Fast and Efficient Titration of Human Cytomegalovirus Stocks with a Self-Excisable Bacterial Artificial Chromosomes Cassette by Flow Cytometry.

Abstract

The study of human cytomegalovirus (HCMV) has for long been challenging due to the inability of clinical strains to efficiently proliferate in vitro until adaptive mutations occur. These mutations lead to strains that differ considerably from clinical isolates, many of them showing altered cell tropism, a decrease in cell association and higher susceptibility to an innate immune response. These problems were recently solved by the use of bacterial artificial chromosome (BAC) vectors that allow for the conservation of an intact HCMV genome. Other characteristics that render HCMV difficult for in vitro study are related to its slow replication rate that leads to some constraints in its titration. During the cloning of HCMV into BAC vectors, many groups additionally inserted a fluorescent tag to facilitate the virus characterization. However, the methods used for titration of HCMV-BAC stocks are still relaying on the standard methods that are expensive and/or time consuming. In this study, we assessed the possibility of viral titration by fluorescence-activated cell sorting (FACS), making use of the fluorescent tags that many of the HCMV-BACs hold. We compared viral titers obtained by immunohistochemistry with FACS, a faster and inexpensive technique. We showed that viral titers are comparable using the techniques already mentioned, and that titration by FACS is an efficient, fast, and cost-effective method. The establishment of viral titration of BAC vectors by FACS can further simplify the study of HCMV.