우리는 Virochip 프로토콜의 초기 개발 및 최적화에 대한 데이비드 왕, 아나톨리 Urisman, 에이미 키슬러, 카엘 피셔, 패트릭 당나라, 그리고 알렉산더 Greninger 감사드립니다. 이 작품은 NIH K08 부여와 애보트 디스커버리 수상 (CC)을하고 하워드 휴스 의학 연구소 (JLD)를 지원합니다.

Virochip 분석의 단계는 임상 샘플에서 (1) 핵산 추출, 추출 RNA (2) 반대로 전사 및 2 차 - 가닥 cDNA 합성, 무작위로 준비하는 cDNA (3) PCR 증폭, (3) Cy3 형광 염료의 정관을 포함 , Virochip microarray로 표시된 자료 (4) 하이브 리다이 제이션, 그리고 (5) 검사 및 분석 (그림 1). 아래의 프로토콜은 독감과 같은 질병을 가진 아이에서 비강 면봉 샘플에 Virochip 분석의 사용을 보여줍니다. 프리머 시퀀스 프라이머 5&#39; - GTTTCCCAGTCACGATA - (N 9) -3 &#39; 프라이머 B 5&#39; - GTTTCCCAGTCACGATA - 3 &#39; 1. 핵산 추출 <ol><li> 이 샘플은 잠재적으로 전염성이 있으므로 임상 시료에서 핵산 추출은 Biosafety 레벨 2 (BSL - 2) 이상의 조건​​ 하에서 수행되어야합니다. </li><li> 비강 면봉 샘플 바이러스성 지주 매체 수송이다. 1.5 ML Eppendorf의 microcentrifuge 관에 샘플 나누어지는 200 μL. </li><li> 옵션 : 0.22 μm의 필터 (바이러스성 입자 정화)을 통과 샘플 </li><li> 선택 사항 : 제조 업체의 지침에 따라 터보 DNAse 키트 (앰비온)를 사용 DNAse (호스트 게놈 DNA를 저하하고 보호 encapsidated 바이러스 핵산에 대한 정화)와 pretreat 샘플 </li><li> Zymo ZR 바이러스성 RNA 추출 키트 또는 제조 업체의 지침에 따라 Qiagen Ultrasens 바이러스 키트를 사용하여 샘​​플을 추출합니다. </li></ol> 2. 역방향 전사 및 2 차 - 스트랜드 CDNA 합성 ( &quot;라운드&quot;) <ol><li> RNA를 추출 4 UL 1 UL 40 μL / pmol 프리머을 추가합니다. 65 열 ° C thermocycler (예 : GeneAmp PCR 시스템 9700) 후 5 분 방 온도 X 5 분 식지. </li><li> 2 μL 배 RT 버퍼, 1 μL 12.5 MM dNTP 1 μL 물, 0.5 μL 0.1M DTT, 0.5 μL 및 SSIII RT (Invitrogen)로 구성된 마스터 믹스 5 μL를 추가합니다. 42 품어 ° C X 60분. </li><li> 프로그램 thermocycler에서 94에 열을 ° 10로 냉각 C X 2 분 (중지 리버스 transcriptase 반응), ° C, 펄스 원심 분리기, 다음 1 μL 배 Sequenase 버퍼, 3.8 μL 물로 구성된 5 μL Sequenase 믹스를 추가 그리고 0.15 μL Sequenase (USB 공사). 2 차 스트랜드 합성 들어, 10 ° 37 C ° C 8 분 이상, 94에 다음 8 분, 열을 보유 ° C X 2 분 (Sequenase를 inactivate)을 10 ° C (보유)에 냉각. 최대 램프 </li><li> 옵션 : 앳는 cDNA 샘플은 -20 ° C.에이 시점에서 저장할 수 있습니다 </li></ol> 3. 의 PCR 증폭이 임의로 준비하는 CDNA ( &quot;라운드 B&quot;) <ol><li> 5 μL 10X PCR 버퍼, 1 μL 12.5 MM dNTP 1 μL 100 pmol / μL 뇌관의 B, 1 μL KlenTaq LA (시그마), 37 μL 물로 구성된 마스터 믹스 45 μL에 드르 5 UL 샘플을 추가합니다. </li><li> 다음과 같이 PCR 프로토콜을 실행 94 ° C, 2 분 → (94의 25주기 ° C, 30 S / 50 ° C, 45 S / 72 ° C, 1 분) → 72 ° C, 5 분 → 10 ° C ( 개최) </li><li> 1.5 % 아가로 오스 전기 영동 젤에 드르 B 샘플을 확인하십시오. 약 200의 얼룩 - 1000 BP는 시각되어야합니다 (그림 2A) </li></ol> 4. Cy3 형광 염료의 설립 <ol><li> 옵션 : 다른 샘플이 Cy5 형광 염색으로 표시하고 동일한 microarray에 hybridized 수 있습니다. </li><li> AA - dNTP를 통합하기 위해, 5 UL 10X PCR 버퍼, 1 UL 12.5 MM AA - dNTP (Invitrogen), 1 UL 100 pmol / UL 뇌관 B, 1로 구성된 마스터 믹스 45 UL에 드르 B 샘플 5 UL을 추가 1 UL KlenTaq LA (시그마), 37 UL 물. PCR 프로토콜 다음과 같이 프로그램을 실행 : 94 ° C, 2 분 → (94의 15주기 ° C, 30 S / 50 ° C, 45 S / 72 ° C, 1 분) → 72 ° C, 5 분 → 10 ° C ( 개최) </li><li> 제조 업체의 지침에 따라 DNA 청소 및 농축기 - 5 키트 (Zymo 연구)로 앳 C 예제를 청소합니다. 