These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다. 표현 의견은 여기에 저자의 견해이며 공식 또는 미 국방부 또는 육군의 부서의 의견을 나타내는 것은 아니다.

재조합 HIV-1 분자 클론 및 성장 경쟁 분석의 건설 : 제시된 프로토콜은 두 가지 주요 부분으로 구성되어있다. 이중 감염된 세포 배양 물에서 두 바이러스와 구별하기 위해, 경쟁 분자 클론을 RT-qPCR에 프라이머 - 프로브 분석으로 또는 생거 시퀀싱에 의해 검출 될 수있는 서열 태그를 포함하는 것이 중요하다. 이 프로토콜은 HIV-1의 NL4-3 VIF 동일한 영역을 점유하고 동일한 아미노산 서열을 인코딩하지만 여섯 동의어 돌연변이 차이 VIFA 및 VIFB 태그를 사용한다. 이러한 돌연변이는 영향을주지 나타냈다 바이러스 복제의 적합성 (20). 이 프로토콜에서 사용하는 PCR 계 클로닝 방법은 제한 부위 기반 복제에 비해 클로닝 사이트를 선택하는데 더 많은 유연성을 제공한다. 그러나, PCR 클로닝의 효율은 인서트의 크기가 증가함에 따라 감소한다. 인서트 크기의 전류 제한은 ~ 5킬로바이트 (34)이다. PCR 기반의 복제 및 사이트 감독 mutagenesi 들어S, 고 충실도 DNA 중합 효소의 사용은 추가적인 돌연변이 가능성을 줄이기 위해 매우 중요하다. 제품의 충분한 양을 산출하기 위해 필요한 PCR 사이클의 최소 수의 사용은 권장된다. 우리의 경험에서, 우리는 여기에 설명 된 PCR 기반의 복제 및 부위 특이 적 변이 단계 후 여분의 돌연변이를 검출하지 않았다. 그럼에도 불구하고 PCR 제품은 원치 않는 돌연변이를 확인하기 위해 서열화한다. 이상적으로, 전체 HIV 코딩 영역을 resequenced해야합니다. 적어도 세 개의 샘플링 시점은 지수 적 성장 단계 9에서 검토되어야한다. 바이러스의 성장 동역학을 먼저 성장 경쟁을위한 적절한 배양 시간 및 샘플링 시간의 포인트를 결정하기 위해 매일 샘플링을 사용하여 설정되어야한다. 바이러스 성 성장 반응 속도에 영향을 미치는 하나의 요인은 감염 (MOI)의 다양성이다. 그것은 더 RO을 수득 하였다 나타낸 바와 같이이 프로토콜은, monoinfection 성장 경쟁 모두 0.005 총 MOI를 사용낮은 MOIs 9보다 흉상 결과. 그럼에도 불구하고, 낮은 MOIs 필요하다면 장기간의 지수 성장기를 얻기 위해 사용되지만, 결과의 일관성을 희생 할 수있다. 이 프로토콜은 바이러스의 체력 차이가 미리 일반적으로 알려지지 않은 것으로 가정하여, 50:50의 초기 감염 비율을 의미한다. 바이러스 복제 속도론에 상당한 차이점을 시사 예비 데이터가있는 경우에는, 동일하지 않은 입력 비율의 사용이 적합하다. 이러한 경우에는, 70:30의 비율은 감염 바이러스가 덜 맞는 초과 9에 배치 큰 차이 니스의 검출을 허용하도록 권장한다. 