이 작품은 아이슬란드어 연구 기금과 연구 기금 아이슬란드 대학에 의해 투자되었다.

1. Transfection 분자 복제는 각각 12킬로바이트 및 4.5 킬로바이트 두 plasmids, p8XSp5 - egfp과 p67r에 포함되어 있습니다. 9,10 <ol><li> 두 plasmids, 즉 p8XSp5 - egfp의 4.4 μg과 transfection 전에 XbaI과 ligate과 p67r의 1.6 μg의 몰 수량 잘라. </li><li> 내고 들어, 16 ° C 하룻밤에 50 μl 반응과 부화에 ligase 1 와이즈 단위를 사용합니다. </li><li> 결합이 완료되면 ° C ligase의 불활 성화 15 분 65 내고 믹스를 품어. 이 단계는 생략할 수 있습니다. </li><li> 이틀 전 transfection, 종자 10 6 SCP의 T25 조직 문화 플라스크에 세포, 그리고 confluency 90 % monolayer에 항생제없이 5 ML DMEM + 10% 양고기 혈청과 2mM의 글루타민에서 이틀 동안 세포를 성장. 세포 이틀 후 confluency 90 %에 도달하지 않은 경우 세포가 하나의 추가 일 성장을 둡니다. </li></ol> 참고 : 양고기 혈청이 태아 소 혈청 (FBS)으로 대체하실 수 있습니다. MVV의 일부 변종은 FBS에 의해 저해되기 때문에 우리는 정기적으로, 양고기 혈청을 사용하여,이 MVV 변형은 그러나, FBS에 의해 저해하지 않습니다. <ol start="5"><li> 세포 transfection에 대한 준비가되면, 항생제없이 1 %의 혈청 (양 혈청 또는 FBS)과 DMEM에 매체를 변경합니다. 우리는 transfection에 대한 Lipofectamin 2000 (Invitrogen)을 사용합니다. </li><li> 500μl Opti - 가상 매체에서 DNA를 희석. </li><li> 500μl Opti - 가상 매체에 20μl Lipofectamin 2000을 희석하고 5 분 실온 (RT)에 둡니다. </li><li> 5 분 부화 후 희석 Lipofectamine 2000 (총 부피 = 1000 μl)로 희석 DNA를 결합. 부드럽게 믹스와 RT에서 20-30 분 알을 품다. </li><li> SCP 세포와 미디어 (5 ML)이있는 T25 술병에 transfection 믹스 (1 ML)를 추가합니다. 부드럽게 락을로 섞는다. </li><li> 37 품어 ° C와 5% CO 밤새 2 (18-24 H). </li><li> 1 %의 묘약, 2mM 글루타민, 100IU 페니실린과 스트렙토 마이신과 100IU DMEM 중간 다음날을 변경합니다. </li><li> 거꾸로 형광 현미경으로 SCP 세포에서 GFP의 생산을 모니터링합니다. 15 % - 낮은 transfection 효율은 보통 5이 기본 세포와 기대하실 수 있습니다. </li><li> 37 더 품어 ° C, 5 %는 술병에있는 바이러스의 확산 있도록 몇 일 동안 CO 2. 그것은 일반적으로 전체 감염 7~14일 소요됩니다. </li><li> Eppendorf 튜브에 뜨는를 aliquoting하여 바이러스를 수확. 세포 파편을 제거하는 5 분 3,000 RPM에서 microfuge에 스핀. 새로운 튜브에 바이러스를 포함하고있는 뜨는을 전송합니다. -80에서 보관 ° C. </li></ol> 2. 바이러스의 적정 <ol><li> 10% 혈청, 2mM 글루타민, 100IU/ml 페니실린과 스트렙토 마이신의 100IU/ml, 당 우물에 96 잘 판과 보충 100 μl DMEM 매체의 씨앗 3x10 4 SCP 세포를. 37 품어 ° C 5 % CO 2. </li><li> 세포 다음날 합류하는 경우, 1 % 혈청, 2mM 글루타민, 100IU/ml 페니실린과 스트렙토 마이신 100IU/ml의 각 물론 100 μl와 DMEM에 매체를 변경합니다. </li><li> 다음과 같은 방법으로 바이러스를 희석 : <ol><li> 2mM 글루타민, 100IU/ml 페니실린과 96 잘 원형 바닥이나 V - 하단 플레이트 4 X 10 우물에 잘 따라 100IU/ml 스트렙토 마이신, 1 %의 혈청 180 μl DMEM을 추가합니다. </li><li> 사통으로 작성된 열배 시리얼 dilutions 들어, 샘플이 완전히 혼합되는 것을 보장하기 위해 아래 판의 첫 ​​번째 열에 4wells 각 바이러스의 20 μl를 추가하고, 4 채널 피펫을 사용하여 최대 피펫합니다. 피펫 팁을 변경하고 다음 네 우물 등 20 μl를 전송. 부정적인 제어를위한 바이러스 않고 마지막 우물을 둡니다. </li></ol></li><li> SCP 세포와 매체 바이러스 dilutions 100 μl를 추가합니다. </li><li> 37 ° C와 5% CO 2를 품어. 두 주 동안 GFP 형광 및 cytopathic 효과를 관찰. Cytopathic 효과 multinuclear 나타나는 세포 및 세포 죽음으로 끝나는, 둥글게 뻗어 프로세스 정상 세포보다 투명되고 세포로 이루어져 있습니다. </li><li> 리드 Muench 방법 11을 사용하여 바이러스 titer (TCID50)를 계산합니다. </li></ol> 3. 감염된 세포의 FACS 분석 참고 : 바이러스의 복제의 신속한 모니터링, 감염된 세포 유동세포계측법 검사를하실 수 있습니다. <ol><li> 감염된 세포를 (CA 10 5) Trypsinize, 500μl의 PBS PBS와 resuspend와 1X 씻는다. </li><li> 30 분 RT에서 품어 500 μl 4 % paraformaldehyde 용액 (2 % 최종 농도)를 추가합니다. </li><li> 5 분 3,000 RPM에서 microfuge의 펠릿 세포. </li><li> PBS 2x5 분들과 씻고 FACScan 분석기에서 분석합니다. 10000 이벤트를 계산합니다. </li></ol> 4. 대표 결과 : 10 7 TCID 50 / ML -이 바이러스는 전염성이 완전히 10 6 titer로 복제되어야합니다. 감염된 세포는 형광되며 det 수 있습니다유동세포계측법 및 형광 현미경에 의해 ected. 일반적으로, GFP는 유동세포계측법 (그림 1)를 사용하여 감염된 세포 배양에서 세포의 60 % 이상에서 발견하실 수 있습니다. 마찬가지로, 형광 현미경에 의해 시각 때, 세포의 대부분은 (그림 2A와 B) 형광 있습니다. <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="그림 1" /> 그림 1. 감염 7 일 후 KV1772 - egfp 바이러스에 감염된 세포의 SCP FACScan 분석. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="그림 2" /> 배양 7 일 (A) 이후 KV1772 - egfp에 감염 SCP 전지의 그림 2. 위상 콘트라스트 현미경. 같은 세포는 형광 현미경 (B)에 의해 시각.