10 μL에 Elute. </li><li> 1M 중탄산염 1 UL (시그마)와 앳 C 예제 1 UL Cy5 (GE 헬스케어)를 추가합니다. </li><li> 어둠 속에 품어 X 1 시간. </li><li> 제조 업체의 지침에 따라 DNA 청소 및 농축기 - 5 키트 (Zymo 연구)와 Cy3 - 라벨 샘플을 청소하십시오. 12 μL에 Elute. </li></ol> 5. Virochip Microarray에 하이브 리다이 제이션 <ol><li> 옵션 : (샘플의 정상화를위한) Nanodrop 분광 광도계에 통합 cDNA 농도와 염료의 양을 확인 1.5 μL를 사용하여 </li><li> PCR 스트립 관에 25 μL의 총 볼륨 (애질런트) 1 μL 25X 조각 버퍼, 5 μL 배 차단 버퍼, Cy3 - 라벨 샘플의 9.5 μL (또는 정상화 금액) 및 물을 추가 </li><li> 2X GEx의 하이브리드화 버퍼 (애질런트) 25 μL를 추가합니다. 거품을 제거하기 위해 간략하게 스핀 다운. </li><li> 가스켓 슬라이드에있는 예제 40 μL를로드합니다. </li><li> 장소 애질런트 - 인쇄 Virochip 배열 (Californi 대학샌프란 시스코 / 애질런트) (아래 &quot;애질런트&quot;표시된 쪽) 개스킷 슬라이드 위에 단단히하고 나사를 고정. </li><li> 65 밤새 잡종 ° C 오븐, 10 RPM 속도 설정. </li><li> 37 ° C.에 배열, 앞서 열을 가하다 버퍼 2 (애질런트)를 씻어 버퍼 1 (애질런트)의 개스킷 슬라이드 / 배열 샌드위치 수중 갈라놓을. 버퍼 1 X 1 분 씻으십시오. preheated 버퍼 2 X 1 분에 씻으십시오. 천천히 (멀리 직접 배열의 적극적인 측면을 만지지 않도록주의하고 여분의 수분 윅에 kimwipe 사용) 거품을 피하기 위해주의하고, 버퍼 2 슬라이드를 제거 </li><li> 슬라이드 홀더에 슬라이드를 삽입하고 배열 ​​스캐너에 넣습니다. </li></ol> 6. Virochip 검색 및 분석 <ol><li> 스캔 Cy3 - 표시된 배열 악기의 표준 스캔 프로토콜 (그림 2B)에 따라 5 μm의 또는 2 μm의에서. </li><li> 육안 검사, 클러스터 분석, 또는 자동 Virochip 분석 프로그램에 의해 Virochip의 microarrays를 분석, 11 E - 예측했다. 이러한 세 가지 방법 중 각각으로 인플루엔자 바이러스가 쉽게 비강 면봉 샘플 (독감과 같은 질병을 가진 아이에서) (그림 2C)에 감지 </li></ol> 7. 대표 결과 : 프로토콜이 제대로되면 얼룩이 드르 B 단계 (그림 2A) 이후의 아가로 오스 겔 전기 영동에서 볼 수 있습니다. 애질런트 플랫폼에 Virochip microarray는 (그림 2B) 시각 분석하기 어려울 수 있지만, 바이러스가 존재한다면 그것은 쉽게 육안 검사, 클러스터 분석, 및 / 또는 E - 예측 (그림 1C)에 의해 식별되어야합니다. 검정에 대한 긍정적인 제어 역할을하는, 샘플은 알려진 바이러스 (박테리오 파지 MS2 예 : 담배 모자이크 바이러스)에서 핵산의 측정할 수량를 타서 수 있습니다. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="그림 1" /> 그림 1. Virochip microarray 처리 및 분석의 개략도. 임상 시료에서 핵산을 추출은 무작위 증폭 형광 염료로 표시하고, Virochip microarray에 hybridized입니다. Microarrays 2 μm의 해상도에서 스캔 및 전자 예측을 포함하여, 전산 다양한 도구를 사용하여 분석하고 있습니다. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="그림 2" /> 그림 2. Virochip 분석의 단계는 임의의 PCR, 200 얼룩에 의해 증폭 후 -. 1000 BP 겔 전기 영동에 의해 시각 수 (A) (B) 8 배열 / 유리 슬라이드의 세 Virochip의 microarrays 아웃은 작은 영역으로 표시됩니다. 고장 접속 오른쪽 하단 모서리에있는 삽입된 페이지에 한 microarray니다. (C) 자동 microarray 바이러스 분석을 사용하여 임상 샘플에서 독감 바이러스의 존재를 드러내 E를 예측했다. 높은 유사성 점수 (S = 0.55)은 이처럼 매우 중요한 예측하고, 제로에 가까운 P - 값에 해당합니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

