Propagation, Quantification, Detection, and Storage of West Nile Virus
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
West Nile virus (WNV) is a member of the Flaviviridae family of enveloped, single?stranded, positive?sense RNA viruses. WNV, an emerging viral pathogen, is transmitted by mosquitoes to birds and mammals and is responsible for an increasing incidence of human disease in North America and Europe. Due to its ease of use in the laboratory and the availability of robust mouse models of disease, WNV provides an excellent experimental system for studying molecular virology and pathogenesis of infection by flaviviruses. Here, we describe common laboratory techniques used to propagate, quantify, detect, and store WNV. We also briefly describe appropriate safety precautions required for the laboratory use of WNV, which is classified as a Biosafety Level 3 pathogen by the United States Centers for Disease Control and Prevention. Curr. Protoc. Microbiol . 31:15D.3.1?15D.3.18. ©2013 by John Wiley & Sons, Inc.
Keywords: West Nile virus; infection; titration; plaque assay; purification
Table of Contents
- Introduction
- Basic Protocol 1: Generation and Purification of West Nile Virus Stocks
- Basic Protocol 2: Quantification of WNV by Plaque Assay
- Basic Protocol 3: Determination of WNV RNA by Quantitative Fluorogenic Reverse‐Transcription PCR
- Basic Protocol 4: Viral Growth Curves
- Alternate Protocol 1: Quantification of WNV by Focus‐Forming Assay
- Support Protocol 1: Culturing BHK21 and Vero cells for WNV Propagation and Titering
- Support Protocol 2: Propagation of C6/36 Insect Cells
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: Generation and Purification of West Nile Virus Stocks
Materials
Basic Protocol 2: Quantification of WNV by Plaque Assay
Materials
Basic Protocol 3: Determination of WNV RNA by Quantitative Fluorogenic Reverse‐Transcription PCR
Materials
Basic Protocol 4: Viral Growth Curves
Materials
Alternate Protocol 1: Quantification of WNV by Focus‐Forming Assay
Materials
Support Protocol 1: Culturing BHK21 and Vero cells for WNV Propagation and Titering
Materials
Support Protocol 2: Propagation of C6/36 Insect Cells
Materials
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Figures
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Figure 15.D0.1 Crystal violet–stained plaque assay plate. BHK21 cells were infected with 10‐fold dilutions of WNV, overlaid, and stained with crystal violet. Plaque counts were as follows: 10−2 to 10−4 , too numerous to count (TNTC); 10−5 , 118 plaques; 10−6 , 21 plaques; 10−7 , 2 plaques. The final viral titer was calculated to be 1.4 × 108 PFU/ml. View Image -
Figure 15.D0.2 Focus‐forming assay. Vero cells were infected with 10‐fold dilutions of WNV. Viral antigen was detected with an anti‐WNV MAb, followed by immunoperoxidase staining (purple). The highest‐concentration samples are in row A, with each row representing a 10‐fold dilution. Six samples were titered in duplicate (in adjacent columns). Viral titers were determined from wells with discrete foci (rows D to F). View Image
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Literature Cited
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