이 작품은 오스트리아 과학 기금 (FWF, P20619 및 BZ에 P22988)에 의해 지원되었다.

전자 지원 샘플 준비 <ol><li> 샘플 준비 하나의 시작 전자 현미경에 대한 자동 전자 조직 프로세서 (Leica EM AMW, Leica 마이크로 시스템즈, 비엔나, 오스트리아)을 프로그램 할 필요가 전에 TEM에 대한 전자 지원 샘플 준비에 대한 다음 프로토콜 (표 1) : <img src="/files/ftp_upload/2950/2950table1.jpg" alt="표 1" /> 표 1. 전자 방사선을 사용하여 샘플 준비를위한 프로토콜. 다른 열 (왼쪽에서 오른쪽으로) 표시 : <ol><li> 유리 병 번호 (비알의 NR은.) 유리 병은 프로세서의 회전 목마에로드되는 순서를 나타냅니다. </li><li> 실제 프로세스의 단계. </li><li> 병에 시약. </li><li> 실제 단계의 기간. </li><li> 전자 조사가 해제되기 전에 병에 도달 최대 온도. </li><li> 전자 방사선 설정 : 연속 = 급속한 온도 increASE, 설정 온도를 개최, 슬로프 = 부드러운 온도 상승, 끝 부분에 도달 최종 온도는, 온도가 5 감소 할 때까지 펄스 = 신속한 온도 상승, 전원이 꺼져 ° C, 전원이 온도에 도달 재개 할 수 있습니다. </li><li> 전자 조사의 최대 전력. </li></ol></li><li> 갓 샘플 준비 프로토콜 (한천 100 에폭시 수지에 1.7 참조​​)에 설명 된 여러 단계의 솔루션을 준비, 프로그래밍 프로토콜 (표 1)에 따라 지정된 병에 넣어 작성, 회전 목마에 튜브를로드 한 다음을 삽입 전자 레인지 조직 프로세서, 그리고에 회전 목마가 마침내 모노 모드 챔버 내로 첫 번째 병을로드합니다. </li><li> 60mM 소렌슨 3 %의 글 루타 알데히드 (한천 과학 (주), 스탠스 테드, 영국)의 드롭 모델링 왁스 판에 면도날과 담배 모자이크 바이러스 (TMV)에 감염된 Nicotiana의 tabacum에서 잎의 작은 섹션 (1mm 2) 잘라 온도는 실온에서 인산 버퍼 (산도 7.2)erature. </li><li> 즉시 약 200μm의 메쉬 폭 지정 바구니에 고급 핀셋으로 섹션을 전송합니다. 서로의 상단에있는 바구니를 스택과 모노 모드 챔버에 삽입. 케어는 샘플을 지속적 아웃 건조하지 않도록로드 및 바구니의 스태킹 동안 정착액 솔루션으로 덮여 있다는주의해야합니다. </li><li> 고정, 탈수와 침투에 대한 이전에 프로그램 된 전자 레인지 지원 샘플 준비 절차를 시작합니다. </li><li> 샘플 준비가 전자 조직 프로세서에 의해 자동으로 수행되는 동안 섹션 3 (부정적인 염색법)에 설명 된대로 나머지 공장 소재 부정적인 착색를 계속합니다. </li><li> 플라스틱 컵에 기입 24g 한천 100, 16g dodecenyl succinic 무수물, 그리고 10g 메틸 nadic 무수물 (모든 구성 요소에 대한 한천 과학 (주), 스탠스 테드, 영국 참조), 열 : 신선 다음과 같은 구성 요소 설명 된대로를 혼합하여 한천 100 에폭시 수지를 준비 거기에40 ° C와 잘 섞는다. 벤질 dimethylamine의 1.2g을 추가하고 철저하게 섞는다. 샘플 준비 프로토콜이 종료 (예 : 표 1의 단계 22시)에있어서 바로 전에 지정된 중합에 한천 100 에폭시 수지를 기입은 형성한다. </li><li> 프로토콜이 완료되면 (표 1에서 단계 22 이후) 모노 모드 챔버에서 회전 목마의 마지막 유리 병에 침투 샘플을 포함하는 스택 바구니를 놓습니다. 전자 장치에서 회전 목마를 제거, 바구니를 제거시켜야하고 지정된 중합 형태로 고급 핀셋을 이용하여로드합니다. 케어는 샘플이 항상 unstacking하고 아웃 건조하지 않도록로드하는 동안 한천 100 에폭시 수지로 덮여 있다는주의해야합니다. </li><li> 서로의 상단에있는 중합 양식을 쌓아. 케어는 샘플을 항상 스택과 그들이 건조하지 않도록로드하는 동안 한천 100 에폭시 수지로 덮여 있다는주의해야합니다. </li><li> 전자 레인지 tiss의 회전 목마에서 이전에 사용 된 튜브를 제거UE 프로세서는 스택 바구니로로드하고 전자 레인지 조직 프로세서에 회전 목마를 삽입합니다. </li><li> 이전에 프로그램 된 중합 프로토콜 (표 1)를 시작합니다. </li><li> 중합은 전자 레인지 조직 프로세서에 의해 자동으로 수행하는 동안 전송 전자 현미경과 부정적인 스테인드 격자를 검토 [예 필립스 CM10 TEM, 페이 (구 필립스), 아인트호벤, 네덜란드]와 섹션 3과 4 (에 설명 된대로 이미지 분석을 수행 부정적인 착색 및 이미지 분석). </li><li> 프로토콜은 제거시켜야한다, 모노 모드 챔버에서 중합 양식을 중합 양식을 제거하고 샘플을 포함 polymerized 블록을 제거 완료되면. 