바이러스 성장 속도론 및 HIV-1 서브 타입 B를 사용하여 경쟁 분석 최적화 된 성장을 수행하기위한 TCID 50을 결정하기위한 프로토콜은, PBMC를 하나의 기증자로부터 NL4-3 COTB-P24 및 PBMC를이 HIV-하는 일관된 감수성을 증명하고있다 체외에서 1 감염. 배양 기간이 프로토콜에서 제공 샘플링 시점에 인간 PBMC를 HIV-1 바이러스 M 군을 연구에 적합한 것으로 보인다. 단일 소스에서 PBMC를 사용하는 것은 매우 바이러스 복제가 다른 공여 세포 (35)에 따라 다릅니다 수있는 일관성있는 결과를 얻을 것을 권장합니다. 교체는 동일한 풀 모든 실험에 걸쳐 사용되는 것을 규정 덜 바람직하지만, 복수의 공여체로부터 풀링 된 PBMC를 사용할 수있다. 또 다른 대안은 세포주의 사용이다. 여기에 제시된 프로토콜 T 세포주 CEMx174 23,36 성공적으로 사용되었다. 그러나, 세포의 파종 번호 재 최적화 일관된 세포 성장을 달성하는 것이 중요하다. 그것이 성장 경쟁 단계에 적절한 샘플링 시점을 결정하기 위해, 상이한 세포주에서 다를 가능성이 바이러스 성 성장 동역학은 또한 재 확립되어야한다. 체력을 계산하는 바이러스 성 비율을 결정하는 두 가지 방법이 protoc에 포함되어 있습니다OL. 첫째는 각 샘플링 시점에서 바이러스의 cDNA 카피 수를 측정하기 위해 RT-qPCR에 사용. 바이러스 복제 적당이어서 cDNA의 사본 번호에 기초하여, 바이러스의 비로부터 계산 하였다. 대안으로, 바이러스 성 비 VifAB 태그 사이트의 크로마토 그램의 피크 높이의 비율에 기초하여 결정될 수있다. 두 가지 방법이 유사한 결과 (도 4)을 수득 하였다. 크로마토 그램의 피크 높이 방법은 서열 태그 조작없이 다른 HIV-1 균주에 적용될 수있다. RT-qPCR의 바이러스 변이체를 구별하기위한 프라이머 또는 프로브의 사용은 제 신중하게 평가되어야 들어 (20 참조). 그럼에도 불구하고, RT-qPCR에 같은 지수 성장기 내의 제 시점에서 그와 같은 바이러스 성 RNA의 소량의 샘플과 더 나은 민감도를 제공한다. 바이러스 cDNA를 직접 측정은 또한 RNA 추출 및 cDNA 합성에서 발생할 수있는 기술적 인 문제를 감지 할 수 있습니다. 서열 태그 분자 클론을 사용하는 것은 비용 효율적인 솔루션을 제공 tRT-qPCR에 방법 O를, 같은 프라이머 및 프로브의 두 쌍은 여러 바이러스를 연구하기 위해 필요하다. 이 전략은 또한 시퀀싱이 연구에서 모든 바이러스에서 동일한 사이트에서 수행되는 것처럼 순서로 피크 높이 변화의 문제, 이웃 기지 (37)에 의한 크로마토 그램 방지 할 수 있습니다. RT-qPCR에와 생거 시퀀싱 생성 된 PCR 제품은 다른 방법과 함께 사용은 개별 클론 12, 13의 대량 순서, HTA 4,7,17,19, 또는 올리고 뉴클레오티드 결찰 분석 (OLA) (38) 바이러스 성 비율을 결정하기 위해 할 수 있습니다 .

