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논문 상세정보


Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.


참고문헌 (11)

  1. 1. Wobus CE Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages PLoS Biol 2004 2 e432 15562321 
  2. 2. Wobus CE Thackray LB Virgin HW Murine norovirus: a model system to study norovirus biology and pathogenesis Journal of virology 2006 80 5104 5112 16698991 
  3. 3. Condit RC Knipe DM Howley PM Ch. 2 Fields Virology 2007 1 Lippincott Williams & Wilkins 25 58 
  4. 4. Thackray LB Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence Journal of virology 2007 81 10460 10473 17652401 
  5. 5. Reed LJ Muench H A simple method for estimating 50% endpoints American Journal of Hygiene 1932 27 493 497 
  6. 6. Chachu KA Antibody is critical for the clearance of murine norovirus infection Journal of virology 2008 82 6610 6617 18417579 
  7. 7. Barron EL Diversity of murine norovirus strains isolated from asymptomatic mice of different genetic backgrounds within a single U.S. research institute PLoS ONE 2011 6 e21435 21738664 
  8. 8. Cox C Cao S Lu Y Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line Virology journal 2009 6 196 19903359 
  9. 9. Cooper PD The plaque assay of animal viruses Adv. Virus Res 1961 8 319 378 13881155 
  10. 10. Hyde JL Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway Journal of virology 2009 83 9709 9719 19587041 
  11. 11. Simmonds P Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses Nucleic acids research 2008 36 2530 2546 18319285 


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