그들은 지금 마이크로톰으로 sectioned 될 준비가 된 것입니다. </li></ol> 2. 트리밍 및 sectioning <ol><li> ultrathin의 홀더에 대해 상단 그걸 고정에 샘플로 1cm를 sectioning에 대해 별도의 샘플 홀더에 하나 이상의 블록을 삽입합니다. </li><li>TEM (예 : Leica 라이 Ultratrim, Leica 마이크로 시스템즈)에 대한 표본 트리머로 블록을 잘라 있도록 최대의 블록 얼​​굴. 길이 (얼굴 크기를 차단 다이아몬드 나이프의 크기에 조정해야 할 수도) 달성 가능한 한 많은 잎 물질을 포함하는 폭 200μm의 1mm. </li><li> 45 ° (예 : Diatome 울트라 45, Gröpl, Tulln, 오스트리아)의 칼 각도로 다이아몬드 칼을 사용하여, ultramicrotome (Leica 마이크로 시스템즈 예 라이 Leica Ultracut S)와 제 블록을. 섹션의 두께는 약 70 ~ 90nm로 조정해야하며 절단 속도는 1mm / s의 주변에 있어야합니다. formvar (한천 과학 (주)) 코팅 구리 또는 니켈 321 평방 메쉬 그리드에 여러 섹션을 선택합니다. </li><li> 일부 2 무료 CO를 만들 NaOH로 가득 배양 접시에 5 분, 리드 구연산 (42ml 더블 증류수와 8ml 1N NaOH에 용해 1.1g 리드 구연산 한천 과학 (주))와 그리드의 섹션을 포스트 얼룩 환경 15 마일에1 % uranyl 아세트산 (한천 과학 (주))와 nutes는 상온에서 증류수에 용해. 각 포스트 착색 단계 사이에 1 분 동안 증류수로 격자를 씻으십시오. 공기는 그리드 상자에 격자를 건조. </li><li> 전송 전자 현미경 (예 : 필립스 CM10 TEM, 페이, 아인트호벤, 네덜란드)와 ​​섹션을 확인합니다. </li></ol> 3. 음성 염색법 <ol><li> TMV에 감염된 잎 소재의 100mg 약 60mM 인산 Søfrensen 버퍼 (산도 7.2)의 100μl의 현미경 슬라이드에 면도날을 2 분 동안 자료를 homogenizing으로 원유 수액을 준비 수확. </li><li> 4 개 이상의 우물 (또는 그 parafilm의 한 부분에 homogenate를 전송하는 것도 가능합니다)와 테플론 코팅 현미경 슬라이드의 잘 처음에 결과 homogenate의 20μl를 전송합니다. </li><li> 드롭 및 incu으로 직면하고있는 formvar (한천 과학 (주)) 코팅 된면으로 homogenate의 상단에 formvar 코팅 그리드를 배치5 분을 덜다. </li><li> 200μl 60mM 인산 소렌슨 버퍼 (산도 7.2) 두 방울의 상단에있는 격자를 배치하여 격자에게 이분 각각에 대한 2 번 씻으십시오. </li><li> 신선하게 조리 된 2% phosphotungstic 산 (한천 과학 (주)) 60mM 인산 소렌슨 버퍼 (산도 6.5)에 솔루션을 1 분 격자를 품다. </li><li> 그리드를 제거하고 그리드 상자에 드라이를 방송 할 수 있습니다. </li><li> 전송 전자 현미경 (; 페이, 아인트호벤, 네덜란드 필립스 등 CM10 TEM)으로 격자를 검사합니다. 21000X 이상의 기본 배율에서 전송 전자 현미경으로 무작위로 선택한 부정적인 스테인드 virions의 적어도 10 이미지를 (적어도 10 명 이상 virions 각 이미지에 표시됩니다)보세요. 주의 모든 이미지가 동일한 배율을 가지고 촬영해야합니다. </li></ol> 4. 이미지 분석 <ol><li> 에서 가져온 micrographs에 최소 100 무작위로 선택된 하나의 바이러스 입자의 길이와 너비를 측정모든 이미지 분석 컴퓨터 소프트웨어를 사용하여 부정적인 스테인드 샘플 입자 분석 도구 또는 Optimas과 [예를 들면 세포 D (올림푸스, 생활과 재료 과학 유럽 GmbH의, 함부르크, 독일) 6.5.1-(미디어 인공 이식 주식회사 메릴랜드 주 베데스다, USA)]. </li><li> 부정적인 착색 방법 및 TEM으로 시각화 바이러스 입자의 평균 길이와 너비를 달성하기 위해 수단과 표준 편차를 계산합니다. </li><li> 명확하게 바이러스 질병을 파악하기 위해 문헌에 알려진 바이러스 질환에 의해 유도 바이러스와 ultrastructural 변경의 크기와 길이와 ultrastructural 기능을 (2.5에서 얻은) 비교합니다. </li></ol> 5. 대표 결과 : 같은 전자 지원 샘플 준비 전형적인 TMV 유발 ultrastructural 변경 한 후 병렬 형태로 정렬 virions을 포함하는 넓은 영역은 감염된 담배의의 세포 기질에 전송 전자 현미경으로 관찰 할 수tabacum 세포 (그림 1A). 