바이러스 복제의 니스는 주어진 환경에서 한 감염성 자손을 생산하는 바이러스의 능력으로 정의하고 2 시간 이상 인구 수준에서 바이러스 변이체의 유병률을 결정하는 중요한 요인이다. 생체으로 피트니스 연구는 HIV-1과 같은 병원성 인간 바이러스, 다양한 vitro와 in와 가능하지 않은 생체 복제 피트니스 분석 약제 내성과 면역 탈출 돌연변이, 상위 성 및 진화에서 발생하는 체력에 미치는 영향을 연구하기 위해 개발되었다 바이러스 성 인구 3-6. 다른 피트니스 분석 중 성장 경쟁 분석은 생체 -1,7,8,8- 발생으로 두 개 이상의 바이러스 변종, 정확하게 동일한 환경 조건 하에서 동일한 세포 인구를위한 경쟁으로, 피트니스 차이에 더 민감하고 유효한 조치를 얻을에 인식하고 있습니다. 성장 경쟁 실험을 시작, 여러 variab 전레 감염의 다양한 다중성 (MOI), 바이러스의 입력 비율 및 분석을위한 샘플링 타이밍의 사용을 포함하여 결정되어야한다. 우리는 바이러스 성 성장 속도론에 경쟁 실험의 결과에 이러한 매개 변수의 효과를 공부하고, 세포 배양 9 HIV-1의 적합성에 강력한 측정에 필요한 핵심 요소를 확인했다. 분석 변수 외에도, 성장 경쟁 실험에서 바이러스 변이체를 정량을위한 다양한 방법이있다. 10,11 또는 벌크 클론 시퀀싱 12,13은 관심 부위 (들)에서 뉴클레오티드 주파수에 기초하여 상기 경쟁 바이러스의 비율을 결정하기 위해 사용되어왔다. 상대 체력은 시간이 지남에 따라이 비율의 변화에​​서 파생됩니다. DNA 시퀀싱 서비스를 광범위하게 사용할 수있는이 방법은 편리하다. 병렬 대립 유전자 특정 염기 서열 (PASS) 방법은 여러 사이트에서 시퀀싱 및 재조합 (14)의 검출을 가능하게 </sup&gt;뿐만 아니라 특별히 개발 시약 및 탐지 시스템을 필요로한다. 본질적으로,이 방법은 관심 영역의 뉴클레오티드 차이의 소수의 바이러스 균주를 개발하기 위해 연구 하였다. 다른 방법은 작은 리포터 유전자를 시퀀싱하여 경쟁 바이러스 heteroduplex를 추적 분석 (HTA) 4,7,17,19 구별 또는 전사 정량 실시간 중합 효소 연쇄 반응을 반대로 태그 15 또는 돌연변이 4,16-18 동의어를 사용 에 관계없이 서열 유사성의 균주 경쟁 연구에 적용 할 수 모두 (RT-qPCR에) 15,16,18,20. 추가 단계는 바이러스의 게놈에 태그를 도입하는 데 필요한 및 RT-qPCR에 분석은 특정 시약 및 장비를 필요로한다. 발명자들은 대량 생거 시퀀싱 수익률 비교 결과 (9). 성장 경쟁에 이어, 바이러스 복제 피트니스는 상대의 적합성, 또는 T 사이의 피트니스 비율로 제공됩니다바이러스 변종 하시다. 바이러스의 상대적인 니스는 접종의 초기 비율에 의해 또는 두 바이러스 간의 경쟁 순 성장 속도 차이로 규격화 바이러스 변이체의 최종 비율로 정의 할 수있다. 우리는 후자의 방법은, 단지 지수 성장기 내에 길이 데이터 포인트를 이용하여, 가장 견고한 결과 9,20 생산하였습니다. 체외에서 피트니스 분석은 생물학적 클론 6-8과 HIV-1 감염 분자 클론을 연구하기 위해 주로 사용된다. 후자는, 유전자 조작 의무가되고, 종종 특정 돌연변이 또는 관심 3-5,21,22의 특정 시퀀스에서 체력에 미치는 영향을 연구하기 위해 사용된다. 다음 프로토콜은 바이러스 성 성장 속도론을, 일련의 태그를 포함하는 HIV-1 벡터를 사용하여 새로운 전체 길이 감염성 HIV-1 분자 클론을 구성하는 관심의 돌연변이를 도입, 바이러스 성 주식을 만들고 구축의 관점에서 워크 플로를 설명상대 체력을 성장 경쟁 분석을 수행하고 계산하는 (그림 1). 우리의 최적화 된 절차를 사용하여, 우리는 세 가지 재조합 HIV-1 돌연변이를 만들어 자신의 복제 체력을 결정했다. 재조합 분자 복제가 먼저 합성 COTB (센터 외과, pNL4-3, HIV-1 실험실 변형 NL4-3의 전체 길이 감염 유전자를 포함하는 플라스미드의 HIV-1 개그-P24 유전자 영역을 대체하여 건설되었다 나무, 서브 타입 B) 개그-P24 시퀀스 (23)는 프로토 타입의 변형을 만들 수 있습니다. 단일 아미노산 변경 (T186M은 T242N 및 I256V는) 다음 세 가지 돌연변이 클론을 생성하기 위해 도입되었다. 각각의 돌연변이는 주어진 유전 적 배경에서 각 돌연변이의 체력에 미치는 영향을 관찰하기 위해 프로토 타입 바이러스에 대해 경쟁했다. 세 가지 돌연변이 프로토 타입 바이러스보다 약간 크게 낮은에서 복제 fsitness 다양한 수준의 보여 주었다. T242N 돌연변이는 이전에 적당한 fitne이보고되었다SS는이 연구에 나타낸 결과와 유사한 24-26, 비용. 다른 두 변이의 피트니스 비용은 이전에보고되지 않았다.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name of Material/ Equipment</td>
			<td style="width:484px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:196px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Construction of recombinant clones</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Chimeric primer/Mutagenic/Sequencing primers</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom DNA oligos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pNL4-3VifA and pNL4-3VifB plasmid</td>
			<td style="width:484px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-Fidelity DNA polymerase</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-fidelity buffer</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems&#174; GeneAmp&#174; PCR System 9700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA loading dye</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td style="width:137px;">R0631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1kb Plus DNA ladder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAquick PCR Purification Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick gel extraction kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">DpnI enzyme</td>
			<td style="width:484px;">New England Biolabs</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">TOP10 chemically competent Escherichia coli</td>
			<td style="width:484px;">Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Luria Broth base powder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">12795-084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Carbenicillin</td>
			<td style="width:484px;">Research Products International</td>
			<td style="width:137px;">C46000-25.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep kit&#160;</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">27104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">12362</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">X-tremeGENE 9 DNA transfection reagent</td>
			<td style="width:484px;">Roche</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HEK 293T-17</td>
			<td style="width:484px;">ATCC</td>
			<td style="width:137px;">CRL-11268</td>
			<td>http://www.atcc.org/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.22um filter top tube</td>
			<td style="width:484px;">VWR International</td>
			<td>89220-716&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#8242;s Modified Dulbecco&#8242;s Medium (IMDM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>31980-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RPMI-1640 media</td>
			<td style="width:484px;">Life Technologies</td>
