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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

Neutralization of viral infection with VIG.Vaccinia Immune Globulin was titrated in 3-fold serial dilutions starting from 1:50 dilution (66.67 IU/mL) and was pre-incubated for 30 minutes at 37°C with test virus (cowpox, vaccinia or monkeypox viruses), or at 35.5°C for variola virus. Pre-incubated mixture was transferred to Vero E6 cells and incubated for 17 hours (vaccinia virus), or 24 hours (cowpox, monkeypox and variola viruses).
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pone.0138836.g005: Neutralization of viral infection with VIG.Vaccinia Immune Globulin was titrated in 3-fold serial dilutions starting from 1:50 dilution (66.67 IU/mL) and was pre-incubated for 30 minutes at 37°C with test virus (cowpox, vaccinia or monkeypox viruses), or at 35.5°C for variola virus. Pre-incubated mixture was transferred to Vero E6 cells and incubated for 17 hours (vaccinia virus), or 24 hours (cowpox, monkeypox and variola viruses).

Mentions: Using vaccinia Wyeth virus as the target of neutralization, we prototyped development of the high content neutralization assay (Fig 5). Based on the results from vaccinia virus infectivity assay, we selected 17 hours incubation post infection with a viral MOI 0.25 that expected to produce infection in approximately 90% of cells in the absence of any neutralizing serum. VIG neutralized vaccinia virus infection at EC50 of 1.59 IU/mL, or at 1:2100 fold dilution of the product. Similar neutralization experiments were conducted with monkeypox, cowpox, and variola viruses, where cells were infected at MOI 0.25 and neutralized with titration of VIG, but allowed to incubate for 24 hours post infection. Since infection dynamics experiment (Fig 1) showed that monkeypox, cowpox and variola viruses grew at a slower rate than vaccinia virus, we selected a longer incubation time, but kept the same MOI concentration. Cowpox and variola virus infections were 50% neutralized at VIG concentrations 0.9 IU/mL (1:3311 fold dilution) and 1.0 IU/mL (1:3295 fold dilution), respectively. Monkeypox virus infection was neutralized at 0.3 IU/mL (1:12603 fold dilutions). The differences in VIG EC50 concentrations are directly associated with observed infection dynamics for all tested viruses. According to the results of monkeypox virus infection dynamics (Fig 1), infection at MOI 0.25 for 24 hours produces approximately 65% infection positive cells, whereas similar conditions for cowpox and variola viruses correspond to approximately 80–85% infected cells, which require higher amounts of VIG for virus neutralization.


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)

Neutralization of viral infection with VIG.Vaccinia Immune Globulin was titrated in 3-fold serial dilutions starting from 1:50 dilution (66.67 IU/mL) and was pre-incubated for 30 minutes at 37°C with test virus (cowpox, vaccinia or monkeypox viruses), or at 35.5°C for variola virus. Pre-incubated mixture was transferred to Vero E6 cells and incubated for 17 hours (vaccinia virus), or 24 hours (cowpox, monkeypox and variola viruses).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g005: Neutralization of viral infection with VIG.Vaccinia Immune Globulin was titrated in 3-fold serial dilutions starting from 1:50 dilution (66.67 IU/mL) and was pre-incubated for 30 minutes at 37°C with test virus (cowpox, vaccinia or monkeypox viruses), or at 35.5°C for variola virus. Pre-incubated mixture was transferred to Vero E6 cells and incubated for 17 hours (vaccinia virus), or 24 hours (cowpox, monkeypox and variola viruses).
Mentions: Using vaccinia Wyeth virus as the target of neutralization, we prototyped development of the high content neutralization assay (Fig 5). Based on the results from vaccinia virus infectivity assay, we selected 17 hours incubation post infection with a viral MOI 0.25 that expected to produce infection in approximately 90% of cells in the absence of any neutralizing serum. VIG neutralized vaccinia virus infection at EC50 of 1.59 IU/mL, or at 1:2100 fold dilution of the product. Similar neutralization experiments were conducted with monkeypox, cowpox, and variola viruses, where cells were infected at MOI 0.25 and neutralized with titration of VIG, but allowed to incubate for 24 hours post infection. Since infection dynamics experiment (Fig 1) showed that monkeypox, cowpox and variola viruses grew at a slower rate than vaccinia virus, we selected a longer incubation time, but kept the same MOI concentration. Cowpox and variola virus infections were 50% neutralized at VIG concentrations 0.9 IU/mL (1:3311 fold dilution) and 1.0 IU/mL (1:3295 fold dilution), respectively. Monkeypox virus infection was neutralized at 0.3 IU/mL (1:12603 fold dilutions). The differences in VIG EC50 concentrations are directly associated with observed infection dynamics for all tested viruses. According to the results of monkeypox virus infection dynamics (Fig 1), infection at MOI 0.25 for 24 hours produces approximately 65% infection positive cells, whereas similar conditions for cowpox and variola viruses correspond to approximately 80–85% infected cells, which require higher amounts of VIG for virus neutralization.

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