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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

Infection dynamics.Infection dynamics were tested in Vero E6 cells for four different types of Orthopoxviruses. Cells were infected with viral titrations starting from MOI 1 (1.7x104 pfu/well), X-axis, followed by a series of two-fold dilutions down to a lowest multiplicity infection of 3.9x10-4 for vaccinia virus, and 9.76x10-4 for variola, cowpox and monkeypox viruses. The Y-axis corresponds to percent of infected cells. Incubation times ranged from 5–28 hours post infection (hpi). NC = Negative control.
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pone.0138836.g001: Infection dynamics.Infection dynamics were tested in Vero E6 cells for four different types of Orthopoxviruses. Cells were infected with viral titrations starting from MOI 1 (1.7x104 pfu/well), X-axis, followed by a series of two-fold dilutions down to a lowest multiplicity infection of 3.9x10-4 for vaccinia virus, and 9.76x10-4 for variola, cowpox and monkeypox viruses. The Y-axis corresponds to percent of infected cells. Incubation times ranged from 5–28 hours post infection (hpi). NC = Negative control.

Mentions: Vero E6 cells were plated in growth medium in black 96-well, clear bottom plates (Perkin Elmer, Whaltham, MA, USA) at 1.7x104 cells per well and were allowed to attach overnight at 37°C, 5% CO2. On the day of infection viral stocks were sonicated three times for one minute with 20-second incubations on ice between sonications and pre-diluted 100-fold, using 2% FBS infection medium. Further viral dilutions were prepared in dilution plates and then transferred onto test plates. At the infection step, growth medium was removed from test plates and replaced with 50uL/well of virus. Infected cells were incubated at 37°C, 5% CO2. For experiments with variola virus, the incubation temperature was 35.5°C. In order to find optimal assay conditions we evaluated growth dynamics for each virus, and prepared multiple plates to test different incubation times. Infectivity assays were established using vaccinia Wyeth virus, which was prepared in a series of nine two-fold dilutions, starting from an intended MOI = 1 (1.7x104 pfu/well), followed by MOIs of 0.5, 0.25, 0.125, etc. (Fig 1). Vero E6 cells were infected with 50uL per well of viral dilutions, in duplicates and incubated for 5, 7, 17 and 24 hours at 37°C. Each plate had 4–6 wells with infection medium only, that were used as uninfected control for data analysis. At the end of each incubation period, cells were fixed with formaldehyde and stained with anti-vaccinia virus polyclonal antibody.


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)

Infection dynamics.Infection dynamics were tested in Vero E6 cells for four different types of Orthopoxviruses. Cells were infected with viral titrations starting from MOI 1 (1.7x104 pfu/well), X-axis, followed by a series of two-fold dilutions down to a lowest multiplicity infection of 3.9x10-4 for vaccinia virus, and 9.76x10-4 for variola, cowpox and monkeypox viruses. The Y-axis corresponds to percent of infected cells. Incubation times ranged from 5–28 hours post infection (hpi). NC = Negative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138836.g001: Infection dynamics.Infection dynamics were tested in Vero E6 cells for four different types of Orthopoxviruses. Cells were infected with viral titrations starting from MOI 1 (1.7x104 pfu/well), X-axis, followed by a series of two-fold dilutions down to a lowest multiplicity infection of 3.9x10-4 for vaccinia virus, and 9.76x10-4 for variola, cowpox and monkeypox viruses. The Y-axis corresponds to percent of infected cells. Incubation times ranged from 5–28 hours post infection (hpi). NC = Negative control.
Mentions: Vero E6 cells were plated in growth medium in black 96-well, clear bottom plates (Perkin Elmer, Whaltham, MA, USA) at 1.7x104 cells per well and were allowed to attach overnight at 37°C, 5% CO2. On the day of infection viral stocks were sonicated three times for one minute with 20-second incubations on ice between sonications and pre-diluted 100-fold, using 2% FBS infection medium. Further viral dilutions were prepared in dilution plates and then transferred onto test plates. At the infection step, growth medium was removed from test plates and replaced with 50uL/well of virus. Infected cells were incubated at 37°C, 5% CO2. For experiments with variola virus, the incubation temperature was 35.5°C. In order to find optimal assay conditions we evaluated growth dynamics for each virus, and prepared multiple plates to test different incubation times. Infectivity assays were established using vaccinia Wyeth virus, which was prepared in a series of nine two-fold dilutions, starting from an intended MOI = 1 (1.7x104 pfu/well), followed by MOIs of 0.5, 0.25, 0.125, etc. (Fig 1). Vero E6 cells were infected with 50uL per well of viral dilutions, in duplicates and incubated for 5, 7, 17 and 24 hours at 37°C. Each plate had 4–6 wells with infection medium only, that were used as uninfected control for data analysis. At the end of each incubation period, cells were fixed with formaldehyde and stained with anti-vaccinia virus polyclonal antibody.

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