This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol>
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The authors have nothing to disclose.

<html> 비리 막에 바이러스 성 스파이크 당 단백질 구조의 기술은 바이러스 복제를 이해하고 치료 및 감염을 예방하는 치료제의 개발에 필수적이다. 단일 입자의 평균 결합 전자 현미경 크라이 스파이크 당 단백질을 포함 엔벨로프 바이러스 입자의 구조를 해결하기 위해 대부분의 이용 방법으로서 떠오르고있다. 그러나,이 방법은 대칭 바이러스를 icosahedrally로 제한됩니다. 여기 Jsubtomo에서 전자 크라이 단층 촬영 subtomogram 평균의 응용 프로그램을 통해, 우리는 다형성에 다른 현재의 구조 생물학 방법 의무가없는 바이러스 당 단백질 스파이크를 포위 결정하기위한 일반적인 프로토콜을 설명했다. 우리의 대표적인 결과이 방법의 해상도가 도메인 아키텍처, 올리고머, 그대로 비리에 당 단백질 스파이크의 고차 조직에 대한 통찰력을 공개하기에 충분 함을 입증. jove_content &quot;&gt;이 프로토콜 내에서 가장 중요한 단계는이 단계의 또 다른.을 성공적으로 실행하는 당 단백질 스파이크 있도록 충분히 큰 너무 밀접하게 서로에 대해 포장하지 않은 것으로 가정 한 통계적으로 독립적이 신뢰할 수있는 초기 모델을 건설하고 개별 스파이크 수 시인 수동 단층 촬영 고른, 2 개의 독립적 인 모델은 평균화 될.이 가능하지 않은 경우, 프로토콜이 변경이 시도 될 수있다. 첫째, 두 개의 독립적 인 랜덤 모델은 먼저 subtomograms 두 임의의 서브 세트를 정의에 의해 구성 될 수있다 이러한 서브 세트 (30) 내에 subtomograms 평균. 둘째, 절연 스파이크의 구조를 X-선 결정학에 의해, 예를 들면, 다른 방법에 의해 도출 된 경우, 이는 초기 모델로서 사용될 수있다. 그러나, 치료가 저 -주의해야 정제의 다음 라운드에서 것이 모델의 결과로서, 저해상도 컷오프 (50-70 A)을 이용하여이 모델에 필터를 통과만이 해상도를 넘어 통계적으로 독립. 이주의해야 할 점으로 인해 이전의 접근 방식을 권장합니다. 이 프로토콜에서 얻을 수있는 해상도는 네 가지 주요 요인에 따라 달라집니다 내가. 데이터 수집 전략 및 입력 데이터의 품질, II. subtomograms 수, III. 배향 subtomograms의 정확성, 및 IV. 구조의 이질성. 제 1 및 제 2 한계가 크게 높은 신호대 직접 전자 CTF 결합 검출기 단층 수정 및 자동 데이터 수집을 사용함으로써 극복 될 수 있지만, 얼라인먼트 정밀도를 더욱 관심 자체의 구조의 크기 및 형상에 의해 영향을 받는다. 눈에 띄는 특징을 결여 작은 스파이크에이 프로토콜을 적용하면, 배향의 정확도 때문에 해상도를 개선하기 위해 31 스파이크의 Fab 단편에 결합하는 것이 유리할 수있다. 마지막으로, 구조는 평균화 할 경우 전시 배좌 여러 서브 단층AM 분류 방법은 별도로 상이한 입체 형태를 평균화하기 위하여 사용될 수있다. 이를 위해 Jsubtomo 강력한 subtomogram 분류 구를 제공하고, 디나모 패키지에 통합하고있다. 상기 프로토콜은 절연 바이러스 당 단백질의 X-선 결정학에 상보. 결정 구조는 비리 막에 대하여 당 단백질의 정확한 배향을 얻기 위해 서브 - 단층 평균에 장착 할 수있다. 이 방법의 적용은 의심 할 여지없이 싸여 바이러스 구조와 pathobiology에 대해 설명해 주실 것입니다.

