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
Introduction on the host cells (Eyer et al., 2017a) . A Phase 1 clinical safety and pharmacokinetics study in 87 healthy subjects has been completed, and at present, Galidesivir is under clinical 88 development as an antiviral drug for treatment of Ebola virus infection (Taylor et al., 2016) . 89
The broad-spectrum antiviral activity makes this drug a promising candidate for 90 development of therapy not only for Ebola virus infection but also other important diseases 91 caused by various RNA viruses, including TBEV. 92
Although Galidesivir has been studied intensively and is known to inhibit a wide 93 range of RNA viruses, there are no published reports of resistance to this compound. frequency is estimated to be 10 −4 to 10 −6 errors per nucleotide (Lauring et al., 2013) . The 98 high mutation frequency and high replication rate of viral RNA copies enable the viruses 99 quickly to adapt to changes in the environment, including the introduction of antiviral drugs. 100
Identification of mutations conferring antiviral resistance provides information not only 101 7 fragments into permissive BHK-21 cells, the following recombinant TBEV strains were 151 successfully rescued: E460D-Rec (E460D substitution in the NS5 gene), Y453H-Rec (Y453H 152 substitution in the NS5 gene), and recombinant wild-type (no introduced mutations). The 153 presence of the E460D and Y453H substitutions in the viral genomes was confirmed by 154 whole-genome sequencing of all recombinant viruses. Despite repeated attempts, the 155 E460D/Y453H-Rec mutant (both mutations E460D and Y453H in the NS5 gene) was not 156 rescued from the transfected BHK-21 cell culture. E460D-Rec was found to be 7.9-fold less 157 sensitive to Galidesivir than engineered wild-type, showing an EC50 value of 6.32 ± 1.01 µM 158 (Table 2) . On the other hand, Y453H-Rec was highly sensitive to Galidesivir; the replication of 159 this mutant strain was completely inhibited at 12.5 µM, showing an EC50 value of 0.81 ± 0.01 160 µM (Table 2) . Thus, the results indicate, that the E460D (not Y453H) substitution is solely 161 responsible for the drug-resistant phenotype of TBEV ( Figure 2A ). 162
The E460D mutation has only a moderate effect on TBEV replication in cell culture 164
To characterize the phenotypic properties of the drug-resistant mutant in vitro, 165 growth kinetics ( Figure 2B ) and plaque morphology ( Figure 2C ) of the recombinant TBEV 166 mutant E460D-Rec were assayed in cultures of PS cells and compared with the wild-type 167 virus. The wild-type virus amplified in the absence of Galidesivir ( Figure 2B , red line), showed 168 a short lag-period within intervals 0 -12 hours p.i. Starting 24 hours p.i., the wild-type TBEV 169 exerted an exponential increase in virus infectivity reaching a peak titre of 3x10 6 PFU/mL 170 within 72 hours p.i. and gradually declining thereafter. In contrast, the presence of 171 Galidesivir (25 µM) completely inhibited replication of the wild type virus ( Figure 2B , violet 172 line). 173
In comparison with the wild-type virus, the E460D-Rec mutant cultured in the 174 absence of Galidesivir ( Figure 2B , blue line) showed a prolonged lag-period within intervals 0 175 -36 hours p.i. However, subsequently, the infectivity of the mutant virus increased 176 exponentially reaching a peak of 2.6x10 6 PFU/mL at 72 hours p.i. After that, the titre 177 gradually declined to 5.5x10 4 PFU/mL. The considerably longer lag-period of the E460D-Rec 178 mutant could be explained by a slightly decreased replication capacity (attenuation) of the 179 mutant, when amplified in PS cell culture. The decrease in replication capacity of E460D-Rec 180 was manifested particularly in the first few hours after cell culture infection and was no 181 longer detecable in the later stages of the infection.
