The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2′-C-methyladenosine, 2′-C-methyladenosine, and 4′-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence. IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia for which there is currently no specific therapy. The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of TBEV and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. In our study, we performed serial in vitro passaging of TBEV in the presence of increasing concentrations of galidesivir (up to 50 μM), which resulted in the generation of two drug-resistant TBEV mutants. The first TBEV mutant was characterized by a single amino acid change, E460D. The other carried two amino acid changes, E460D and Y453H. Both mutations mapped to the active site of the viral RNA-dependent RNA polymerase (RdRp). Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2′-C-methyladenosine, 2′-C-methyladenosine, and 4′-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. By contrast, infection with wild-type virus resulted in fatal infections for all animals. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.

19The adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has 20 entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under 21 clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also 22 inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other 23 medically important flaviviruses. Until now, studies of this antiviral agent have not yielded 24 resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of 25 TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell 26 culture. Stochastic molecular simulations indicate that the steric freedom caused by the 27 E460D substitution increases close electrostatic interactions between the inhibitor and the 28 interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an 29 incorrect/modified nucleotide. Galidesivir-resistant TBEV exhibited no cross-resistance to 30 structurally different antiviral nucleoside analogues, such as 7-deaza-2´-C-methyladenosine, 31 2´-C-methyladenosine and 4'-azido-aracytidine. Although, the E460D substitution led only to 32 a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly 33 attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. 34Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, 35 flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to 36 flavivirus neurovirulence. 37 38 attenuation 40 41 Importance 42Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections 43 in large areas of Europe and Asia and for which there is currently no specific therapy. We 44 have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, 45which is under clinical development for treatment of Ebola virus infection, has a strong 46 antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant 47 mutants to understand how the drug works. Here, we produced TBEV mutants resistant to 48 Galidesivir and found that the resistance is caused by a single amino acid substitution in an 49 3 active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for 50 replication of viral RNA genome. Although, this substitution led only to a subtle decrease in 51 viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse 52 model. Our results contribute to understanding the molecular basis of Galidesivir antiviral 53 activity. 54 4 55 Introduction 56Flaviviruses (family Flaviviridae, genus Flavivirus) cause widespread human morbidity 57 and mortality throughout the world. These viruses are typically transmitted to humans by 58 mosquito or tick vectors. Tick-borne encephalitis virus (TBEV) is a typical flavivirus 59 transmitted by Ixodes spp. ticks. TBEV is a causative agent of tick...