이 문서의 출판은 테네시 대학에서 연구의 사무실에서 교부금에 의해 지원되었다. 저자는 이러한 절차를 수정하기 위해 노력하는 학생과 연구자의 이전 세대 주셔서 감사합니다. 연구는 국립 과학 재단 (NSF - 0851113, NSF - 0825405 및 NSF - 0550485)의 보조금에 의해 지원되었다.

1. &quot;바이러스 무료&quot;물 (빌헬름 &amp; Poorvin 2001)를 생성하기 위해 해수의 Ultrafiltration <ol><li> 해수 / lakewater 약 20L가 가능한 무균 수집됩니다. </li><li> 워터는 순차적으로 142 - mm 직경의 폴리 카보 네이트를 통해 지역 사회의 분석을 위해 -20 ° C에서 보관 수있는 0.8 μm의 필터를 prefiltered입니다. 큰 기공 크기 필터는 매우 생산적인 시스템에 대해이 단계 이전에 사용할 수 있습니다. </li><li> 30 KDA - 커토프 나선형 카트리지가 모두 바이러스를 제외하는 데 사용됩니다 Amicon M12 시스템 (Millipore), 심지어는 작은 RNA 바이러스의 ultrafiltered 물을 얻을 수 있습니다. </li><li> 샘플은 backpressure의 ~ 15-16 kPa로 처리 ~ 25 %의 속도로 집중되고있다. </li><li> 물 ~ 500 ML의 나머지 샘플은 바이러스 무료 물의 나머지 19.5 L가 바이러스성 생산 assays에 사용됩니다 동안 집중 바이러스 커뮤니티 (어떤 다른 실험을 위해 저장될 수도 있습니다)가 포함됩니다. </li><li> 이용의 각 하루를 보낸 후, Amicon M12 시스템은 필터 카트리지의 멤브레인 손상을 방지하기 위해 청소해야합니다. </li><li> 이 해수와 함께 작업하는 경우 30~45분에 대한 0.1N NaOH 용액과 함께 세탁 다음 밀리 Q 물 최소한 6L와 멤브레인을 씻어. </li><li> 다시 밀리 Q 물 최소한 6L와 카트리지를 씻어. </li><li> M12 시스템을 이용하여 작업이 완료되면 나선형 카트리지 4 0.05MH 3 PO 4 용액 ° C.에 보관해야 </li></ol> 2. 바이러스성 생산을위한 바이러스 감소 방법 (빌헬름 외. 2002) <ol><li> 최대 두 호스트 및 바이러스가 0.2 - μm의 공칭 기공 크기 낮은 단백질 바인딩 그것에 배치 필터 (예 : Durapore.) 함께 취득하고 sterifilter 단위에 배치됩니다와 해수 / lakewater 샘플 500 ML에 </li><li> 지속 필터에 집중에서 박테리아 세포를 억제하는 살균 전송 피펫을 사용하여 샘​​플 resuspending하면서 예제는 부드럽게 &lt;200 mmHg로 압력을 진공이다. </li><li> 천천히 ultrafiltrate 세 볼륨 크게 샘플 무료 바이러스의 수를 줄이기 위해 세균 현탁액에 추가됩니다. </li><li> 세균성 분수는 바이러스가없는 물 500 ML로 희석 세 나누어 150 ML 각 복제 맑은 250ml 폴리 카보 네이트 병에 배치됩니다있다. </li></ol> 3. 바이러스성 생산을위한 접선 흐름 여과 (TFF) 방법 (Weinbauer 외. 2002 윙젯 외. 2005) TFF는 바이러스 감소 방법에 대한 대안적인 접근 방식을 나타냅니다. <ol><li> 위에서 설명한대로 자연 시료의 약 500 ML이 수집됩니다. </li><li> 이 예제는 0.2 - μm의 공칭 기공 크기의 접선 흐름 여과 시스템을 사용 집중되어 있습니다. </li><li> 세균성 분율이 약 10-15 ML로 감소되면, ultrafiltered, 바이러스 무료 물을 추가하고 위와 같이 배포됩니다. </li><li> 복제 병은 환경 챔버를 사용하여 현장 조건에 incubated 수 있습니다. <ol><li> 라이트 수준은 빛의 강도를 감소 NET 심사와 푸른 적용된 아크릴 또는 취소 아크릴을 사용하여 표면 조건 변경됩니다. </li><li> 주위 표면 온도는 종종 흐르는 해수 데크 배양기를 사용하여 얻을 수 있습니다. </li></ol></li><li> 박테리아와 바이러스성 풍부한 견적에 대한 샘플은 cryovials에 추가 2.0-2.5 % 살균 글루 타 알데히드의 최종 농도와 시간을 0에서 찍은 있습니다. 이 샘플은 즉시 액체 질소로 냉동 플래시하고 처리하기 전까지 냉동 저장됩니다. <ol><li> 액체 질소를 사용할 수없는 경우, 현미경 슬라이드는 (아래 절차를 참조) 준비하고 즉시 처리할 수 있습니다 </li></ol></li><li> Subsamples은 위에서 설명한 방법으로 적어도 10 시간 동안 매 2.5 시간을 수집하고 있습니다. <ol><li> 이 시간에 물을 정량 PCR 분석을 위해 수집될 수 있습니다. 최대 샘플 5 ML하기는 액화 질소에 즉시 플래시 냉동로없이 정착액 에이전트 cryovial에 추가할 수 있습니다. </li></ol></li></ol> 4. 바이러스성 생산 현미경 (노블 &amp; 퍼먼 1998, 웬 외. 2004) <ol><li> 0.02 - μm의 현미경에 대한 필터링하는 냉동 샘플은 얼음에서 해동해야합니다. </li><li> 멸균 물 주식 솔루션 1시 10분을 diluting하여 SYBR 녹색의 주식 솔루션을 준비합니다. 다음 주식 솔루션에서 멸균 물 39 ML에 대한 재고 솔루션의 1mL를 추가하여 작업 솔루션을 준비합니다. 50 % 글리세롤, 50 % 인산이 호수 솔루션을 버퍼 (PBS, 0.05 M 나 2 HPO 4, 0.85 % NaCl, 산도 7.5) 및 P - phenylenediamine의 신선한 2.5 % 주식 솔루션은 또한 시작하기 전에 준비를해야합니다. 