Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrAwas addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrAwt-treatedmice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-alpha R (IFNAR)(-/-) animals were additionally treated with anti- TNF-alpha (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR(-/-) and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-alpha-treated IFNAR(-/-) animals. No effect of AdrA wt was seen in STING- deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-alpha are both required and act synergistically.