전자 크라이 단층 복잡한 생물학적 시료의 3 차원 (3D)의 재구성 연산을 허용하는 전자 크라이 현미경 이미징 기술이다. 적절한 표본은 전체 원핵 세포 5 및 전체 진핵 세포 6 심지어 얇은 지역에 정제 된 거대 분자 복합체 일, 필라멘트 2, 코팅 된 소포 3, 다형성 막 바이러스 4 이르기까지 다양합니다. 틸트 계, 3 차원 단층 볼륨, 또는 단층 촬영 데이터의 컬렉션 후, Bsoft 7 및 8 IMOD 포함한 여러 확립 소프트웨어 패키지를 이용하여 계산 될 수있다. 전자 크라이 단층 제한 대응 단층 볼륨의 생물학적 해석에 의한 생체 시료의 연구에 내재 두 측면. 인해 상당한 방사선 손상을 도입하기 전에 생체 재료에 적용 할 수있는 제한된 전자 도즈, 시그나하려면 우선단층 촬영 데이터에 L 잡음비은 일반적으로 아주 낮다. 둘째, 데이터를 수집하는 동안 샘플로 경사 형상의 결과로서, 객체의 몇몇보기는 단층 부피 소위 &#39;누락 쐐기&#39;이슈 선도 유지 부재. 단층 볼륨이 성공적 일 수 9-12의 평균 수와 같은 거대 분자 복합체로서 동일한 구조를 반복 포함한다면, 이러한 제한을 모두 극복 할 수있다. 단층 복원에서 구조를 평균에 앞서, 관심 객체 발견하고 같은 방향으로 정렬되어야합니다. 이러한 구조를 찾는 것은 종종 주형으로 13 일치 함 접근법을 사용하여 단층 부피 주형 구조의 상호 - 상관에 의해 달성 될 수있다. 이 매칭 처리에 사용되는 템플릿은 극저온 전자 현미경 또는 3D 재구성 결합 전자 크라이 단층로부터 유도 될 수 있거나 밀도에서 시뮬레이션지도가 될 수있다원자 구조. 몇몇 연산 패키지 (11)는 이들 작업을 수행하기 위해 개발되었다. 이러한 HIV-1과 같은 막 바이러스의 당 단백질 스파이크의 평균, 그 구조 14-16 공부에 특히 성공적으로 접근하고있다. 구조에 대한 이해는 바이러스 호스트 상호 작용의 두 분자 기초를 공개하고 바이러스 백신 디자인 개발을 안내하기위한 필수입니다. 고분자 결정학 개별 바이러스 당 단백질과 그 복합체의 구조 분석 (4보다 일반적으로 더 나은) 고해상도에 대한 선택의 기술은 있지만,이 방법에 의한 X-선 구조 비리에 천연 멤브레인 환경에서 분리 된 단백질 아르 . 따라서, 그러한 비리 온의 맥락에서 바이러스 당 단백질의 고차 구조 등의 중요한 사항, 부족한 남아있다. 한편, 전자 현미경 및 크라이 단일 입자 재건전체 싸여 바이러스의 면체 대칭 (17, 18)와 비리로 제한됩니다. 서브 볼륨 조정과 함께 전자 cryotomography 따라서 현장에서 불규칙한 모양, 다형성 바이러스의 당 단백질 스파이크의 연구를 허용하는 상호 보완적인 기술로 떠오르고있다. 우리는 Jsubtomo 단층 서브 볼륨의 감지, 정렬 및 평균화 (www.opic.ox.ac.uk/jsubtomo)라는 소프트웨어를 개발했다. Jsubtomo은 세포 및 바이러스 성 구조 19-26의 번호의 구조 결정에 이용되었다. 여기서는 판정 바이러스 표면 스파이크 구조 있도록 상세한 프로토콜을 설명합니다. 소음의 상관 관계에 의해 과도하게 세분화 평균 구조를 회피하기 위해, &#39;황금 표준&#39;정제 방식은 10,27을 채용하고 있습니다. 마지막으로, 시각화 및 전형 결과의 해석을위한 전략을 논의한다.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

