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Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC50 values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV–envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 108 copies/mL in vehicle control. The dose–response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC50 values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC90 values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.