The E460D-Rec mutant cultured in the prensence of Galidesivir (25 µM) ( Figure 2B , 183 green line) also exerted an extended lag-period within intervals 0 -36 hours p.i.; there was 184 even a moderate decrease in viral titers after 36 hours p.i. Starting 48 hours p.i., the mutant 185 showed an exponential infectivity and reached a peak titre of 3.9x10 5 PFU/mL at day 96 186 hours p.i. The results clearly indicate, that the resistance of TBEV to Galidesivir is only partial; 187 the growth of the mutant strain in the presence of Galidesivir was partially inhibited 188 compared to wild-type ( Figure 2B In general, mutant genotypes rapidly revert to the wild-type in the absence of the 227 selection agents. Indeed, after three passages in PS cells in the absence of Galidesivir a pool 228 comprising mutated and wild-type genotypes was detected. Moreover from passage 5 229 onwards, the wild-type genotype dominated in the infected PS cell culture ( Figure 1E ). 230
Interestingly, the codon GAT encoding an aspartic acid in the E460D mutant had changed to 231 the codon GAA in the revertant, not to GAG as seen in mock-selected wild-type virus; both 232 codons, GAA (in the revertant) and GAG (in the wild-type), are synonymous and encode a 233 glutamic acid residue ( Figure 1F ). In contrast, after 6 passages in the presence of Galidesivir, 234 at concentrations up to 25 µM, the E460D substitution was retained; even at low 235 concentrations of Galidesivir (6.25 µM) and did not result in reversion to the wild-type 236 genotype (data not shown). 237 238 Galidesivir migration towards the TBEV RdRp active site 239
The deduced position of Galidesivir bound within the TBEV RdRp active site is shown 240 in Figure 4A with its phosphate tail forming close contacts with the manganese cofactors. 241
The E460D (and Y453H) mutation is located at motif F of the flavivirus RdRp finger domain. 242
The flavivirus RdRp motif F is highly flexible and occludes the NTP-tunnel in the nucleotide 243 bound RdRp structure (Valdés et al., 2016) . Therefore, the apo-structure was used for 244 stochastic simulations to compare and contrast how Galidesivir approaches the TBEV active 245 site in the wild type and mutant RdRps. At the active site of the wild type TBEV RdRp, the 246 ribose of Galidesivir is at a 5.7 Å distance from amino acid (aa) position 460. The missing 247 carbon in Asp increases this distance (7 Å) compared to Glu. This increased distance, caused 248 by the mutation, results in more steric freedom for Galidesivir as it approaches the active 249 site ( Figure 4B,C) . 250
There is a ~6.7 Å distance cutoff by Galidesivir approaching the wild type TBEV RdRp 251 active site that is explored closer (<5 Å) in both mutant types ( Figure 4B ). The single RdRp 252 mutation, E460D, however, has lower enthalpic ligand binding values compared with the 253 double mutant, E460D/Y453H. These lower enthalpic values indicate a favorable ligand 254 binding and conformation. The distinct differences in Galidesivir exploration between the 255 wild type and mutant RdRps are shown by the average COM distance/enthalpy in Figure 4B , 256 specifically those by the single E460D mutation (wild type: 11.3 Å  4.6 Å/-179.9 kcal/mol  257 121.9 kcal/mol, E460D: 9.5 Å  4.9 Å/-257.3 kcal/mol  121.2 kcal/mol; E460D/Y453H: 10.6 Å 258 Data are expressed as means ± SD, and the significance of differences between 521 groups was evaluated using the Mann-Whitney U test or ANOVA. Survival rates were 522 analysed by log-rank Mantel-Cox test. All tests were performed using GraphPad Prism 5.04 523 (GraphPad Software, Inc., San Diego, CA, USA). P-values < 0.05 were considered to be 524 statistically significant. The reverse genetics method used in this study was based on the generation of infectious 707 Table 1 . Unmodified and mutated primers used to generate two overlapping amplicons of 720 fragment III (fragment IIIa and IIIb), i.e. to produce recombinant wild-type and mutated and cultivated at Galidesivir concentrations ranging from 0 to 50 µM for three days.