			<td>61870-036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phytohemagglutinin (PHA)</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>R30852801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Human Interleukin-2</td>
			<td style="width:484px;">Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum</td>
			<td style="width:484px;">JR Scientific</td>
			<td style="width:137px;">43640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicillin and Streptomycin</td>
			<td style="width:484px;">Corning Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">73520-906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">62407-338</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat-bottomed plate</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td style="width:137px;">T-3015-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well round-bottomed plate</td>
			<td style="width:484px;">BD Falcon</td>
			<td style="width:137px;">353077</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 ml Microcentrifuge Tube</td>
			<td style="width:484px;">Mt. Baker Bio</td>
			<td style="width:137px;">MBD-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 mL tubes w/ O-ring</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">89004-290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">50 mL conical tube</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td>C-3317-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Triton-X 100</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phosphate buffer saline (PBS)</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum (FBS)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>F0392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">glycerol</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td style="width:137px;">G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tween 20</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-HIV-1 SF2 p24 antiserum</td>
			<td style="width:484px;">NIH AIDS Reagent Program</td>
			<td style="width:137px;">4250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit HRP</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">474-1516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMB Microwell Peroxidase Substrate</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">52-00-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">H2SO4</td>
			<td>Fisher Scientific</td>
			<td>A300-500</td>
			<td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 minutes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HIV p24 standard</td>
			<td style="width:484px;">AIDS and Cancer Virus program (NCI-Frederick)</td>
			<td style="width:137px;">N/A</td>
			<td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microplate reader</td>
			<td style="width:484px;">Molecular Devices</td>
			<td style="width:137px;">N/A</td>
			<td>VMax Kinetic ELISA Microplate Reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA isolation cDNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxtractor</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">N/A</td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAamp Viral RNA Mini Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">52906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">SuperscriptIII</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">First-strand buffer&#160;</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Rnase inhibitor</td>
			<td style="width:484px;">Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RnaseH</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18021-071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Real-time qPCR machine</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems 7300 Real-Time PCR&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan&#174; Gene Expression Master Mix</td>
			<td style="width:484px;">Life Technologies</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Forward, reverse primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Probe</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">optical 96 well reaction plate</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">I19N3Q216</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR+sequencing</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Taq DNA polymerase (Biolase)</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NH4 buffer&#160;</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Sequencing primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxcel</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;"></td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA sequencing service provider</td>
			<td>http://www.htseq.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GRC web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/grc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChromatQuan web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

