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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

MicroChem treatment experiment.Treatment of cells with disinfectant after completion of staining procedure produced similar signal intensity as the non-treated control. The Y-axis shows the percent of infected cells for each condition tested. The list of different conditions is shown on X-axis. MicroChem treatment at the end of staining procedure produced comparable signal as control (No MicroChem).
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pone.0138836.g003: MicroChem treatment experiment.Treatment of cells with disinfectant after completion of staining procedure produced similar signal intensity as the non-treated control. The Y-axis shows the percent of infected cells for each condition tested. The list of different conditions is shown on X-axis. MicroChem treatment at the end of staining procedure produced comparable signal as control (No MicroChem).

Mentions: To evaluate the effect of MicroChem-Plus disinfecting reagent on signal intensity from the virus stained with anti-vaccinia polyclonal detection antibody, 5% solution of MicroChem-Plus was applied at different steps of staining protocol. MicroChem-Plus treatment had a minimum effect on vaccinia virus signal intensity when incorporated at the end of the staining protocol. Signal from the wells that received MicroChem-Plus was comparable to the signal of control wells (Fig 3). However, signal intensity decreased when MicroChem-Plus was introduced after fixation or permeabilization step. Due to the drop in signal intensity when MicroChem-Plus treatment occurred prior to staining, all immunostaining steps for experiments with variola virus were conducted within the BSL- 4 and MicroChem-Plus treatment occurred at the end of the staining procedure.


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)

MicroChem treatment experiment.Treatment of cells with disinfectant after completion of staining procedure produced similar signal intensity as the non-treated control. The Y-axis shows the percent of infected cells for each condition tested. The list of different conditions is shown on X-axis. MicroChem treatment at the end of staining procedure produced comparable signal as control (No MicroChem).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g003: MicroChem treatment experiment.Treatment of cells with disinfectant after completion of staining procedure produced similar signal intensity as the non-treated control. The Y-axis shows the percent of infected cells for each condition tested. The list of different conditions is shown on X-axis. MicroChem treatment at the end of staining procedure produced comparable signal as control (No MicroChem).
Mentions: To evaluate the effect of MicroChem-Plus disinfecting reagent on signal intensity from the virus stained with anti-vaccinia polyclonal detection antibody, 5% solution of MicroChem-Plus was applied at different steps of staining protocol. MicroChem-Plus treatment had a minimum effect on vaccinia virus signal intensity when incorporated at the end of the staining protocol. Signal from the wells that received MicroChem-Plus was comparable to the signal of control wells (Fig 3). However, signal intensity decreased when MicroChem-Plus was introduced after fixation or permeabilization step. Due to the drop in signal intensity when MicroChem-Plus treatment occurred prior to staining, all immunostaining steps for experiments with variola virus were conducted within the BSL- 4 and MicroChem-Plus treatment occurred at the end of the staining procedure.

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