또한 TMV에 감염된의 원유 SAP에서 TMV 입자가 부정적인 착색 후 flexuous, 막대 모양의 구조 (그림의 1B)로 관찰 할 수 둡니다. 100 바이러스 입자의 이미지 분석은 폭 길이와 17nm에서 280nm (그림 2)의 TMV에 대한 평균 크기를 발표했다. TMV에 감염된 세포와 본 연구에서 관찰 virions의 크기에 ultrastructure는 TMV 입자의 담배 크기의 범위에서 TMV 유발 ultrastructural 특성에 따라 이전에 문학 9-15에보고 된 것으로되었다. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="그림 1" /> TMV에 감염된 잎 세포와 virions의 그림 1. 전송 전자 micrographs. A) 이미지 Nicotiana tabacum의 전자 레인지 지원 식물 샘플 준비 후의 TMV에 감염된 mesophyll 잎 세포의 ultrastructure을 보여줍니다. 세포 기질에 축적 된 병렬 정렬 virions의 넓은 지역을 확인합니다.C 전분과 = 엽록체 (ST), M = mitochondrion, N = 핵, V = 액포, 바 = 2μm. B) 이미지가 감염된 잎의 수액에 부정적인 염색법에 의해 감지 된 virions을 보여줍니다. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="그림 2" /> 그림 2. virions의 상대 크기입니다. 길이와 부정적인 착색는 전자 현미경에 나타난 것처럼 감염된 잎의 수액 후 TMV-입자의 폭이 상대적 배포. (길이 / 폭 ± 표준 편차를 의미) 값을 평균 100 virions에서 계산되었다.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+tobacco+mosaic+virus+strains+on+the+ultrastructure+of+tobacco+leaf+parenchymal+cells.">Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells.</a> Biol. Bull. 33, 409-415 (2006).</li><li>Sachse, C., Chen, J. Z., Coureux, P. D., Stroupe, M. E., F&#228;ndrich, M., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+electron+microscopy+of+helical+specimens:+a+fresh+look+at+tobacco+mosaic+virus.">High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus.</a> J. Mol. Biol. 371, 812-835 (2007).</li><li>Smith, M. L., Lindbo, J. A., Dillard-Telm, S., Brosio, P. M., Lasnik, A. B., McCormick, A. A., Nguyen, L. V., Palmer, 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=Modified+tobacco+mosaic+virus+particles+as+scaffolds+for+display+of+protein+antigens+for+vaccine+applications.">Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications.</a> Virology. 348, 475-488 (2006).</li><li>Zechmann, B., Zellnig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+TEM+diagnosis+of+plant+virus+diseases.">Rapid TEM diagnosis of plant virus diseases.</a> J. Virol. Meth. 162, 163-169 (2009).</li><li>Giberson, R. T., Demaree, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+processing+techniques+for+electron+microscopy:+a+four-hour+protocol.">Microwave processing techniques for electron microscopy: a four-hour protocol.</a> Meth. Molec. Biol. 117, 145-158 (1999).</li></ol>