여기에 설명한 Virochip 프로토콜은 복잡하고 세심한과 숙련된 연구 기술자가 필요합니다. 정확한 시약의 농도와 PCR, 라벨에 대한 조건과 하이브 리다이 제이션이 중요합니다. Virochip 프로토콜은 쉽게 같은 핵산 추출에 대한 TRIzol (Invitrogen)와 같은 조직 추출 방법의 사용에 의해 질병 조직의 분석을 수용하기 위해 수정할 수 있습니다. 프로브를 추가하거나 원하는 스펙트럼 및 범위의 광범를 얻을 필요 삭제할 수 있습니다, 예를 들어 &quot;- the - fly 방식에서&quot;같은 박테리아 및 곰팡이 등 nonviral 타겟의 검출을위한 프로브가 설계 될 수 있으며 추가됩니다. 현재 개발중인 Virochip 분석의 일부 응용 프로그램은 임상 실험실, 확산 조사, 약물 및 백신의 순도 검사, 그리고 새로운 바이러스성 병원체 발견에 광범위한 스펙트럼 바이러스 진단을 포함합니다.

<table border="1"><tbody><tr><th> 시약 / 재료의 이름 </th><th> 시약 / 재료의 이름 </th></tr><tr><td> 프리메라 </td><td> 5&#39; - GTTTCCCAGTCACGATA - (N9) -3 &#39; </td></tr><tr><td> 프리머 B </td><td> 5&#39; - GTTTCCCAGTCACGATA - 3 &#39; </td></tr><tr><td rowspan="5"> RT 마스터 믹스 (5 μL) </td><td> 2 μL 배 RT 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 물 </td></tr><tr><td> 0.5 μL 0.1M DTT </td></tr><tr><td> 0.5 μL SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase 믹스 (5 μL) </td><td> 1 μL 배 Sequenase 버퍼 </td></tr><tr><td> 3.8 μL 물 </td></tr><tr><td> 0.15 μL Sequenase (USB 공사) </td></tr><tr><td rowspan="5"> 차 B PCR 마스터 믹스 (45 μL) </td><td> 5 μL 10X PCR 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 100 μL / pmol 프리머 B </td></tr><tr><td> 1 μL KlenTaq LA (시그마) </td></tr><tr><td> 37 μL 물 </td></tr><tr><td rowspan="5"> 앳 C PCR 마스터 믹스 (45 μL) </td><td> 5 UL 10X PCR 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 100 μL / pmol 프리머 B </td></tr><tr><td> 1 μL KlenTaq LA (시그마) </td></tr><tr><td> 37 μL 물 </td></tr><tr><td rowspan="4"> 하이브 리다이 제이션 버퍼 (25 μL) </td><td> 1 μL 25X 조각 버퍼 (애질런트) </td></tr><tr><td> 5 μL 5 배 차단 버퍼 (애질런트) </td></tr><tr><td> Cy3 - 라벨 샘플의 9.5 μL (또는 정상화 금액) </td></tr><tr><td> 25 μL의 총 볼륨 물을 추가 </td></tr><tr><td> 애질런트 Virochip microarray </td><td> 애기 실험실, UCSF에서 제공; 보류 라이센스 </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