4 50 % glycerol/50 % PBS 솔루션을 유지 ° C 및 P - phenylenediam-20에서 오프라인 재고 ° 시작 때까지 어둠 속에 C. 오른쪽 필터링하기 전에 Antifade 솔루션으로 사용되는 0.1 %의 최종 농도 50 % glycerol/50 % PBS로 P - phenylenediamine를 추가합니다. </li><li> 0.45 - μm의 MicronStep, cellulosic 후원 필터의 상단에 0.02 - μm의 Anodisc 필터 25mm를 놓습니다. 완전히 건조까지 20 pKa에 Anodisc과 진공의 상단에 고정 샘플 850 μL를 추가합니다. 플레이스 100 멸균 페트리 접시에 SYBR 그린 작업 솔루션의 μL, 그리고 여전히 진공으로 조심스럽게 SYBR 그린 필터 탑과 장소에서 Anodisc를 제거합니다. 20 분 상온에서 어둠의 샘플을 품어. 조심스럽게 SYBR 그린 솔루션에서 필터를 제거하고 모든 잔류 염료를 제거하는 Kimwipe와 필터의 뒷면 윅. 원하는 경우, 타워에 필터를 반환하고 여분의 얼룩을 씻어하는 필터를 통해 0.02 - μm의 여과 물 또는 멸균 미디어 800 μL까지 전달합니다. </li><li> 현미경 슬라이드에 antifade 솔루션의 작은 방울을 추가하고 위에 덮개 용지를 넣으십시오. 커버 슬립을 제거하고 antifade 솔루션와 젖은 현미경 슬라이드에 말린 필터를 추가합니다. 다시 커버 슬립에 antifade 솔루션의 작은 금액을 추가하고 천천히 그 양식을 수있는 모든 거품을 제거해야하고, 필터의 상단에 배치하십시오. </li><li> 필요가 (이가 변색 방지하기 위해 몇 개월 이내에 사용되며, 바이러스 카운트를 낮춘한다)까지 즉시 -20 ° C에서 슬라이드를 동결 </li><li> 바이러스는 다양한 파란색 필터 세트 (λ 예 = 450-490 nm의, λ λ 엠에서 억제 필터 = 510 NM = 510 nm의)와 (과 우리의 경우 Leica DMRXA 현미경) 형광 현미경을 사용하여 열거됩니다. 각 필터는 필터 멤브레인 전체 바이러스의 균등을 보장하기 위해 각 분야의 그리드에서 전체 바이러스를 계량해야하고, 계산보기 최소한 20 필드를합니다. </li><li> 세 독립에서 바이러스 reoccurrence의 평균 가격은 다음 계산하고 표준 편차가 생산 가격에서 결정됩니다 복제합니다. </li></ol> 5. 대표 결과 연구자에 의해 수집된 원시 데이터는 바이러스 풍요의 reoccurrence 속도를 생성하기 위해 최소한의 수학적 처리를 필요로합니다. 본 연구의 결과 기본 데이터 집합은 incubations에서 subsamples에서 바이러스 풍요의 reoccurrence 요금입니다. 이러한 결과는 샘플의 각 바이러스 어번던스 대 시간의 독립 regressions를 형성하고 있습니다. 각 샘플에 대해 개별 incubations이 연구원은 가격뿐만 아니라 변화의 추정 (예 : 표준 편차) (그림 1 참조). 계산할 수있는 세 가지 복제 - incubations을 작성하여 이렇게 하나의 치료 역할 이 과정 중 하나 주의해야 할 점은 바이러스 어번던스 불변의 감소는 바이러스 부담을지고있는 예제에서 호스트 세포의 감소로 연결한다는 것입니다. 소스 (필터없는 해수 또는 호수)와 T = 0 배양 샘플 모두에서 손실, 세균성 풍요의 열거를 상쇄하기 위해서 필요합니다. 이 정보가 손실 세포의 비율을 위해 계정을 사용할 수 있습니다 바이러스 풍부한를 줄이는 과정이 또는 미생물 지역 사회의 모든 구성원에 대한 선택이 아니라고 가정하면,이 요소는 다음 바이러스의 현장 생산 가격 추정하는 데 사용할 수 있습니다 . <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="그림 1" /> 그림 1. epifluorescence 현미경을 사용하여 현장 조건에서 10 시간 보육을 통해 입자와 같은 바이러스. 샘플의 생산은 2008 년 9 월 뉴질랜드의 해안에서 피는 식물성 플랑크톤을 통해 수집된되었습니다. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="그림 2" /> 그림 2. 바이러스 생산을 시금위한 워크플로우 프로세스의 개략도 그림.이 과정은 바이러스 무료 물을 생성하는 샘플 물 ultrafiltration 시작합니다. 이 ultrafiltration 시스템을 사용하여 완료됩니다. 미생물 커뮤니티 (감염되지 않은 감염된 세포의 혼합물을 포함) 유지하는 동안 병렬 물 샘플에서는 필터를 통해 전달되는 동일한 사이트 및 무료 바이러스로부터 수집하고 있습니다. 이 커뮤니티는 다음 바이러스 무료로 물에 resuspended 및 현장 조건에 따라 incubated입니다. 바이러스의 reoccurrence 요금은 다음 바이러스 생산 속도를 결정하기 위해 앞으로 10 시간 동안 감시하고 있습니다.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangential+flow+diafiltration:+an+improved+technique+for+estimation+of+virioplankton+production.">Tangential flow diafiltration: an improved technique for estimation of virioplankton production.</a> Aquat Microb Ecol. 41, 221-232 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

