scholarly journals Development and Use of a Vaccinia Virus Neutralization Assay Based on Flow Cytometric Detection of Green Fluorescent Protein

2003 ◽  
Vol 77 (19) ◽  
pp. 10684-10688 ◽  
Author(s):  
Patricia L. Earl ◽  
Jeffrey L. Americo ◽  
Bernard Moss
Keyword(s):  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Recombinant Vaccinia Virus ◽  
Neutralization Assay ◽  
Flow Cytometric Assay ◽  
Virus Neutralization Assay ◽  
Flow Cytometric ◽  
Green Fluorescent

ABSTRACT A rapid and sensitive neutralization assay is required to evaluate alternative smallpox vaccines. Here we describe the development and use of a 96-well plate, semi-automated, flow cytometric assay that uses a recombinant vaccinia virus expressing enhanced green fluorescent protein and which would be applicable to other viruses.

scholarly journals Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

2004 ◽  
Vol 11 (2) ◽  
pp. 406-410 ◽  
Author(s):  
Antonio Cosma ◽  
Silja Bühler ◽  
Rashmi Nagaraj ◽  
Caroline Staib ◽  
Anna-Lena Hammarin ◽  
...  
Keyword(s):  
Immune Response ◽  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Safety Concern ◽  
Neutralization Assay ◽  
Modified Vaccinia Virus Ankara ◽  
Green Fluorescent

ABSTRACT Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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