121. Adenovirus Titers Determined by Quantitative Real-Time PCR Correlate with Infectious Titers

The accurate determination of adenovirus titers is crucial because as little as a three-fold difference in the amount of virus administered can dramatically affect the outcome of an experiment, especially when high doses are used in animal models. The plaque assay is the standard method for determination of infectious titers, while physical titers (infectious plus non-infectious virus) are typically determined by optical absorbance at 260 nm. Although the plaque assay is a well-established method, there can be considerable variability in titers depending on the cell line used, the passage of the cells, the researcher performing the assay, and the laboratory protocol. For example, in our hands, the plaque assay titers determined in A549 cells are typically several-fold higher than those obtained on HEK 293 cells. In addition, the plaque assay requires as many as four weeks to complete, particularly for vectors which replicate slowly, such as those in which the adp gene is deleted. We have developed a unique quantitative real-time PCR assay for rapid adenovirus quantification. Our method includes a DNase treatment prior to capsid disruption to eliminate non-encapsidated DNA. Also, the real-time PCR primers are located in the E4 region at the right end of the genome, distant from the packaging signals at the left end of the genome. With this design, only full-length encapsidated adenovirus DNA should be measured. Our assay has been used to titer a wide variety of adenovirus stocks, including viruses with various point mutations, insertions, and deletions. In addition, we have assayed both cesium chloride-banded preparations and crude lysates, as well as stocks which have been stored in different buffers, at different temperatures, and for different lengths of time (ranging from recently prepared stocks to those prepared 20 years ago). Regardless of these differences, we have found this assay to produce rapid and consistent titers. Interestingly, the titers determined by real-time PCR correlated with infectious titers determined by the plaque assay and the endpoint dilution assay. This method can be utilized to verify the previously determined titer of virus stocks as well as to rapidly predict the infectious titer of new virus stocks.A portion of the research was funded by a biotechnology company named VirRx, Inc. AET and WSMW have equity in VirRx. The accurate determination of adenovirus titers is crucial because as little as a three-fold difference in the amount of virus administered can dramatically affect the outcome of an experiment, especially when high doses are used in animal models. The plaque assay is the standard method for determination of infectious titers, while physical titers (infectious plus non-infectious virus) are typically determined by optical absorbance at 260 nm. Although the plaque assay is a well-established method, there can be considerable variability in titers depending on the cell line used, the passage of the cells, the researcher performing the assay, and the laboratory protocol. For example, in our hands, the plaque assay titers determined in A549 cells are typically several-fold higher than those obtained on HEK 293 cells. In addition, the plaque assay requires as many as four weeks to complete, particularly for vectors which replicate slowly, such as those in which the adp gene is deleted. We have developed a unique quantitative real-time PCR assay for rapid adenovirus quantification. Our method includes a DNase treatment prior to capsid disruption to eliminate non-encapsidated DNA. Also, the real-time PCR primers are located in the E4 region at the right end of the genome, distant from the packaging signals at the left end of the genome. With this design, only full-length encapsidated adenovirus DNA should be measured. Our assay has been used to titer a wide variety of adenovirus stocks, including viruses with various point mutations, insertions, and deletions. In addition, we have assayed both cesium chloride-banded preparations and crude lysates, as well as stocks which have been stored in different buffers, at different temperatures, and for different lengths of time (ranging from recently prepared stocks to those prepared 20 years ago). Regardless of these differences, we have found this assay to produce rapid and consistent titers. Interestingly, the titers determined by real-time PCR correlated with infectious titers determined by the plaque assay and the endpoint dilution assay. This method can be utilized to verify the previously determined titer of virus stocks as well as to rapidly predict the infectious titer of new virus stocks. A portion of the research was funded by a biotechnology company named VirRx, Inc. AET and WSMW have equity in VirRx.