INTRODUCTION 46 growing threat to public health, but at present no approved antiviral therapies are available to 48 treat any flaviviral infection (1-3). Drug repurposing offers the fastest route to move anti-49 flaviviral drugs into the clinic (4), and to this end we have recently demonstrated that three FDA-50 approved fluoroquinolones, enoxacin, difloxacin, and ciprofloxacin, suppress replication of six 51 flaviviruses, including DENV and ZIKV, in cultured HEK-293 (human embryonic kidney) cells 52 (5). Additionally, we found that treatment with enoxacin suppressed replication of ZIKV in the 53 testes of interferon-deficient A129 mice in vivo, although it did not impact ZIKV titer in the 54 serum, brain, or liver in these mice (5). Further, Xu et al. (6) recently reported that enoxacin also 55 suppresses ZIKV replication in human neural progenitor cells (hNPCs) and brain organoids (6). 56
Thus, fluoroquinolones offer a promising candidate for a repurposed therapy for flavivirus 57 infections. However, several key aspects of the fluoroquinolone-flavivirus interaction must be 58 elucidated before these drugs can move forward on the path to the clinic, particularly: (i) the 59 barrier to resistance to these drugs, (ii) patterns of cross-resistance among fluoroquinolones, (iii) 60 the fitness consequences of evolution of drug resistance, (iv) the mechanism(s)-of-action of 61 fluoroquinolones against flaviviruses. 62 6 mosquito vector (a trade-off) (51-53). As both fluoroquinolone-resistant and media-control 116 DENV-4 had been passaged in HEK-293 cells, we predicted that these lineages would both have 117 lower fitness in mosquitoes than the parent DENV-4, and that the ciprofloxacin-resistant virus 118 would be more disadvantaged than the media-control virus. In sum, these studies seek to move 119 the evaluation of FDA-approved drugs for antiviral repurposing beyond the demonstration of 120 efficacy to consider the consequences of resistance. 121 122
Dengue virus serotype 4 (rDEN-4 Dominica p4) was derived from full-length clone p4 125 (54) and passaged four times in Vero cells. Replicate vials of a working stock were collected in 126 1X SPG (2.18 mM sucrose, 38 mM potassium phosphate [monobasic] , 72 mM potassium 127 phosphate [dibasic] , 60 mM L-glutamic acid), clarified by centrifugation, and stored at -80 °C. 128 HEK-293 (human embryonic kidney) cells were obtained from ATCC (CRL-1573); Vero, HuH-129 7, and C6/36 cells were obtained from the lab of Dr. Stephen Whitehead (NIAID, NIH). HEK-130 293 cells were maintained at 37°C with 5% CO2 in Dulbecco's minimum essential medium 131 with 5% CO2 in MEM supplemented with 10% heat-inactivated FBS, 2mM L-glutamine, 2mM 139 nonessential amino acids (Gibco), and 0.05 mg/mL gentamycin. Viral titers were determined via 140 serial dilution onto HEK-293, Vero, HuH-7 or C6/36 cells followed by immunostaining using 141 previously described methods (5,55,56). Briefly, each virus was serially diluted ten-fold and 142 inoculated onto confluent cells in 24-well plates. After two hours of incubation at 37°C with 143 occasional rocking, infected cells were overlaid with 1% methylcellulose in OptiMEM (Gibco) 144 that had been supplemented with 2% FBS, 2mM L-glutamine, and 0.05 mg/mL gentamycin. were collected on the day of infection as well as 1, 2, 3, 4, 6, and 8 days post-infection (dpi). For 262 replication curves with HuH-7 and Vero cells, the time points for collection were extended to 263 include 9, 10, 11, and 12 dpi as the plateau viral titer was not detected by 8 dpi. At each time 264 point, 1 ml of the media was removed from each flask and 1 ml of fresh media was added back 265 to each flask. The viral supernatants were clarified by centrifugation at 1200 rpm for 10 minutes 266 at 4˚C, stored at -80 °C in 1X SPG, and viral titers were determined using the same cell line as 267 that used for the replication dynamics (HEK-293, HuH-7, Vero, or C6/36). 268 269
To detect differences in virion stability between the ciprofloxacin-resistant and media 271 control DENV-4, 7 T25 flasks of 80% confluent HEK-293 cells were infected at MOI: 0.1 with 272 each of the ciprofloxacin-resistant DENV-4 or media control DENV-4 lineages (both from 273 passage seven) or the parent DENV-4. After 2 hours of incubation with occasional rocking, the 13 flasks were washed with 1X PBS and 5ml of cell-specific media was added to each flask. On the 275 expected day of peak viral titer. day 3, the viral supernatants were collected on ice then clarified 276 by centrifugation at 1200 rpm for 10 minutes at 4˚C. Immediately after centrifugation, 45 µL of 277 each clarified supernatant was added to cryotubes with 405 µL Leibovitz L15 media (Gibco, Life 278
