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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

Assay Concordance plot.Bivariate scatter correlation plot between vaccinia virus infectivity measured by high-content analysis based infectivity assay (HC-IA) (Y-axis) and the plaque assay (X-axis) presented in log 10 scale. Potencies of thirteen stocks of vaccinia virus were measured in plaque assay and HC-IA. Results were analyzed using GraphPad Prizm software showing significant correlation between image-based and standard plaque counting assays (Pearson r—0.95, P value—0.0001, R2—0.9, slope 0.83). Dashed line represents the ideal linear curve with slope of 1, while solid line is an observed concordance curve.
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pone.0138836.g004: Assay Concordance plot.Bivariate scatter correlation plot between vaccinia virus infectivity measured by high-content analysis based infectivity assay (HC-IA) (Y-axis) and the plaque assay (X-axis) presented in log 10 scale. Potencies of thirteen stocks of vaccinia virus were measured in plaque assay and HC-IA. Results were analyzed using GraphPad Prizm software showing significant correlation between image-based and standard plaque counting assays (Pearson r—0.95, P value—0.0001, R2—0.9, slope 0.83). Dashed line represents the ideal linear curve with slope of 1, while solid line is an observed concordance curve.

Mentions: We evaluated the correlation between the HC-IA and standard plaque assay using vaccinia Western Reserve and vaccinia Wyeth virus strains (Fig 4). Thirteen vaccinia virus stocks were tested in duplicate with both assays. The majority of viral stocks had titers between 108−109 pfu/mL and were tested in high content infectivity assays starting at a 1:1000 dilution, followed by two-fold serial dilutions. With these starting dilutions, a few low titer viral stocks infected less than 50% of cells per well, instead of the targeted 90–95%, after 17 hours of incubation. These stocks were re-tested with starting dilution at 1:200, followed by eleven 2-fold dilutions, which resulted in 90–95% of cells per well detected as infected at the lowest dilution tested. For future experiments we decided to follow the rule that if stock is expected to have potency at 108−109 pfu/mL, then the starting dilution should be at 1:1000, but for any stocks with lower or unknown potency, a starting dilution at 1:200 dilution, followed by eleven 2-fold dilutions was used.


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)

Assay Concordance plot.Bivariate scatter correlation plot between vaccinia virus infectivity measured by high-content analysis based infectivity assay (HC-IA) (Y-axis) and the plaque assay (X-axis) presented in log 10 scale. Potencies of thirteen stocks of vaccinia virus were measured in plaque assay and HC-IA. Results were analyzed using GraphPad Prizm software showing significant correlation between image-based and standard plaque counting assays (Pearson r—0.95, P value—0.0001, R2—0.9, slope 0.83). Dashed line represents the ideal linear curve with slope of 1, while solid line is an observed concordance curve.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g004: Assay Concordance plot.Bivariate scatter correlation plot between vaccinia virus infectivity measured by high-content analysis based infectivity assay (HC-IA) (Y-axis) and the plaque assay (X-axis) presented in log 10 scale. Potencies of thirteen stocks of vaccinia virus were measured in plaque assay and HC-IA. Results were analyzed using GraphPad Prizm software showing significant correlation between image-based and standard plaque counting assays (Pearson r—0.95, P value—0.0001, R2—0.9, slope 0.83). Dashed line represents the ideal linear curve with slope of 1, while solid line is an observed concordance curve.
Mentions: We evaluated the correlation between the HC-IA and standard plaque assay using vaccinia Western Reserve and vaccinia Wyeth virus strains (Fig 4). Thirteen vaccinia virus stocks were tested in duplicate with both assays. The majority of viral stocks had titers between 108−109 pfu/mL and were tested in high content infectivity assays starting at a 1:1000 dilution, followed by two-fold serial dilutions. With these starting dilutions, a few low titer viral stocks infected less than 50% of cells per well, instead of the targeted 90–95%, after 17 hours of incubation. These stocks were re-tested with starting dilution at 1:200, followed by eleven 2-fold dilutions, which resulted in 90–95% of cells per well detected as infected at the lowest dilution tested. For future experiments we decided to follow the rule that if stock is expected to have potency at 108−109 pfu/mL, then the starting dilution should be at 1:1000, but for any stocks with lower or unknown potency, a starting dilution at 1:200 dilution, followed by eleven 2-fold dilutions was used.

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