바이러스 RNA, NP 구성을하고, 중합 효소 복합체 PB1 / PB2 / PA). 들어오는 바이러스 초기 엔도 좀 (EES) 6.5 (이하)의 pH에​​ 노출 레 약 5.0의 pH에​​서의 바이러스 퓨즈까지 계속 될 때이 프로세스는 시작된다. <p class="jove_content"...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

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 ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K 조회수.  ETH Zurich. 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.

비디오: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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 ...

연구

면역학 및 감염병학

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 ...

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, ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

인간의 두 그라데이션 원심 분리하여 단핵구와 테프론 코팅 세포 배양 가방에서 대 식세포로 분화의 분리

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

인플루엔자 A 바이러스의 핵 단백질과 마우스 MX1 단백질의 공동 면역 침전

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

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 ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

유동 세포 계측법에 의해 A549 세포의 부착 및 국제화 인플루엔자의 바이러스를 측정

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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.