많은 전염성 질병의 바이러스 원인의 진단은 바이러스의 고유한 시퀀스 다양성뿐만 아니라 전통적인 방법에 의해 감지되지 SARS coronavirus 2009 유행성 H1N1 독감 바이러스로 소설 바이러스 병원균의 지속적인 출현으로 인해 어렵습니다. 이러한 문제를 해결하기 위해, 우리는 이전에 개발하고 보존 순서 상동 1을 바탕으로 모든 알려진 바이러스뿐 아니라 소설 변종을 감지할 수있는 능력과 Virochip라는 판 - 바이러스 microarray 플랫폼을 확인했습니다. Virochip를 사용하여, 우리는 동등하거나 기존의 임상 테스트를 2-5로 뛰어난 감도로 입원한 환자에 설명할 수없는 중요한 질병의 경우를 포함한 호흡기 감염,과 관련된 바이러스의 전체 스펙트럼을 확인했습니다. Virochip 또한 SARS coronavirus 6,7, 소설 리노 바이러스의 clade 5, XMRV (전립선암에 연결된 레트로 바이러스) 8, 조류 bornavirus (앵무새의 낭비 질병의 원인) 9 포함한 새로운 바이러스를 식별하는 데 사용되었습니다 호흡기 질환과 diarrheal 10 어린이와 소설 cardiovirus. Virochip의 현재 버전은 애질런트 microarray 플랫폼에 포팅 2009 년 12 월 현재 GenBank에 ~ 이상 1500 바이러스로부터 파생된 ~ 36,000 프로브로 구성되어있다. 여기 샘플 핵산 추출, 임의 primers를 사용하여 PCR 증폭, 형광 염료 정관 및 microarray의 하이브리드화, 검사 및 분석을 포함하여 (~ 24 시간 처리 시간) 처음부터 끝까지 Virochip 분석 처리에 관련된 단계를 보여줍니다.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

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

Virochip 동시에 보존 순서 상동에 기초하여 모든 알려진 바이러스뿐 아니라 소설의 바이러스를 검출하기위한 판 - 바이러스 microarray입니다. 여기 알려진 알려지지 않은 두 바이러스의 존재에 대한 임상 샘플을 분석 Virochip 분석을 실행하는 방법을 보여줍니다.

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 ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

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

18.8K 조회수.  University of California, San Francisco. Virochip 동시에 보존 순서 상동에 기초하여 모든 알려진 바이러스뿐 아니라 소설의 바이러스를 검출하기위한 판 - 바이러스 microarray입니다. 여기 알려진 알려지지 않은 두 바이러스의 존재에 대한 임상 샘플을 분석 Virochip 분석을 실행하는 방법을 보여줍니다.

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

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

임상 항체 샘플 Phagocytic 활동 결정

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

연구

면역학 및 감염병학

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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

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

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

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

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

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

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략<em&gt; Vermamoeba의 vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

효소 결합 렉틴 분석에 의해 인플루엔자 뉴 라미니다 아제 억제 항체가를 측정

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Biocontainment 내 바이러스 샘플의 음성 염색을위한 캡슐의 활용

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

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

요약

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이 비디오의 챕터

임상 항체 샘플 Phagocytic 활동 결정

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지

호스트 세포 RNA의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략

효소 결합 렉틴 분석에 의해 인플루엔자 뉴 라미니다 아제 억제 항체가를 측정

Biocontainment 내 바이러스 샘플의 음성 염색을위한 캡슐의 활용

관련된 바이러스 및 세포질 단백질의 Id에 대 한 바이러스 성 DNA의 정화

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

요약

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임상 항체 샘플 Phagocytic 활동 결정

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략

효소 결합 렉틴 분석에 의해 인플루엔자 뉴 라미니다 아제 억제 항체가를 측정

Biocontainment 내 바이러스 샘플의 음성 염색을위한 캡슐의 활용

관련된 바이러스 및 세포질 단백질의 Id에 대 한 바이러스 성 DNA의 정화

호스트 세포 RNA의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여