바이러스가 해양 미생물 커뮤니티에 영향을 어떻게 이해 핵심 구성 요소는 바이러스 입자가 생성되는 속도를 결정하는 것입니다. abundances이 (빌헬름 &amp; 셔틀 1999 Weinbauer 2004) 대부분의 시스템에서 정적 (자세한 이하)이며, 그 바이러스가 수중 시스템에 신속하게 제거 또는 비 전염성이 렌더링되는 것을 감안할 때 (빌헬름 외. 1998), 다음의 생산 가격이어야합니다 상대 빠른 손실 입자를 교체합니다. 사망률 바이러스는 미생물 커뮤니티 원인이 예측하는 것은 바이러스가 세포를 (이하 &quot;버스트 크기&quot;) lyses 때마다 생산 얼마나 많은 바이러스의 지식이 필요합니다. 자연 샘플에서 바이러스 버스트 크기는 크게 다를 수 있습니다. 버스트 크기는 전송 전자 현미경 (예 : Weinbauer &amp; Peduzzi 1994)에 의해 직접적으로 결정하지만, 이것은 항상 실용적이 아닌 주어진 실험실의 기능 넘어 자주하거나 수 있습니다. 그들이 경험적으로 결정되지 않을 수있는 상황에서, lytic 이벤트 당 24 바이러스의 문학 값은 담수 시스템 해양 시스템 34에 사용할 수 있습니다 (파라다 외. 2006). 바이러스 생산의 비율이 나눈 경우, 결과는 매일 바이러스에 의해 파괴 볼륨 당 세포의 풍부이다. 가치를 lysed 미생물 그런 다음 해당 시스템에 대한 바이러스 유도 사망률의 결과 세균성 풍요의 서 주식으로 나눌 수있다 : 기존 추정 범위 몇 %에서 거의 전체 인구와 자주 문제가 시스템의 다른 요소에 의존 (빌헬름 &amp; 메이트슨 2008). 총 사망률의 비율을 확인하려면이 번호는 종종 두 (세포의 50 %가 복제에 가서 세포의 50 %, Weinbauer 2004 년 손실 가정에서 작업) 곱한 것입니다. 되어 양분과 추적 요소 생체 이용율 (예, N, P, 철) 주요 생산성의 속도를 제한하고, 이러한 탄소 유량으로 수중 시스템을 통해,이 과정에서 바이러스 기반의 미생물 사망률의 역​​할의 이해 수 감안할 때 해양 geochemists에 관심. 몇몇 견적은 지금 (2009. 2008, 히긴스 외. 로우 외) 바이러스가 매일 물 열로 영양 요소의 상당 농도를 공개 제안 존재하고 이러한 요소들이 빠르게 미생물 지역 사회에 의해 동화되는 (Poorvin 동부 표준시 알. 2004 Mioni 외. 2005). 환경에 영양 흐름의 속도는 영양 당 셀 (표시된 &quot;할당량&quot;)의 금액에 의해 파괴 세포의 개수를 곱하여 결정하실 수 있습니다. 이 정보는 미생물의 식품 거미줄이 해양 시스템에서 작동하는 방법에 대한 우리의 이해에 중요한 구성 요소를 제공할 수 있습니다. 지속적인 발전 : 연구 그룹의 일련의 현재 노력은 특정 생물이 바이러스 활동에 의해 영향을 방법을 결정하기 위해, 같은, 지역 사회 내의 특정 바이러스를 열거할 수 위의 전략을 채택하고 포함됩니다. 위해이 연구팀은 전체 바이러스 공동체의 추정에 병렬로 특정 바이러스의 그룹이나 가족의 풍부한을 예측할 수있는 정량 중합 효소의 연쇄 반응 (qPCR)를 사용합니다. 결과는 다음 직접 특정 플랑크톤 그룹에 대한 바이러스 사망률, 영양 매출 등 견적을 제공하기 위해 적용됩니다. 이 강력한 새로운 접근 방식은 향후 몇 년 동안 연구자가 바이러스의 생태와 관련된 프로세스에 더 깊게 조사하고, 처음으로, 실험실 시스템의 제약없는 특정 바이러스 호스트 커뮤니티의 상호 작용을 수치하실 수 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

