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Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

Gates I, Olson V, Smith S, Patel N, Damon I, Karem K - PLoS ONE (2015)

Bottom Line: In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein.However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing.Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

View Article: PubMed Central - PubMed

Affiliation: Atlanta Research and Education Foundation, Decatur, Georgia, United States of America.

ABSTRACT
Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

No MeSH data available.


Related in: MedlinePlus

Variola virus infection in Vero E6 cells at different MOI at 24 hours post infection.Cell images were collected at 5X magnification with 9 fields per well. Variola virus (Solaimen) was titrated at different MOI concentrations. Wells with uninfected control cells were included on each plate to be used for gating purposes during image analysis. Cells were identified by nuclear staining (pseudo-colored blue) and virus presence was detected with indirect staining (pseudo-colored green). Images show infected cells in one field per well at different MOI after 24 hours infection.
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pone.0138836.g002: Variola virus infection in Vero E6 cells at different MOI at 24 hours post infection.Cell images were collected at 5X magnification with 9 fields per well. Variola virus (Solaimen) was titrated at different MOI concentrations. Wells with uninfected control cells were included on each plate to be used for gating purposes during image analysis. Cells were identified by nuclear staining (pseudo-colored blue) and virus presence was detected with indirect staining (pseudo-colored green). Images show infected cells in one field per well at different MOI after 24 hours infection.

Mentions: Fig 2 represents variola virus infection at different MOI concentrations, detected at 24 hours post infection. The number of infected cells is the highest at MOI 1 and gradually decreasing with virus titrations, allowing to identify an individual foci.


Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

Gates I, Olson V, Smith S, Patel N, Damon I, Karem K - PLoS ONE (2015)

Variola virus infection in Vero E6 cells at different MOI at 24 hours post infection.Cell images were collected at 5X magnification with 9 fields per well. Variola virus (Solaimen) was titrated at different MOI concentrations. Wells with uninfected control cells were included on each plate to be used for gating purposes during image analysis. Cells were identified by nuclear staining (pseudo-colored blue) and virus presence was detected with indirect staining (pseudo-colored green). Images show infected cells in one field per well at different MOI after 24 hours infection.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591290&req=5

pone.0138836.g002: Variola virus infection in Vero E6 cells at different MOI at 24 hours post infection.Cell images were collected at 5X magnification with 9 fields per well. Variola virus (Solaimen) was titrated at different MOI concentrations. Wells with uninfected control cells were included on each plate to be used for gating purposes during image analysis. Cells were identified by nuclear staining (pseudo-colored blue) and virus presence was detected with indirect staining (pseudo-colored green). Images show infected cells in one field per well at different MOI after 24 hours infection.
Mentions: Fig 2 represents variola virus infection at different MOI concentrations, detected at 24 hours post infection. The number of infected cells is the highest at MOI 1 and gradually decreasing with virus titrations, allowing to identify an individual foci.

Bottom Line: In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein.However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing.Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

View Article: PubMed Central - PubMed

Affiliation: Atlanta Research and Education Foundation, Decatur, Georgia, United States of America.

ABSTRACT
Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

No MeSH data available.


Related in: MedlinePlus