싸여 바이러스는 숙주 세포에 진입을 중재하기 위해 표면에 막 당 단백질을 사용합니다. 이러한 당 단백질 &#39;스파이크&#39;의 입체 구조 해석은 종종 바이러스 성 병인을 이해하고 약물 디자인 기술적 도전하지만 중요하다. 여기서, 프로토콜은 전자 크라이 단층 촬영 데이터의 계산 평균을 통해 바이러스 스파이크 구조 결정을 위해 제공됩니다. 전자 극저온 단층 촬영은 거의 네이티브, 냉동 수화 상태에서 막 바이러스와 같은 다형성 생물 표본의 3 차원 단층 촬영 볼륨 복원 또는 단층 촬영을 유도하는 데 사용되는 전자 현미경의 기술입니다. 이러한 단층 촬영 낮은 해상도에서이기는하지만, 세 가지 차원에 대한 관심의 구조를 알 수있다. 서브 볼륨, 또는 하위 단층 촬영 전산 평균, 이러한 바이러스 당 단백질 스파이크 같은 구조적 모티프를 반복의 높은 해상도 세부 사항을 얻을 필요가있다. 알리에 대한 자세한 계산 방법gning 및 Jsubtomo 소프트웨어 패키지를 이용하여 서브 - 단층 촬영을 평균하는 개략되어있다. 이 접근법은 비리 막에 고차 스파이크 투 스파이크 상호 작용의 연구 (A)의 20 ~ 40 범위의 해상도와 연구 바이러스 성 스파이크 당 단백질의 구조의 시각화를 가능하게한다. 일반적인 결과는 Bunyamwera 바이러스, 가족 Bunyaviridae에서 포락선 바이러스되게됩니다. 이 가족은 인간과 동물의 건강에 위협이 병원균의 구조적 다양한 그룹입니다.

우리는 24를 설정 이전에 게시 된 데이터를 사용하여 Bunyamwera 바이러스 (Orthobunyavirus, Bunyaviridae)의 엔벨로프 당 단백질 복합체에 대한 위에서 설명한 서브 단층 평균 워크 플로우의 응용 프로그램을 보여줍니다. 데이터 수집 및 정제 파라미터는 표 1에 나열되어 있습니다. 대표하는 하나의 단층는 그림 2와 같다. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 매개 변수 (단위) </td><td> 값 </td></tr><tr><td> 데이터 수집 </td><td></td></tr><tr><td> 전압 (kV의) </td><td> 300 </td></tr><tr><td> 보정 배율 (X) </td><td> 111000 </td></tr><tr><td> 픽셀 크기 (A) </td><td> 5.4 </td></tr><tr><td> 예상 용량 (전자 - / 2) </td><td> 100 </td></tr><tr><td> 언더 포커스 (μM) </td><td> 4.0-4.5 </tD&gt; </tr><tr><td> 제 CTF 제로 (A) </td><td> 26-30 </td></tr><tr><td> 틸트 범위 (°) </td><td> -60-60 </td></tr><tr><td> 틸트 샘플링 (°) </td><td> 3 </td></tr><tr><td> 데이터 및 개선 </td><td></td></tr><tr><td> 단층 촬영 </td><td> 11 </td></tr><tr><td> 비리 </td><td> 29 </td></tr><tr><td> 비리 당 씨앗 </td><td> (106) </td></tr><tr><td> 씨앗의 총 수 </td><td> 3074 </td></tr><tr><td> 스파이크 검출 </td><td> 1346 </td></tr><tr><td> 중복을 제거한 후 스파이크 </td><td> 1401 </td></tr><tr><td> 교차 상관 관계를 기반으로 선택 후 스파이크 </td><td> 1022 </td></tr><tr><td> 스파이크는 최종 평균에 포함 </td><td> 1022 </td></tr><tr><td> 대칭 </td><td> C3 </td></tr><tr><td> 각도 샘플링 (°) </td><td> 8 </td></tr><tr><td> 해상도정제에 사용되는 범위 (A) </td><td> 42-334 </td></tr><tr><td> 최종 해상도 추정 (A) (B) </td><td> 35 </td></tr></tbody></table> 표 1 : Bunyamwera 데이터 수집 및 정제 통계. CTF, 콘트라스트 전달 함수. B 0.143의 임계 값이 독립적으로 정제 된 구조물 사이 푸리에 쉘 상관을 사용하여 계산. <img alt="그림이" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 그림 2 :. Bunyamwera 비리의 단층 촬영을 통해 조각 각 비리의 주변에서 분명 여러 스파이크 측면보기는 화살촉으로 표시됩니다. 단층 촬영 (60)에 로우 패스 필터링하고 있습니다. 스케일 바 100 nm의. 첫째, 우리는 205 수동 고른 스파이크를 사용 초기 모델 (정제 <strong&gt; 그림 3). 가운데 가장 스파이크의 3 배 대칭은 대칭 (그림 3B)를 적용하지 않고 분명했다 및 개선의 다음 라운드 (그림 3C)에 부과되었다. 비리 온의 표면 상에 자동적으로 모든 스파이크를 검출하기 위해, 우리는 43 내지 20도 (도 4A)의 간격의 반경마다 비리 대 106 씨앗 생성 반복적 막 (도 4b)에 대하여 자신의 위치를 정제. <img alt="그림 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 그림 3 : 초기 템플릿 구조의 제련. (A) 원통형 스파이크의 수동으로 정의 위치에서 건설 (C100) 템플릿을 평균. (B) &#39;평균 밀도를 정제의 5 라운드 후 모든 대칭없이 (C1) 3 배에 표시에게 스파이크를 부과대칭 기능을 제공합니다. 모델의 해상도는 48 Å이다. 스파이크 (C) 평균이 3 배 대칭 정제의 5 라운드 이후 41 Å에서 해결되었습니다. <img alt="그림 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 그림 4 :.. 씨의 제련 AB) (전 씨의 집합)과 후 (B) 정제는 그림 2에서 한 비리 밀도 (B)의 씨앗을 나타내왔다 색으로 각각의 크로스를 기반으로 상관 계수 (파란색, 낮은 상관 관계, 빨강, 높은 상관 관계). (중복을 제거한 후 상위 75 %; ~ 1000 스파이크) 최상의 스파이크 당 단백질 상호 관련 패치는 최종 평균을 계산하는 데 사용 하였다. 평균은 35 Å 해상도 (그림 5)로 확인되었다. 이 글은, 중간에 삼량 스파이크 구조를 밝혀여섯 이웃 스파이크에서 약간의 기여뿐만 아니라. 알려진 위치에 구조를 배치하여 계산 된 비리의 복합 모델은 비리 표면 (그림 6A)에 스파이크의 위치를 한 것으로 밝혀졌습니다. 때때로, 스파이크의 로컬 주문 패치 (그림 6B) 분명했다. <img alt="그림 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 그림 5 : 템플릿 매칭 후 당 단백질 스파이크 층의 패치의 정제 구조. (AB) &#39;도&#39;와 &#39;홀수&#39;, 데이터의 두 부분에서 재구성 된 두 개의 맵이 표시됩니다. 이지도는 접근 방식의 validness을 확인, 유사의 놀라운 수준을 보여줍니다. 이지도는 서로 완전히 독립적으로 재구성 된대로 스파이크 긴 축을 중심으로 방향이 다르다. 이지도의 (C) 평균에 표시됩니다35 Å의 최종 해상도. <img alt="그림 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 그림 6 : Bunyamwera 비리에 스파이크를 배치. (A) 비리의 복합 모델입니다. 보기 벡터 (스틱) 스파이크의 방향을 나타냅니다. 색상은 각각의 스파이크와 템플릿 구조 (파란색, 낮은 상관 관계, 빨강, 높은 상관 관계) 사이의 상호 상관을 나타냅니다. 스파이크의 정렬 된 패치의 (B) 근접.