관심 없음 충돌이 선언 없습니다.

TEM에 대한 전자 지원 식물 샘플 준비는 몇 시간 3,4,16 내에 신속하고 신뢰할 수있는 ultrastructural 데이터를 제공하기 위해 입증되었습니다. 본 연구에 사용 된 방법으로 달성 세포 소기관과 멤브레인의 미세 구조 보존 샘플 고정의 대규모 감소의 장점과 5,15 기존과 cryofixed 샘플과 비슷했고 약 2 시간에 3 일 이상에서 시간을 삽입. 이것은 문학에서 현재 TEM에 대한 가장 빠른 샘플 준비 프로토콜을 나타냅니다. 본 연구에서 설명하는 방법은 TMV 유발 ultrastructural 변경 및 바이러스 에이전트 자체의 명확하고 신속한 식별 할 수 부정적인 염색 방법과 TEM에 대한 전자 지원 식물 샘플 준비를 결합. TMV 유도 ultrastructural 변경은 전송 elec에 고정의 시작 후 약 4 시간 이내에, 트리밍 sectioning 및 사후 착색 한 후에 조사 할 수TRON 현미경. 완전히 자동으로 표본 준비 모드를 사용하면 바이러스 대리인의 크기와 폭을 결정하기 위해 중간에 제외 착색을 수행 할 연구원을 해제. 따라서, 우리는이 방법은 공장 phytopathology의 농업과 과학 실험 향후 사용하기 위해 매우 중요하다 절반 정도 하루에 식물 바이러스 질환의 명확하고 신속한 진단을 할 수 있다는 결론을 내릴 수 있습니다. 이 방법은 또한 동물과 인간의 질병의 급속한 진단을 위해 사용될 수로 의료 및 수의학 병리의 미래 응용 프로그램에 대한 큰 가능성이 있습니다.

<table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 글 루타 알데히드 </td><td> 한천 과학 (주) </td><td> R1312 </td><td></td></tr><tr><td> 오스뮴의 tetroxide </td><td> 한천 과학 (주) </td><td> R1022 </td><td></td></tr><tr><td> 한천 100 수지 </td><td> 한천 과학 (주) </td><td> R1043 </td><td></td></tr><tr><td> Dodecenyl succinic 무수물 </td><td> 한천 과학 (주) </td><td> R1051 </td><td></td></tr><tr><td> 메틸 nadic 무수물 </td><td> 한천 과학 (주) </td><td> R1081 </td><td></td></tr><tr><td> 벤질 dimethylamine </td><td> 한천 과학 (주) </td><td> R1060 </td><td></td></tr><tr><td> 구연산 리드 </td><td> 한천 과학 (주) </td><td> R1210 </td><td></td></tr><tr><td> Uranyl 아세테이트 </td><td> 한천 과학 (주) </td><td> R1260A </td><td></td></tr><tr><td> Phosphotungstic 산 </td><td> 한천 과학 (주) </td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td> 한천 과학 (주) </td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Leica EM AMW </td><td> Leica 마이크로 시스템즈 </td><td></td><td></td></tr><tr><td> Leica (라이) Ultratrim </td><td> Leica 마이크로 시스템즈 </td><td></td><td> 최근 모델을 사용할 수 있습니다 </td></tr><tr><td> Leica (라이) Ultracut S </td><td> Leica 마이크로 시스템즈 </td><td></td><td> 최근 모델을 사용할 수 있습니다 </td></tr><tr><td> Diatome 울트라 45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td> 필립스 CM10 TEM </td><td> 페이 (구 필립스) </td><td> &lt;/ TD&gt; <td> 최근 모델을 사용할 수 있습니다 </td></tr><tr><td> 셀 D </td><td> 올림푸스 주식회사 </td><td></td><td></td></tr><tr><td> Optimas 6.5.1 </td><td> 미디어 인공 이식 주식회사 </td><td></td><td> 최신 버전을 사용할 수 있습니다 </td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