<ol>
	<li>Sigurdsson, B., Palsson, P. A., Grimsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna,+a+demyelinating+transmissable+disease+of+sheep.">Visna, a demyelinating transmissable disease of sheep.</a> J. Neuropathol. Exp. Neurol. 14, 389-403 (1957).</li><li>Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J., Narayan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovine+lentivirus+is+macrophagetropic+and+does+not+replicate+productively+in+T+lymphocytes.">Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.</a> J. Virol. 66, 2679-2688 (1992).</li><li>Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+genetic+differences+between+lung-+and+brain-derived+isolates+of+maedi-visna+virus.">Biological and genetic differences between lung- and brain-derived isolates of maedi-visna virus.</a> Virus Genes. 16, 281-293 (1998).</li><li>Georgsson, G., Houwers, D. J., Palsson, P. A., Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+viral+antigens+in+the+central+nervous+system+of+visna-infected+sheep:+an+immunohistochemical+study+on+experimental+visna+induced+by+virus+strains+of+increased+neurovirulence.">Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence.</a> Acta Neuropathol. 77, 299-306 (1989).</li><li>Turelli, P., Guiguen, F., Mornex, J. F., Vigne, R., Querat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dUTPase-minus+caprine+arthritis-encephalitis+virus+is+attenuated+for+pathogenesis+and+accumulates+G-to-A+substitutions.">dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.</a> J. Virol. 71, 4522-4530 (1997).</li><li>Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna+virus+dUTPase+is+dispensable+for+neuropathogenicity.">Visna virus dUTPase is dispensable for neuropathogenicity.</a> J. Virol. 72, 1657-1661 (1998).</li><li>Sigurdsson, B., Thormar, H., Palsson, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+visna+virus+in+tissue+culture.">Cultivation of visna virus in tissue culture.</a> Arch Gesamte Virusforsch. 10, 368-380 (1960).</li><li>Gudmundsdottir, H. S., Olafsdottir, K., Franzdottir, S. R., Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+an+infectious+molecular+clone+of+maedi-visna+virus+that+expresses+green+fluorescent+protein.">Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.</a> J. Virol. Methods. 168, 98-102 (2010).</li><li>Andresson, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+and+biological+properties+of+a+pathogenic+proviral+molecular+clone+of+neurovirulent+visna+virus.">Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.</a> Virology. 193, 89-105 (1993).</li><li>Skraban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mutations+within+39+amino+acids+in+the+envelope+glycoprotein+of+maedi-visna+virus+alter+the+neutralization+phenotype.">Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.</a> J. Virol. 73, 8064-8072 (1999).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li><li>Berkowitz, R. D., Ilves, H., Plavec, I., Veres, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+systems+derived+from+Visna+virus:+analysis+of+virus+production+and+infectivity.">Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.</a> Virology. 279, 116-129 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 제시 MVV의 분자 클론을 표현 GFP는 호스트 세포의 상호 작용과 체외 및 생체내 두 MVV의 pathogenicity을 분석하는 데 유용합니다. 복제 7~14일 후 얻은 바이러스는 의심할 여지없이 반전 transcriptase의 부정확으로 인해 일부 변이를 인수했다. 그러나, 단일 사이클 벡터로 복제의 사용에 제한이 있습니다. 하나는 복제의 사용이 양 염소 원산지 세포로 제한되어 있기 때문입니다. 우리의 구조를 사용?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 10938025 </td></tr><tr><td> Opti - 가상 </td><td> Gibco </td><td> 51985018 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668019 </td></tr></tbody></table>

propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Maedi - visna 바이러스 (MVV) 천천히 진보 간질성 폐렴 및 뇌염 1 원인, 양 lentivirus입니다. 생체내의 MVV의 주요 대상 세포는 단핵구 혈통의 2로 간주됩니다. MVV의 일부 변종 그러나 3,4, 다른 세포 유형에 복제할 수 있습니다. 녹색 형광 단백질은 살아있는 세포에 lentiviruses 공부를 위해 일반적으로 사용되는 마커입니다. 우리는 MVV의 dUTPase에 대한 유전자에 egfp 유전자를 삽입했습니다. dUTPase 유전자는 잘 양과 염소의 대부분 lentivirus의 변종에 보존하고 CAEV 5 복제에 중요하다고 표시되었습니다. 그러나, dUTPase는 체외에서 생체내 6 모두 본 연구에 사용된 MVV의 분자 클론의 복제 소모품으로 표시되었습니다. MVV 복제는 엄격히 양이나 염소 원산지 세포에 국한됩니다. 우리는 transfection 및 바이러스 7 전파에 대한 양의 맥락막 얼기 (SCP 세포)에서 기본 세포 라인을 사용합니다. 형광 MVV 완전히 전염성이며 EGFP 표현은 적어도 6 구절 8 이상의 안정이다. TCID 50 EGFP 측정 사이 좋은 상관 관계가있다. 이 바이러스는 따라서 세포 tropism와 체외에서 생체내 8 pathogenicity 연구에 감염된 세포의 신속한 검출에 유용합니다.

Watch this Scientific Journal Video about Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein at JoVE.com

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

우리는 GFP를 표현하고 완전히 전염성이있다 maedi - visna 바이러스의 분자 클론을 설명합니다. 이 바이러스의 복제는 형광 현미경과 유동세포계측법를 사용하여 검색하실 수 있습니다.

전파와 녹색 형광 단백질을 표현 Maedi - visna 바이러스의 감염성 분자 클론을 검출

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

propagating-detecting-an-infectious-molecular-clone-maedi-visna-virus

Research

JoVE Journal

Immunology and Infection

9.8K 조회수.  University of Iceland. 우리는 GFP를 표현하고 완전히 전염성이있다 maedi - visna 바이러스의 분자 클론을 설명합니다. 이 바이러스의 복제는 형광 현미경과 유동세포계측법를 사용하여 검색하실 수 있습니다.

비디오: Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

그린 형광 단백질을 표현 레브 종속 Lentiviral 벡터를 사용하여 HIV - 1 양성 세포의 특정 마킹

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

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 ...

Visualizing Dengue Virus through Alexa Fluor Labeling

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

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

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

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

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

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Alphaherpes 바이러스 선행 성 전송 및 확산의 라이브 셀 이미징

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

인플루엔자 A 바이러스의 핵 단백질과 마우스 MX1 단백질의 공동 면역 침전

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축<em&gt; 녹농균</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika 바이러스 감염 세포 배양 시스템과<em&gt; 체외</em인터페론의&gt; 예방 효과

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

새로운 세계 Zika 바이러스 전염성 클론으로부터 재조합 바이러스의 구조 및 특성 규명

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...