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo을 사용하여 전자 Cryotomography 재건에서 바이러스 성 봉투 ​​당 단백질 스파이크의 평균화

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K 조회수.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

비디오: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

바이러스성 병원균에 대한 임상 샘플을 화면에 판 - 바이러스성 Microarray 분석 (Virochip)를 사용

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

연구

면역학 및 감염병학

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Cryo - 전자 Tomography 및 자동 하위 tomogram 평균을 사용하여 HIV의 봉투 Glycoproteins의 분자 구조 결정

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

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 ...

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

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

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 ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

바이러스 변이 주파수의 RNA 바이러스 및 특성화의 피델리티 변종의 분리

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

히드록 시아파 타이트 크로마 토그래피를 사용하여 환경 바이러스성 커뮤니티에서 단일 좌초 DNA, 더블 좌초된 DNA와 RNA의 분리

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids에서 바이러스성 DNA의 준비

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

셀 기반의 ELISA 분석을 사용하여 HIV-1 삼량 봉투 당 단백질의 구조적 평가

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

의 탐지를위한 동결 건조 루프 - 매개 등온 증폭 시약의 개발<em&gt; Coxiella burnetii의</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Jsubtomo을 사용하여 전자 Cryotomography 재건에서 바이러스 성 봉투 ​​당 단백질 스파이크의 평균화

Jsubtomo을 사용하여 전자 Cryotomography 재건에서 바이러스 성 봉투 당 단백질 스파이크의 평균화