Technologies, Grand Island, NY) and placed into a 37 °C incubator, one tube for each virus and 279 for each time point (0 hours post-incubation, 2, 4, 6, 8, 12, 24, and 30). At each time point, the 280 cryotube was removed from the incubator and 50 µL of 10X SPG was added before the tube was 281 snap frozen on a dry ice-methanol bath. Frozen samples were stored at -80 °C and viral titers 282 were determined using HEK-293 cells as described above. 283 284
Eggs from Aedes aegypti (Rockefeller strain) were hatched and reared as described in 286 (66). Infectious bloodmeals were prepared and mosquito feeding was conducted as described in 287 (56). Briefly, 1 mL of virus was mixed with 2 mL of washed red blood cells from defibrinated 288 rabbit blood (Hemostat, Dixon, CA, USA) in 10% sucrose; 75 µl of 120 mM ATP was added to 289 each meal immediately prior to feeding. Sealed cartons containing ~30 mosquitoes were starved 290 for 24 hours, placed under parafilm-covered feeders warmed to 37°C, and allowed to feed for 20 291 minutes. Engorged mosquitoes were separated into new cartons and incubated at 27 °C with 292 80% relative humidity on a 12-hour light and 12-hour dark cycle. The mosquitoes were provided 293 10% sucrose in cotton pledgets ad libitum. After 10 days, mosquitoes were cold-killed and 294 stored at -80 °C, after which whole mosquitoes were homogenized in Hank's balanced salt 295 solution (Gibco) supplemented with 10% FBS, 250µg/mL amphotericin B (Gibco), 1% ciprofloxacin, and 150 µg/mL clindamycin and viral titer was determined as described above in 297 C6/36 cells with the addition of 5 µg/mL amphotericin B to the methylcellulose overlay media. 298 299
Viral titer data were log transformed, assessed for normality and then compared using t-301 tests or two-way ANOVAs as appropriate. Repeated measures ANOVA was used to detect 302 differences in replication kinetics and percent change in viral titer from the stability assay 303 between the ciprofloxacin-resistant and media control viruses. The parent DENV-4 was not 304 included in the repeated measures ANOVA as there was only one parental replicate. Differences 305 in variant frequency, Shannon entropy, and mosquito mortality (%) were detected by ANOVA. 306 Differences in variant frequency within genes and between treatments were identified using a 307 two-way ANOVA. Differences in the counts of mosquito bodies positive for DENV-4 infection 308 was detected using contingency table analyses. Tukey-Kramer or pairwise t-tests, as specified, 309 were used to ascertain post hoc pairwise differences. Statistics were conducted in R using 310 packages: tidyverse, plyr, dplyr, lme4, car, emmeans, lsmeans, and pbkrtest. 311
In this study, resistance was defined as a lack of a difference between the mean viral 315 titers of the media-passaged control viruses cultured in media and viruses passaged in a specific 316 fluoroquinolone cultured in the presence of that fluoroquinolone. After seven passages with 317 increasing concentration of ciprofloxacin (Figure 1a, 1b) , DENV-4 resistance to ciprofloxacin 318 was detected (Figure 2a ). An interaction between the passage condition (media or ciprofloxacin) and resistance test condition (media or ciprofloxacin) was found with a two-way ANOVA (F 320 (1,8): 40.7, P = 0.0002). Treatment of both media-passaged virus and ciprofloxacin-passaged 321 virus with 39.4µM ciprofloxacin (2x the EC50) resulted in a significant decrease in titer for both 322 virus lineages relative to treatment with media (Tukey pairwise comparisons, P < 0.05), but the 323 decrease in viral titer of the media-control viruses after ciprofloxacin treatment was greater than 324 that of the ciprofloxacin-passaged viruses (4.2 log decrease vs 1.9 log decrease). There was no 325 significant difference in mean viral titers between the media-passaged viruses treated with media 326 and the ciprofloxacin-passaged viruses treated with ciprofloxacin (Tukey pairwise comparison, P 327 < 0.05). In the absence of ciprofloxacin, replication of the ciprofloxacin-passaged viruses was 328 not different than the media-passaged viruses (Tukey pairwise comparison, P > 0.05), and in the 329 presence of ciprofloxacin, the ciprofloxacin-passaged viruses replicated to significantly higher 330 levels (2.8 log increase) than the media-passaged viruses (Tukey pairwise comparison, P < 0.05). 331 After ten passages (Figure 1c the concentration of difloxacin was tested ( Figure S1 ). In the resistance test with the lower 334 concentration of difloxacin, an interaction was detected between passage and resistance test 335 conditions using a two-way ANOVA (F (1,8) = 13.7, P = 0.006) (Figure 2b ) that was absent in 336 the resistance test at the higher concentration of difloxacin (two-way ANOVA F (1,8) = 0.07, P = 337 0.8) ( Figure S1 ). When the media-passaged viruses were treated with 10.1 µM difloxacin a 338 significant reduction in viral titer occurred compared to the media treatment that was not 339 detected when the difloxacin-passaged viruses were treated with difloxacin or media (Figure 2b ; 340 Tukey pairwise comparison, P < 0.05). Media and difloxacin-passaged viruses were both not a significant difference between the mean viral titers of the media-passaged viruses treated 343 with media and the difloxacin-passaged viruses treated with difloxacin ( Figure 2b ; Tukey 344 pairwise comparison, P > 0.05). 345
