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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 reproducibility.Z-factor experiment was performed to evaluate assay reproducibility, where 20% of a plate had wells infected with vaccinia virus at MOI 0.250, and the rest of the plate had wells that were infected (MOI 0.250) and neutralized with pooled human serum at 1:100 dilution Calculated Z-factor value is 0.63 indicating that the developed assay is robust and reproducible.
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pone.0138836.g006: Assay reproducibility.Z-factor experiment was performed to evaluate assay reproducibility, where 20% of a plate had wells infected with vaccinia virus at MOI 0.250, and the rest of the plate had wells that were infected (MOI 0.250) and neutralized with pooled human serum at 1:100 dilution Calculated Z-factor value is 0.63 indicating that the developed assay is robust and reproducible.

Mentions: To evaluate assay reproducibility we applied Z-factor statistical method (Fig 6). Vero cells were either treated with vaccinia virus at MOI 0.250 (“Infected”), or a mixture of virus and pooled human serum from previously immunized individuals at 1:100 dilution (“Neutralized”). Mean and standard deviations of “Infected” and “Neutralized” wells were calculated and used to determine Z-factor value. According to the results, the calculated Z-factor is 0.63, which falls within the range for a robust and reproducible assay.


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 reproducibility.Z-factor experiment was performed to evaluate assay reproducibility, where 20% of a plate had wells infected with vaccinia virus at MOI 0.250, and the rest of the plate had wells that were infected (MOI 0.250) and neutralized with pooled human serum at 1:100 dilution Calculated Z-factor value is 0.63 indicating that the developed assay is robust and reproducible.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g006: Assay reproducibility.Z-factor experiment was performed to evaluate assay reproducibility, where 20% of a plate had wells infected with vaccinia virus at MOI 0.250, and the rest of the plate had wells that were infected (MOI 0.250) and neutralized with pooled human serum at 1:100 dilution Calculated Z-factor value is 0.63 indicating that the developed assay is robust and reproducible.
Mentions: To evaluate assay reproducibility we applied Z-factor statistical method (Fig 6). Vero cells were either treated with vaccinia virus at MOI 0.250 (“Infected”), or a mixture of virus and pooled human serum from previously immunized individuals at 1:100 dilution (“Neutralized”). Mean and standard deviations of “Infected” and “Neutralized” wells were calculated and used to determine Z-factor value. According to the results, the calculated Z-factor is 0.63, which falls within the range for a robust and reproducible assay.

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