Although the molecular mechanisms by which host cells defend themselves against viral infection have been studied in great depth, and countermeasures viruses employ to suppress such defensive responses have been widely documented, relatively little attention has been devoted toward elucidating how such interactions between virus and host are resolved over multiple rounds of infection. Here we describe the design, synthesis, and validation of a dual-color fluorescent reporter system to study how viral infections spread through a host cell monolayer and how the cellular innate immune system mounts an antiviral response. We employed recombinant, red fluorescent protein (RFP) expressing mutants of a prototypical RNA virus, vesicular stomatitis virus (VSV) to enable identification and tracking of infected cells. Further, we generated stable reporter cells that use green fluorescent protein (GFP) to report on the expression of IFIT2, an interferon stimulated gene (ISG) involved in the interference of viral protein translation, and a marker of antiviral defense activation. The presence of the fluorescent protein reporters had minimal effects on the normal behavior of the cells or viruses. Moreover, expression of the virus and cell reporters correlated with the kinetics of viral replication and activation of an anti-viral response, respectively. This two-color system enabled us to track and quantify in live cells how viral replication and activation of host defensive responses play out over multiple rounds of infection. Initial study of propagating infections demonstrated that antiviral activation over multiple rounds was critical for slowing and ultimately halting the spread of infection.

Although virus release from host cells and tissues propels the spread of many infectious diseases, most virus particles are not infectious; many are defective, lacking essential genetic information needed for replication. When defective and viable particles enter the same cell, the defective particles can multiply while interfering with viable particle production. Defective interfering particles (DIPs) occur in nature, but their role in disease pathogenesis and spread is not known. Here, we engineered an RNA virus and its DIPs to express different fluorescent reporters, and we observed how DIPs impact viral gene expression and infection spread. Across thousands of host cells, co-infected with infectious virus and DIPs, gene expression was highly variable, but average levels of viral reporter expression fell at higher DIP doses. In cell populations spatial patterns of infection spread provided the first direct evidence for the co-transmission of DIPs with infectious virus. Patterns of spread were highly sensitive to the behavior of initial or early co-infected cells, with slower overall spread stemming from higher early DIP doses. Under such conditions striking patterns of patchy gene expression reflected localized regions of DIP or virus enrichment. From a broader perspective, these results suggest DIPs contribute to the ecological and evolutionary persistence of viruses in nature.

BackgroundUpon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread. These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.ResultsWe have developed an antiviral assay based on replication inhibition of an engineered fluorescent vesicular stomatitis virus reporter strain on A549 human lung epithelial cells. Our assay provides a quantitative functional readout of human type I, II, and III interferon activities, and it provides better sensitivity, intra-, and inter-assay reproducibility than the traditional crystal violet based assay. Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement.ConclusionsA dsRed2-strain of vesicular stomatitis virus that is sensitive to type I, II, and III interferons was used to develop a convenient and sensitive assay for interferon antiviral activity. We demonstrate use of the assay to quantify the kinetics of paracrine antiviral signaling from human prostate cancer (PC3) cells in response to viral infection. The assay is applicable to high-throughput screening for anti-viral compounds as well as basic studies of cellular antiviral signaling.