After DENV-4 was passaged 10 times in the presence of enoxacin (Figure 1e , 1f) the 346 interaction between the passage condition (media or enoxacin) and resistance test condition 347 (media or enoxacin at 3.8 µM or 7.6 µM) was not significant (two-way ANOVA F (1,2): 1.9, P = 348 0.19; Figure 2c ). The mean viral titer for media-passaged viruses treated with media was not 349 significantly different from the mean viral titer for the enoxacin-passaged viruses treated with 3.8 350 µM, or half the EC50, but was significantly different from the enoxacin-passaged viruses treated 351 with the EC50 for enoxacin (7.6 µM) (Tukey pairwise comparison, P < 0.05; Figure 2c ). These 352 data indicate a trend toward enoxacin resistance but did not satisfy our a priori criteria for 353 resistance. 354
Only two coding mutations became predominant (> 50% of all reads) in the 357 ciprofloxacin-resistant but not in the media control viruses; these included a V15L mutation in 358 domain I of the envelope glycoprotein (E) in two replicates of ciprofloxacin-resistant viruses and 359 an E417A mutation in domain III of E in one replicate of the ciprofloxacin-resistant viruses 360 (Table 1) . One of these mutations, E417A, was also found in one replicates of difloxacin-361 resistant viruses (Table 2 ). Other coding mutations in the E, NS2B and NS4B genes and non-362 coding mutations in the untranslated regions (UTRs), many of which had been previously 363 identified in the literature as HEK-adapting mutations, were found in at least a subset of both 364 drug-resistant and control viruses (Table 1) Table S1 . 367
To assess amino acid conservation across the flaviviruses at positions 15 and 417 in E, 368
we generated a multiple sequence alignment of mosquito-borne, tick-borne, no known vector, 369 and insect-only flaviviruses ( Figure S2 frequency of the ciprofloxacin-resistant populations was higher than that of the parent in the 5' 392 UTR (5.1-fold increase), envelope (12-fold increase), and NS4B (3.8-fold increase), but was 393 only also significantly higher than the media control in NS4B (2.1-fold increase) ( Figure 5 , Table  394 S2). The mean variant frequency for the ciprofloxacin-resistant populations was also higher than 395 the media control population in NS2B but was not different than the parent ( Figure 5 , Table S2 ). 396 397
As shown in Figure lineage and treatment in the resistance test was detected using a two-way ANOVA (F (2,12) = 404 8.7, P = 0.005). Viral replication for both lineages decreased after treatment with enoxacin and 405 difloxacin at their EC50 values, 7.6µM and 10.1µM, respectively (Tukey pairwise comparisons, P 406 < 0.05). However, after difloxacin treatment, the ciprofloxacin-resistant DENV-4 replicated to 407 the same level as the media control viruses treated with media. Viral replication after treatment 408 with enoxacin remained 14x lower than the media control viruses treated with media, a highly 409 significant difference (t = -4.3, df = 4. P = 0.01). viral lineage and treatment in the resistance test was not significant (two-way ANOVA F (2,12) 412 = 0.8, P = 0.48; Figure 6b ). The mean viral titer for the viruses passaged with media and tested 413 against media were almost 100-fold higher than the mean viral titer for the difloxacin-resistant 414 viruses tested with enoxacin, indicating that enoxacin still suppresses replication of DENV-4 that 415 is resistant to difloxacin (Tukey pairwise comparison, P < 0.05). Yet, the mean viral titer of the 416 difloxacin-resistant viruses tested with ciprofloxacin was not different from the media-passaged 417 viruses treated with media demonstrating that DENV-4 resistance to difloxacin can result in 418 cross-resistance to ciprofloxacin Tukey pairwise comparison, P > 0.05). (Table 3 ; c 2 = 22.0, df = 2, P < 0.0001). Of the infected mosquitoes that fed on 468 bloodmeals spiked with parent virus, 45% of the heads were also infected with an average titer of 469 2.69 log10PFU/head (Table 3) We recently demonstrated the efficacy of three fluoroquinolones, ciprofloxacin, 475 difloxacin and enoxacin, against a panel of flaviviruses in cultured cells and A129 mice (5). In 476 the current study, we investigated the barrier to evolution of resistance and cross-resistance to 477 these drugs, their mechanisms-of-action, and the fitness consequences of resistance. DENV-4 was passaged in triplicate in the presence or absence of each drug until resistance was detected or 479 to a predetermined endpoint of 10 passages. 480
