Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model

Abstract

Zika virus (ZIKV) is currently undergoing pandemic emergence. While disease is typically subclinical, severe neurologic manifestations in fetuses and newborns after congenital infection underscore an urgent need for antiviral interventions. The adenosine analog BCX4430 has broad-spectrum activity against a wide range of RNA viruses, including potent in vivo activity against yellow fever, Marburg and Ebola viruses. We tested this compound against African and Asian lineage ZIKV in cytopathic effect inhibition and virus yield reduction assays in various cell lines. To further evaluate the efficacy in a relevant animal model, we developed a mouse model of severe ZIKV infection, which recapitulates various human disease manifestations including peripheral virus replication, conjunctivitis, encephalitis and myelitis. Time-course quantification of viral RNA accumulation demonstrated robust viral replication in several relevant tissues, including high and persistent viral loads observed in the brain and testis. The presence of viral RNA in various tissues was confirmed by an infectious culture assay as well as immunohistochemical staining of tissue sections. Treatment of ZIKV-infected mice with BCX4430 significantly improved outcome even when treatment was initiated during the peak of viremia. The demonstration of potent activity of BCX4430 against ZIKV in a lethal mouse model warrant its continued clinical development.

Keywords: AG129 mice; Antiviral; BCX4430; Flavivirus; Ribavirin; ZIKV; Zika virus.

Figure 1

Reduction of virus yield by BCX4430 or Ribavirin in Vero76 (a), Huh-7 (b) and RD (c) cells as determined using a single test in each cell line.

Figure 2

Survival of ZIKV-infected (n=9) or sham-infected (n=4) AG129 mice (a). Weight change of individual mice measured between 0 and 21 dpi (b) and disease signs (c) of AG129 mice infected with ZIKV. Time-course of ZIKV RNA in serum (d), spleen (e), brain (f), liver (g), kidney (h), urine (i), testis (j) and uterus (k) collected at various times after infection.

Figure 3

Histopathologic lesions of ZIKV-infected mice include neutrophilic encephalitis with neuronophagia (a, arrow) and neuronal necrosis (b, arrows) in the brain stem of infected mice. Neutrophilic myelitis (c, arrow) with neuronal necrosis (c, arrowhead) was observed in spinal cord sections. Motor neurons (d, arrow) stained green with NeuN, a neuronal marker, in the ventral horn of spinal cord were infected with ZIKV. Astrocytes in the brainstem (e, arrows), identified by staining with the astrocyte marker GFAP, co-localized with ZIKV. No ZIKV was detected in brain stem sections of a sham control mouse (f) co-stained with anti-ZIKV and anti-GFAP antibodies (scale bar = 50μm). An infectious virus assay was performed using immunohistochemical staining for ZIKV after co-cultivation of tissue homogenates with the indicator Vero 76 cells to verify the presence of ZIKV. Male reproductive tissues, including epididymis (g) and testis (h) were positive for ZIKV, particularly in the Leydig cells (h, arrow). Testis from sham-infected males were included (i). Homogenates from testis (j), brain (k) and kidney (l) were assayed. Kidney homogenates from a sham-infected mouse served as negative controls (m) and ZIKV-infected Vero 76 cells served as a positive control for the assay (n).

Figure 4

BCX4430 significantly improved survival (a), time course weight change between 0 and 19 dpi (b), weight change between 7 and 13 dpi (c) and viral RNA in pooled (n=4 per data point) serum on 5 dpi (d) in ZIKV-infected (n=8) or sham-infected (n=3) AG129 mice, while ribavirin treatment did not improve any disease parameter. Surviving mice (n=7) from the initial treatment study with BCX4430 (a) were re-challenged with ZIKV 28 days after the initial viral challenge. Naïve mice that were sham-infected in the first study and treated with either BCX4430 (n=3) or vehicle (n=2) were also challenged. Surviving mice did not succumb to the secondary challenge, while naïve animals had a similar mortality curve to the initial study (e). High neutralizing antibody titers were present just prior to re-challenge in the serum of mice that were treated with BCX4430 (n=7) and survived the initial virus challenge (f). Treatment with BCX4430 was initiated at various times after ZIKV challenge. Survival (g), average weight change between 0 and 28 dpi (h), individual weight change between 7 and 13 dpi (C), mean weight change between 0 and 28 dpi (i), viral RNA from serum on 5 dpi (j) and disease signs (k) observed in BCX4430-treated (n=8) or vehicle treatment controls (n=10) infected mice or uninfected, untreated normal control (n=3) mice were used as disease parameters. Treatment beginning as late as 7 days after virus challenge provided some benefit to ZIKV-infected mice, although survivors were only observed in mice treated beginning 1 or 3 days after ZIKV challenge (***P<0.001, **P<0.01, *P<0.05, as compared with vehicle control).