HIV-1 개그-P24 단일 아미노산 변화의 체력에 미치는 영향을 연구하기 위해, 우리는 HIV-1 COTB-P24 유전자 23,32,33를 포함하는 pNL4-3 플라스미드에 돌연변이를 소개하는 올리고 뉴클레오티드 특이 적 변이를 사용했다. 바이러스 주식 293T 세포의 형질 감염에 의해 생성 된, 48 시간 후에 수거 하였다. 우리는 리드 - MUENCH 방법 (31)에 의해 각각의 바이러스 주식의 50 % 조직 배양 감염 용량 (TCID 50)을 추정했다. 프로토 타입의 TCID (50)와 돌연변이 바이러스는 104에서 105 IU / ㎖ (그림 3A)였다. 재조합 바이러스의 성장 반응 속도는 0.005의 MOI에서 단일 기증자 PBMC를 설립했다. RT-qPCR의 여섯 일 동안 매일 바이러스 cDNA의 사본 수를 측정 하였다. 모든 돌연변이 및 프로토 타입 바이러스는 &lt;바이러스 RNA의 카피 수 증가의 감소 기울기 (로 표시된 바와 같이 바이러스 성 성장이 둔화 하루 2, 4 일, 다음 사이에 기하 급수적으로 증가강한&gt; 그림 3b). cDNA의 감소는 (접종에 해당) 0 일 및 2 일 사이의 수는 세포에 바이러스의 흡수 하루 1 세척 (단계 5.1.6)에 의해 언 바운드 비리의 제거로 인한 복사합니다. 지수 적 성장 단계에서, 모든 세 돌연변이 바이러스 원형 (도 3c)보다 느린 성장 속도 (g)를 가졌다. 세 가지 돌연변이 0.005 총 MOI에서 성장 경쟁 분석에서 프로토 타입 바이러스에 대해 경쟁했다. 이중 감염된 문화 바이러스 성 성장 속도론은 monoinfection의 그것과 유사 하였다; 바이러스 성 지수 성장기는 2 일 및 4 사이이고, 바이러스 성장은 5 일 (도 4a)의 주위에 도달 고원. 바이러스 성장 속도의 차이는 시간에 따른 바이러스의 비율의 변화로부터 유래 하였다. 비 바이러스의 cDNA 복사본 참조 번호 및 RT-qPCR에를 사용하여 돌연변이 바이러스와 비교하여 피크의 높이에 의해 산출했다시퀀스에서 두 바이러스 (도 4B)를 구분하는 뉴클레오티드 부위에서 크로마토 그램. 피크 높이와 바이러스 RNA 카피 수의 방법을 이용하여 결정 성장 속도의 차이는 유사한 결과 (도 4C)을 수득 하였다. 세 가지 돌연변이가 가장 낮은 피트니스 (그림 4C)을 갖는 돌연변이 I256V와 프로토 타입 바이러스보다 낮은 복제 체력을 가지고 있었다. <img alt="그림 1" src="/files/ftp_upload/52610/52610fig1.jpg" /> 그림 1 : 본 논문에서 제시 한 프로토콜의 흐름도 바이러스 주식의 바이러스 준비 프로토콜 우려 HIV-1 재조합 클론의 건설 및 생성.. 피트니스 분석 실험 프로토콜은 바이러스 성 성장 속도론을 수립하고 관련 바이러스의 적합성을 결정하기위한 것입니다. 점선은 프로토콜에 대한 다른 흐름을 나타냅니다. 예를 들어, 돌연변이 HIV-1 NL4-3로 직접 도입 될 수있다재조합 복제를 생성하지 않고 분자 클론. <img alt="그림 2" src="/files/ftp_upload/52610/52610fig2.jpg" /> 그림 2 :. 오버랩 확장 PCR (A)를 사용하여 HIV-1 NL4-3 COTB 개그-P24 재조합 분자 클론의 건설 키메라 프라이머를 디자인합니다. 