We found that DENV-4 evolved resistance to ciprofloxacin within seven passages and 481 difloxacin within ten passages but the virus failed to evolve resistance to enoxacin within ten 482 passages, suggesting a higher barrier to resistance against enoxacin than the other two drugs. 483
The variation in the rate of evolution of resistance could be due to stochasticity in evolution, 484 particularly as our definition of resistance required that all three replicates within a treatment 485 evolve resistance, but variation in resistance evolution could also reflect differences among the 486 drugs in in mechanism-of-action. In our previous study of these drugs, time-of-addition assays 487 with other antiviral compounds that are not similar in structure and do not share a mechanism-of-498 action may mitigate these concerns. 499
To gain further insight into the antiviral mechanism-of-action, we sequenced the whole parent virus. We discovered two mutations, both in the envelope glycoprotein, that were unique 502 to the ciprofloxacin-resistant viruses: V15L which arose in two replicates and E417A which 503 arose in one replicate. E417A was also detected in one replicate of the difloxacin-resistant 504 viruses, but neither mutation was detected in the enoxacin-passaged viruses. Ciprofloxacin-505 resistant viruses were sequenced by Illumina while difloxacin-resistant and enoxacin-passaged 506 viruses were analyzed via Sanger sequencing, thus it is possible that the mutations detected in the 507 ciprofloxacin-resistant lineages occurred in these two groups at frequencies below the level of 508 detection. Also, resistance to difloxacin was not as complete as resistance to ciprofloxacin, as 509 resistance was confirmed at the EC50 (10.1µM), but not at 2x EC50 (20. conditions, viral titers of ciprofloxacin-resistant viruses were greater than those of the media 566 control viruses in HEK-293 and HuH-7 cells. This gain in fitness in the absence of ciprofloxacin 567 contradicts our hypothesis that resistance evolved due to a specific drug evasion. Intriguingly, 568 replication of the three virus lineages were indistinguishable in Vero cells, a cell line that lacks 569 an interferon response (92,93). It is not clear from our data whether the gain in fitness seen in artifact of adaptation to human cells. Importantly, we also do not know how this gain in fitness 572 will impact infection in vivo. 573
Further, fitness of the ciprofloxacin-resistant and media-control viruses were equivalent, 574 but lower than that of the parent virus, in C6/36 cells, a mosquito cell line that lacks a functional 575
RNAi response (94) and both lineages failed to infect live Ae. aegypti mosquitoes fed on 576 artificial bloodmeals spiked with these viruses. The failure of the passaged viruses to infect 577 mosquitoes is likely due to the suite of mutations in NS4B that they accrued; some of these 578 mutations have previously been associated with mammalian adaptation and loss of mosquito 579 infectivity (40,70-73). This finding is consistent with the trade-off hypothesis, which states that 580 a gain in fitness in the mammalian host will negatively impact fitness in the mosquito vector 581 (51-53). Our data underscore the complexity of viral fitness and how the patterns are likely 582 influenced by a combination of drug resistance-associated and cell culture adaptation mutations. 583
Fluoroquinolones show some promise for repurposing as anti-flavivirals, but this study 584 also suggests several lines of inquiry that should be pursued to further evaluate the consequences 585 of evolution of fluoroquinolone resistance. While the barrier to resistance to enoxacin was 586 relatively high, DENV-4 evolved resistance to ciprofloxacin and difloxacin within ten passages 587 at or near the EC50 of each drug. Furthermore, resistance to either ciprofloxacin or difloxacin 588 confer cross-resistance to the other drug. Unexpectedly, mutations associated with resistance 589 occurred in the envelope gene of the virus; the phenotypic impact of these E mutations should be 590 formally assessed using reverse genetics in a future study. These mutations were associated with 591 substantial gains in virion stability and viral fitness, but the degree to which the increase in 592 fitness is attributable to the envelope mutations associated with resistance and how the increase 593 in fitness in human cells will play out in vivo remains unknown. FFuture experiments to 594 interrogate evolution of resistance to these fluoroquinolones in vivo are needed to better assess 595 their potential and perils as antiflaviviral therapies. 