[논문]Measurement of Antiviral Activities Using Recombinant Human Cytomegalovirus

Song, Byung-Hak; Lee, Gyu-Cheol; Lee, Chan-Hee

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For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral ...

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

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문제 정의

The purpose of this study is to develop a rapid and sensitive assay system using luciferase reporter gene comparable to plaque assays. Hence the earliest time for reasonably high luciferase gene expression in HFF cells infected with HCMV/pp28-luc was determined.
Although the luciferase activity was maximal at 6 days after virus mfection, we chose the time for the luciferase assay at 4 days after infection, since virus production was the highest at 4 days and a relatively high level of luciferase activity was observed at this time. Moreover, the purpose of this study is to develop a rapid assay system for measuring antiviral activity.

제안 방법

In this study, we used a recombinant HCMV (HCMV/pp28-luc) containing firefly luciferase gene driven by HCMV late gene pp28 promoter for measuring antiviral activities of three compounds and the results were compared with results from the plaque assay. We found that use of HCMV/pp28-luc could be a rapid and sensitive alternative method for measuring antiviral activities of potential antiviral agents.
In this study, recombinant HCMV containing luciferase genes under the control of HCMV late pp28 gene promoter was used and the level of luciferase gene expression was compared with the level of production of infectious virus. The reason for choosing HCMV late gene pp28 was that the purpose of this study was to compare plaque assay and luciferase assay in quantifying infectious virus as a result of virus replication in the cell in the presence of potential antiviral agents. If certain antiviral agents affect immediate early or early gene expression, this may not correlate with inhibition of the production of infectious virus.
GCV was used in this study to compare the sensitivity and fidelity of luciferase assays with the sensitivity and fidelity of plaque assays. GCV inhibited HCMV multiplication in a dose-dependent manner in the concentration range of 0.
As mentioned earlier, inhibition of MIE gene expression may not linearly correlate with inhibition of virus multiplication. The recombinant HCMV described in this report based on the luciferase gene driven by HCMV late pp28 promoter directly measure the effect of antiviral agents on virus replication. The luciferase-based assays can also be used not only for antiviral assays as shown above but also used for the study of HCMV gene expression.

대상 데이터

A). Acyclovir(ACV) was purchased from Burroughs Welcome (Research Triangle Park, NC, U.S.A.). GCV and ACV were dissolved in water.

성능/효과

Therefore luciferase assay has been used when the sensitivity is the prime concern. In this study, recombinant HCMV containing luciferase genes under the control of HCMV late pp28 gene promoter was used and the level of luciferase gene expression was compared with the level of production of infectious virus. The reason for choosing HCMV late gene pp28 was that the purpose of this study was to compare plaque assay and luciferase assay in quantifying infectious virus as a result of virus replication in the cell in the presence of potential antiviral agents.

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[국내논문] Measurement of Antiviral Activities Using Recombinant Human Cytomegalovirus 원문보기

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[국내논문] Measurement of Antiviral Activities Using Recombinant Human Cytomegalovirus 원문보기

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