Repurposing FDA-approved compounds could provide the fastest route to alleviate the burden of disease caused by flaviviruses. In this study, three fluoroquinolones, enoxacin, difloxacin and ciprofloxacin, curtailed replication of flaviviruses Zika (ZIKV), dengue (DENV), Langat (LGTV) and Modoc (MODV) in HEK-293 cells at low micromolar concentrations. Time-of-addition assays suggested that enoxacin suppressed ZIKV replication at an intermediate step in the virus life cycle, whereas ciprofloxacin and difloxacin had a wider window of efficacy. A129 mice infected with 1 × 105 plaque-forming units (pfu) ZIKV FSS13025 (n = 20) or phosphate buffered saline (PBS) (n = 11) on day 0 and treated with enoxacin at 10 mg/kg or 15 mg/kg or diluent orally twice daily on days 1–5 did not differ in weight change or virus titer in serum or brain. However, mice treated with enoxacin showed a significant, five-fold decrease in ZIKV titer in testes relative to controls. Mice infected with 1 × 102 pfu ZIKV (n = 13) or PBS (n = 13) on day 0 and treated with 15 mg/kg oral enoxacin or diluent twice daily pre-treatment and days 1–5 post-treatment also did not differ in weight and viral load in the serum, brain, and liver, but mice treated with enoxacin showed a significant, 2.5-fold decrease in ZIKV titer in testes relative to controls. ZIKV can be sexually transmitted, so reduction of titer in the testes by enoxacin should be further investigated.

Genes in the family encode cytidine deaminases that provide a barrier against viral infection and retrotransposition. Of all genes in humans, () is the most polymorphic: some haplotypes encode stable and active A3H proteins, while others are unstable and poorly antiviral. Such variation in human A3H affects interactions with the lentiviral antagonist Vif, which counteracts A3H via proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions, as well as its evolution in Old World monkeys, we characterized A3H variation within four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of function was partially related to protein expression levels but was also influenced by amino acid mutations in the N-terminus. Moreover, we demonstrate that the evolution of A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests that activity of A3H is evolutionarily dynamic and may have a negative effect on host fitness, resulting in its recurrent loss in primates. Adaptation of viruses to their hosts is critical for transmission of viruses between different species. Previous studies had identified changes in a protein from the APOBEC3 family that influenced species-specificity of simian immunodeficiency viruses (SIVs) in African green monkeys. We studied the evolution of a related protein in the same system, APOBEC3H, which has experienced a loss of function in humans. This evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken place during primate evolution suggesting that APOBEC3H places a fitness cost on hosts. The variability of APOBEC3H activity between different primates highlights the differential selective pressures on the gene family.

177 16Importance: 104 17 Text: 5,544 18Figures: 6, Tables: 2 19 20 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/311878 doi: bioRxiv preprint first posted online May. 1, 2018; 2 Abstract 21Genes in the APOBEC3 family encode cytidine deaminases that provide a barrier 22 against viral infection and retrotransposition. Of all APOBEC3 genes in humans, 23 APOBEC3H (A3H) is the most polymorphic: some haplotypes encode stable and active 24 A3H proteins, while others are unstable and inactive. Such variation in human A3H 25 affects interactions with the lentiviral antagonist Vif, which counteracts A3H via 26 proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions 27 as well as its evolution in Old World monkeys, we characterized A3H variation within 28 four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic 29 in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of 30 function was partially related to protein expression levels but was also influenced by 31 amino acid mutations in the N-terminus. Moreover, we demonstrate that the evolution of 32 A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests 33 that activity of A3H is evolutionarily dynamic and may have a negative effect on host 34 fitness, resulting in its recurrent loss in primates. 35 36 Importance 37Adaptation of viruses to their hosts is critical for transmission of viruses between 38 different species. Previous studies had identified changes in a protein from the 39 APOBEC3 family that influenced species-specificity of simian immunodeficiency viruses 40 (SIVs) in African green monkeys. We studied the evolution of a related protein in the 41 same system, APOBEC3H, which has experienced a loss of function in humans. This 42 evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken 43 place during primate evolution suggesting that APOBEC3H places a fitness cost on 44

Antiviral therapies are urgently needed to treat infections with flaviviruses such as Zika (ZIKV) and dengue (DENV) virus. Repurposing FDA-approved compounds could provide the fastest route to alleviate the burden of flaviviral diseases. In this study, three fluoroquinolones, enoxacin, difloxacin and ciprofloxacin, curtailed replication of flaviviruses ZIKV, DENV, Langat (LGTV) and Modoc (MODV) in HEK-293 cells at low micromolar concentrations. Time-of-addition assays revealed that enoxacin suppressed ZIKV replication when added at 6 hours post-infection, suggesting inhibition of an intermediate step in the virus life cycle, whereas ciprofloxacin and difloxacin had a wider window of efficacy of 2, 6, and 8 hours post-infection for difloxacin and 2 to 8 hours post-infection for ciprofloxacin. The efficacy of enoxacin to suppress ZIKV replication in 5-week-old A129 mice was evaluated in two experiments. First, mice were infected with 1×105 plaque-forming units (pfu) ZIKV FSS13025 (n=20) or PBS (n=11) on day 0 and subsets were treated with enoxacin at 10mg/kg or 15mg/kg or diluent orally twice daily on days 1-5. Treated and control mice did not differ in weight change or virus titer in serum or brain. Mice treated with enoxacin showed a significant, 5-fold decrease in ZIKV titer in testes relative to controls. Second, mice were infected with 1×102 pfu ZIKV (n=13) or PBS (n=13) on day 0 and subsets were treated with 15mg/kg oral enoxacin or diluent twice daily on days 0 (pre-treatment) and 1-5. Mice treated with enoxacin showed a significant, 2.5-fold decrease in ZIKV titer in testes relative to controls, while weight and viral load in the serum, brain, and liver did not differ between treated and control mice. Enoxacin efficacy in cultured murine Sertoli cells was not enhanced compared to efficacy in HEK-293 cells. ZIKV can be sexually transmitted, so reduction of titer in the testes by enoxacin should be further investigated.Author SummaryFlaviviruses such as Zika and dengue virus pose a significant threat to public health worldwide, and there are currently no antiviral therapies to treat any flaviviral infection. Repurposing FDA-approved drugs as anti-flaviviral therapies can accelerate clinical use. We demonstrated that fluoroquinolone antibiotics exhibit anti-flaviviral efficacy, suppressing flavivirus replication in cultured human cells. Additionally, we found that the fluoroquinolone enoxacin suppressed Zika virus replication in mouse testes. While Zika virus is primarily transmitted via mosquitoes, the virus also undergoes sexual transmission. The importance of sexual transmission for the overall epidemiology of the virus remains unclear; nonetheless all routes of potential transmission to pregnant women are of concern as fetal infection in utero can have devastating effects. Thus, our data indicate that fluoroquinolones hold promise for treatment of flaviviral infections, particularly infection of the testes by Zika virus, and that this class of drugs warrants further study.