바이러스에 의해 유도 ultrastructural 변화 조사는 종종 명확 식물 바이러스 질병을 식별 할 수 필요합니다. 전송 전자 현미경 (TEM)에 대한 기존의 샘플 준비 등 조사가 며칠 1,2 걸릴 수 있으므로 식물 바이러스 질환의 급속한 진단에 적합하지 않습니다. 전자 고정이 대폭 통상 샘플 준비 3-5 후 관찰과 유사한 ultrastructural 결과와 TEM 조사를위한 샘플 준비 시간을 단축 할 수 있습니다. 여러 가지 맞춤 제작 전자 장치는 성공적으로 고정 및 TEM 조사 5-8에 대한 생물학적 시료의 삽입을 위해 사용할 수있는 현재 사용할 수 있습니다. 이 연구에서 우리는 담배 모자이크 바이러스에 설명 (TMV)는 T에 대한 전자 지원 샘플 준비를 사용하여 절반 정도 하루에 잎의 ultrastructural 변경을 진단하는 것이 가능하다는 것을 Nicotiana tabacum의 식물을 감염EM. 그 많은 단계를 수동으로 6-8을 수행해야하므로 더 많은 시간과 노동력이 소요있는 다른 사용 가능한 장치에 대조적으로 거의 완벽하게 자동으로 5 샘플 준비를 수행으로 우리는 상업적으로 이용 가능한 전자 장치와 본 연구를 수행하도록 선택했습니다. 샘플 준비가 나머지 TMV에 감염된 잎과 ultrastructure 및 크기의 다음 시험의 수액에서 바이러스 입자의 완전 자동으로 부정적인 염색법을 수행으로 고정하고 삽입하는 동안 수행 할 수 있습니다.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

이 연구는 전송 전자 현미경과 부정적인 착색 방법에 대한 전자 지원 식물 샘플 준비의 조합을 사용하여 절반 정도 하루에 식물 바이러스 질병의 신속하고 명확한 진단을 할 수있는 방법을 설명합니다.

전자 레인지 전송 전자 현미경으로 식물 바이러스 질병의 신속한 진단을 도와

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K 조회수.  University of Graz. 이 연구는 전송 전자 현미경과 부정적인 착색 방법에 대한 전자 지원 식물 샘플 준비의 조합을 사용하여 절반 정도 하루에 식물 바이러스 질병의 신속하고 명확한 진단을 할 수있는 방법을 설명합니다.

비디오: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

야생 조류에서 조류 독감 바이러스의 신속한 진단 : 현장에서 휴대용 RRT - PCR 및 정지 - 건조 시약의 사용

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

연구

면역학 및 감염병학

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

베로 세포 배양 분석하여 필드 수집 모기에서 감염성 바이러스의 검출

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo - 전자 현미경에 의해 HIV - 1 Capsid 어셈블리의 구조와 반복 나선형 실제 공간 재건

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

시스테인 잔류 물을 용매 접근성에 대한 분석<em&gt; 옥수수 rayado fino 바이러스</em에서 생산&gt; 바이러스 같은 입자<em&gt; Nicotiana benthamiana</emVLPs에 펩티드의&gt; 식물과 교차 연결

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

고해상도 전자 현미경<em&gt; 헬리코박터 파이로리</em철 가용성의 다양한 조건에서 생산&gt; CAG 유형 IV 분비 시스템 필리

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronic Minigenomes를 포함하는 바이러스 입자와 바이오 안전성 수준이 조건에서 에볼라 바이러스의 수명주기를 모델링

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

유학을위한 방법론<em&gt; B. 서브 틸리</em작은 분자 바이오 필름 억제제를 특성화 모델로&gt; 바이오 필름

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

코튼 면화씨 벌레의 Densovirus의 호스트 피트니스에 호스트 조직 배포, 전송 모드 및 효과 조사를위한 프로토콜

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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 ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

잉크젯 프린터 기반으로 테스트 자동화 된 바둑판 배열 및 수동 시간 죽 일 방법 항균 시너지

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...