Abstract

Due to long-term chemoprevention, the risk of immune compromised patients to suffer from an infection of therapy-resistant herpes simplex virus (HSV) or adenovirus (Ad) is increasing. Therefore, fast and reliable tests for drug resistance of these viruses become more and more important in clinical praxis. Additionally, because of limitation of the therapeutic target range for antiviral therapy of HSV and Ad infections, there is an unmet need for new antivirals improving the infectious mortality in this patient group. Here, we constructed a new conditional expression system that is based on the adeno-associated virus (AAV) genome replication. This system provided an appropriate reporter system for replication of large DNA viruses, for testing their drug resistance and for screening of new antiviral substances. The multiplication of AAV is dependent on super-infection with other viruses such as herpes- or adenoviruses. The helper functions provided by these viruses induce the replication of the previously silent AAV turning its lytic gene expression on. We employed this natural switch to regulate the expression of a reporter gene, replacing the AAV Cap gene, depending on the infection with herpes or adenoviruses. By replacing the coding sequences for the structural AAV proteins with a Gaussia luciferase (GLuc) or a green fluorescence protein (GFP) open reading frame, we constructed a genetic element. This genetic element retained the extremely low basal activity in absence, and inducibility in the presence of helper virus infection. Instead of AAV production, reporter gene expression is turned on dependent on this infection. We coined this genetic element as AAV replicon in analogy to similar virus replication-based reporter systems, which were established for assessing RNA virus replication. After successful introduction of the AAV replicon into permissive cells the reporter gene expression was specifically activated upon infection with different human Ad serotypes, HSV 1, HSV 2, and human cytomegalovirus (HCMV). Almost no induction of the signal was measured without infection. In this study, we characterized the responsiveness of the AAV replicon system using different delivery methods and target cell lines and tested feasibility of different applications such as resistance testing, trans-complementation and drug screening. A fast diagnosis of drug resistance of patient isolates towards certain antiviral drugs is absolutely essential for the decision on effective therapy. Therefore, a phenotypical drug resistance test using the AAV replicon vector system was established for HSV. By testing 21 clinical isolates of HSV we showed that the AAV replicon-based test can differentiate between acyclovir sensitive and resistant strains already after 24 hours (for HSV-1) and 48 hours (for HSV-2) after virus isolation, compared to the gold standard plaque reduction assay, which takes 5-7 days. Furthermore, the AAV replicon vector system was used to generate a drug susceptibility test system, which can be applied in a high-throughput screening format. In a first approach, 24 kinase inhibitors were tested for their ability to inhibit Ad and HSV. The system was reproducibly detecting known inhibitors and could even identify new interesting compound groups.