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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

Bivariate scatter correlation plot between two imaging assays.New Orthopoxvirus neutralization assay (HC-OVNA), Y-axis, and GFP-expressing vaccinia virus (HCS-GFP), X-axis, presented in log 10 scale. Correlation between two assays was evaluated using 49 human serum samples from smallpox vaccine study (CDC IRB protocol 3349). Mean EC50 values from neutralization curves from HC-OVNA and HCS-GFP assays were plotted against each other (log scale) and analyzed using GraphPad Prizm software, showing significant correlation between two assays (Pearson r—0.9, P value—0.0001, R2–0.8).
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pone.0138836.g007: Bivariate scatter correlation plot between two imaging assays.New Orthopoxvirus neutralization assay (HC-OVNA), Y-axis, and GFP-expressing vaccinia virus (HCS-GFP), X-axis, presented in log 10 scale. Correlation between two assays was evaluated using 49 human serum samples from smallpox vaccine study (CDC IRB protocol 3349). Mean EC50 values from neutralization curves from HC-OVNA and HCS-GFP assays were plotted against each other (log scale) and analyzed using GraphPad Prizm software, showing significant correlation between two assays (Pearson r—0.9, P value—0.0001, R2–0.8).

Mentions: The mean EC50 values of serum titrations for three runs were collected for both assays and were plotted against each other (Fig 7). Samples that did not neutralize viral infection (Category A and C days 0, day 7) at 1:40 dilution were excluded from correlation analysis. The correlation analysis between HCS-GFP and HC-OVNA was performed using ID50 values (HCS-GFP) and EC50 results (HC-OVNA) for 43 samples and appears to be significant with correlation coefficient = 0.9, and coefficient of determination = 0.8 (p value < 0.0001).


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)

Bivariate scatter correlation plot between two imaging assays.New Orthopoxvirus neutralization assay (HC-OVNA), Y-axis, and GFP-expressing vaccinia virus (HCS-GFP), X-axis, presented in log 10 scale. Correlation between two assays was evaluated using 49 human serum samples from smallpox vaccine study (CDC IRB protocol 3349). Mean EC50 values from neutralization curves from HC-OVNA and HCS-GFP assays were plotted against each other (log scale) and analyzed using GraphPad Prizm software, showing significant correlation between two assays (Pearson r—0.9, P value—0.0001, R2–0.8).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g007: Bivariate scatter correlation plot between two imaging assays.New Orthopoxvirus neutralization assay (HC-OVNA), Y-axis, and GFP-expressing vaccinia virus (HCS-GFP), X-axis, presented in log 10 scale. Correlation between two assays was evaluated using 49 human serum samples from smallpox vaccine study (CDC IRB protocol 3349). Mean EC50 values from neutralization curves from HC-OVNA and HCS-GFP assays were plotted against each other (log scale) and analyzed using GraphPad Prizm software, showing significant correlation between two assays (Pearson r—0.9, P value—0.0001, R2–0.8).
Mentions: The mean EC50 values of serum titrations for three runs were collected for both assays and were plotted against each other (Fig 7). Samples that did not neutralize viral infection (Category A and C days 0, day 7) at 1:40 dilution were excluded from correlation analysis. The correlation analysis between HCS-GFP and HC-OVNA was performed using ID50 values (HCS-GFP) and EC50 results (HC-OVNA) for 43 samples and appears to be significant with correlation coefficient = 0.9, and coefficient of determination = 0.8 (p value < 0.0001).

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