바이러스는 해양 및 담수 시스템의 전반 구성 요소이며, 미생물의 사망률의 중요한 대리인으로 알려져 있습니다. 우리가 그 미생물 지역 사회의 구조와 기능의 더 나은 모델을 개발뿐만 아니라 바이러스가 수상 biogeochemical주기를 변경하는 작업 방법을 우리의 이해를 사전에 수이 과정의 양적 추정을 개발하는 것은 중요합니다. 바이러스 감소 기술은 연구자 바이러스 입자가 발병 미생물 커뮤니티에서 공개되는 속도를 추정 할 수 있습니다. 미생물 커뮤니티 가까운 주위 농도 유지하는 동안 간단히, 무료 (세포) 바이러스의 풍부한는 샘플로 줄어 듭니다. 미생물 커뮤니티는 다음 무료로 바이러스의 부재와 바이러스 정량 (지역 사회의 이미 감염된 회원의 용해를 통해) 샘플에 reoccur epifluorescence 현미경에 의해 계량 수 또는 특정 바이러스의 경우, 어떤 속도에서 incubated입니다 PCR. 이 요금은 다음 바이러스 매개 세포 용해에 의한 미생물의 사망률의 속도를 추정하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

해양 및 담수 시스템에 바이러스의 매출 비율은 감소하고 reoccurrence 기술로 예상하실 수 있습니다. 데이터는 연구자들이 해양 시스템에서 바이러스 중재의 미생물 사망률의 속도를 추측할 수 있습니다.

아쿼틱 시스템에서 바이러스 생산 요금 예측

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.6K 조회수.  University of Tennessee. 해양 및 담수 시스템에 바이러스의 매출 비율은 감소하고 reoccurrence 기술로 예상하실 수 있습니다. 데이터는 연구자들이 해양 시스템에서 바이러스 중재의 미생물 사망률의 속도를 추측할 수 있습니다.

비디오: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

연구

면역학 및 감염병학

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

아데노 바이러스 파생 시스템의 도움으로 효율적인 재조합 파르 보 바이러스 생산

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

신경 침윤성 Encephalitic 바이러스의 생체 이미징 시스템 (IVIS) 검색에서

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

코딩 및 비 코딩 RNA 클래스의 식별 표시 돼지에서 전 혈

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

기능 리포터 시스템으로 특정 진 균의 오 번역 비율 측정

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

체외 목욕 발효 시스템을 사용하여 내분비병제와 인간 장내 미생물 간의 상호 작용 분석

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...