5 &#39;프라이머의 절반은 NL4-3 벡터 시퀀스를 포함하고 3&#39;반쪽 삽입 시퀀스의 끝을 포함한다. (B) 키메라 프라이머는 삽입 조각, COTB 개그-P24를 증폭하는 데 사용됩니다. (C) 삽입 단편은 재조합 플라스미드를 생성하기 위해 두 번째 PCR 용 프라이머로 사용된다. <img alt="그림 3" src="/files/ftp_upload/52610/52610fig3.jpg" /> 그림 3 :. PBMC를에서 바이러스 성 성장 특성 (A) 로그 (10) TCID (50) </sUB&gt; 바이러스 주식. (B) MOI = 0.005에서 시작 monoinfections에서 바이러스 성 성장 속도론. 패널 (B) 유래의 지수 성장기 (2-4 일) 등 6 일 (C) 바이러스 성장율. 도시 된 값들은 하나로부터 3 회 반복 실험의 평균을 나타낸다. 오차 막대는 95 % 신뢰 구간을 나타낸다. <img alt="그림 4" src="/files/ftp_upload/52610/52610fig4.jpg" /> 그림 4 : 바이러스 성 성장과 상대의 적합성 결정 (A) 이중으로 감염 PBMC 문화에서 바이러스 성 성장.. T242N 및 프로토 타입 바이러스 (B) 비는 RT-qPCR에 또는 시퀀싱 피크 높이 법을 사용하여 측정. (A)에 도시 된 바와 같이, (C) 및 이중 돌연변이 감염된 배양에서 프로토 타입 바이러스 간의 순수한 바이러스 성장율 차이 (d). D 값은 f를 계산했다(B)에 도시 된 비 바이러스 성 데이터를 ROM. 도시 된 값들은 하나로부터 3 회 반복 실험의 평균을 나타낸다. 오차 막대는 95 % 신뢰 구간을 나타낸다.

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

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JoVE Journal

Immunology and Infection

11.0K 조회수.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

비디오: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination 분석을 사용하여 혈청의 반응성 및 Titre를 결정

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

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면역학 및 감염병학

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

xCELLigence 시스템으로 CR51 릴리스 분석을 비교하여 인간 Her2neu 특정한 CD8 T 세포의 최적 세포 독성 활동 결정

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

결정 토양을 전송 기생충 감염 현황 및 학교 세 어린이의 물리적 휘트니스

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

제한 효소 기반의 복제 방법을 평가하기 위해<em&gt; 시험관</em&gt; HIV-1 서브 타입 C 개그 - MJ4 키메라 바이러스의 복제 능력

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.