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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

Screening of immune response to monkeypox virus challenge in prairie dogs.A. Serum samples from the monkeypox virus challenge study in prairie dogs were tested in high-content Orthopoxvirus neutralization assay at four-fold serial dilutions, starting from 1:40 dilution. B. Evaluation of antibody production in prairie dogs post monkeypox virus challenge using ELISA.
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pone.0138836.g009: Screening of immune response to monkeypox virus challenge in prairie dogs.A. Serum samples from the monkeypox virus challenge study in prairie dogs were tested in high-content Orthopoxvirus neutralization assay at four-fold serial dilutions, starting from 1:40 dilution. B. Evaluation of antibody production in prairie dogs post monkeypox virus challenge using ELISA.

Mentions: In this experiment we compared the results of the functional immune response measured by HC-OVNA to the total amount of anti-Orthopoxvirus IgG antibodies measured by ELISA. Serum samples from two prairie dogs intranasally challenged with monkeypox virus (strain MPXV-USA-2003-044 at 5 x103 pfu) were tested in high-content Orthopoxvirus neutralization assay using Wyeth vaccinia virus. Serum samples were collected on days 3, 6, 13, 20, 24, and 31 post-challenge. No neutralization activity was observed on days 3 and 6 for PD 8141 (Fig 9A). However, the first signs of virus neutralization were detected from serum collected on day 13, followed by a gradual increase in virus inhibition from the samples collected on days 20, 24 and the day animal was euthanized (day 31). In PD 8124, neutralization of vaccinia virus infection was first detected from samples collected on day 20 and continuously increased until the day of euthanasia (day 31) (Fig 9A). The results of the neutralization assay were compared with previously performed ELISA data (Fig 9B) [11] and found two assays correlated. Similarly to HC-OVNA immune response to MPXV challenge was detected in ELISA on day 13 for PD 8141 and on day 20 for PD 8124 (day 17 serum samples were not available for HC-OVNA experiments). While ELISA evaluates the level of specific anti-viral IgG antibodies that bind, HC-OVNA is a functional assay that measures neutralizing activity of generated serum antibodies against tested Orthopoxviruses, that could be used as a complementary assay in virus challenge and vaccine studies.


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)

Screening of immune response to monkeypox virus challenge in prairie dogs.A. Serum samples from the monkeypox virus challenge study in prairie dogs were tested in high-content Orthopoxvirus neutralization assay at four-fold serial dilutions, starting from 1:40 dilution. B. Evaluation of antibody production in prairie dogs post monkeypox virus challenge using ELISA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g009: Screening of immune response to monkeypox virus challenge in prairie dogs.A. Serum samples from the monkeypox virus challenge study in prairie dogs were tested in high-content Orthopoxvirus neutralization assay at four-fold serial dilutions, starting from 1:40 dilution. B. Evaluation of antibody production in prairie dogs post monkeypox virus challenge using ELISA.
Mentions: In this experiment we compared the results of the functional immune response measured by HC-OVNA to the total amount of anti-Orthopoxvirus IgG antibodies measured by ELISA. Serum samples from two prairie dogs intranasally challenged with monkeypox virus (strain MPXV-USA-2003-044 at 5 x103 pfu) were tested in high-content Orthopoxvirus neutralization assay using Wyeth vaccinia virus. Serum samples were collected on days 3, 6, 13, 20, 24, and 31 post-challenge. No neutralization activity was observed on days 3 and 6 for PD 8141 (Fig 9A). However, the first signs of virus neutralization were detected from serum collected on day 13, followed by a gradual increase in virus inhibition from the samples collected on days 20, 24 and the day animal was euthanized (day 31). In PD 8124, neutralization of vaccinia virus infection was first detected from samples collected on day 20 and continuously increased until the day of euthanasia (day 31) (Fig 9A). The results of the neutralization assay were compared with previously performed ELISA data (Fig 9B) [11] and found two assays correlated. Similarly to HC-OVNA immune response to MPXV challenge was detected in ELISA on day 13 for PD 8141 and on day 20 for PD 8124 (day 17 serum samples were not available for HC-OVNA experiments). While ELISA evaluates the level of specific anti-viral IgG antibodies that bind, HC-OVNA is a functional assay that measures neutralizing activity of generated serum antibodies against tested Orthopoxviruses, that could be used as a complementary assay in virus challenge and vaccine studies.

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