Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model

Abstract

<jats:p>Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an<jats:italic>in silico</jats:italic>model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.</jats:p>

Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection

“…E1A starts the viral gene expression and genome replication programs, which drive viral immune escape and, ultimately, the release of progeny viruses from the nucleus upon cell lysis (26), yet the clinical oncolytic efficacy of adenoviruses and other virus-derived oncolytic vectors has been modest (27,28). This is paralleled by a recent observation from 2-dimensional cell cultures showing that inefficient viral transmission correlates with low-level lytic infection events (29). The results presented here show that induction of the UPR through the inositol-requiring enzyme 1 (IRE-1) sensor and the X box binding protein 1 (XBP-1) transcription factor leads to enhanced viral cytotoxicity in primary human cancer cells.…”

“…Viral oncolysis also crucially depends on efficient intratumoral transmission of the oncolytic agents and the ability of the virus to overcome innate immunity (67). Notably, the spreading of HAdV-C occurs by cell-free viruses after lysis of infected cells, yet spreading and oncolysis are limited both in cell cultures and in organisms (29,68). Our data raise the possibility that viral oncolysis can be chemically tuned by manipulating the UPR and that this can be applied for cancer treatment.…”

“…The involvement of the IRE-1/XBP-1 branch in the GCA infection boost was further tested by live cell assays measuring the spread of infection, where comets of infected cells are formed by replicating HAdV-C2-dE3B_GFP (29). Comets are the equivalent cells.…”

“…DAPI stain was used to mark the cell nucleus, and a custom-made script (Matlab; Mathworks, USA) or a custom-made CellProfiler (version 2.0) pipeline (as described previously [43]) was used to quantify the average nuclear intensity of the GFP and protein VI signals, which were used as measures of infection efficiency. The spreading of HAdV-C2-dE3B_GFP was analyzed by time-lapse fluorescence microscopy as described previously (29). A detailed description of the imaging procedures is available on request.…”

“…Nonreplicating HAdV-C5-dE1_GFP (30,32) and HAdV-B3-dE1_GFP (39) containing GFP under the control of the cytomegalovirus (CMV) major immediate early promoter were grown in 911 cells. The formation of HAdV-C2-dE3B_GFP progeny was as described previously (29,40). Cell viability was measured by resazurin as described previously (41).…”