92 Assessment of ANKLE2 as a mediator of Zika associated microcephaly in a murine KO model

Mutations in Ankle2 are known to cause microcephaly. Disruption of the gene’s function through protein-protein interactions with viral proteins then may lead to a similar phenotype. We have previously shown that mutations in Ankle2 affect cell division in the brain, causing microcephaly in Drosophila, and that ANKLE2 interacts with the ZIKV protein NS4A. Thus we hypothesize that the NS4A-ANKLE2 associations may explain how congenital ZIKV infection leads to microcephaly in mammals, and tested it in a novel murine genetic model. Timed matings of C57BL/6J Ankle2 -/+ and wild type controls enabled infection of pregnant females (n= 4 WT, 3 HT) with four doses of 1x104 PFU first passage ZIKV or a mock control (n= 3 WT, 1 HT) on embryonic days 6 through 9 (Fig A). To promote ZIKV susceptibility, an anti-IFNI1 antibody was administered S.Q. on days 5 and 7. Animals were monitored daily, and pups delivered by Caesarean section on day 18, and fetus’ examined. There was no significant difference in the number of liveborn pups (p= 0.3) between treated and control animals with an average of 8.3 (SD 1.8) live pups per litter with a range of 0 to 4 in utero demises per litter. However, a two way ANOVA revealed significant changes in pup weight by genotype (p<0.0001) but not by ZIKV infection with no significance of the interaction (Fig B). Ankle2 -/+ heterozygous pups were significantly smaller than WT regardless of maternal genotype (p<0.004, Tukey’s). H&E stain and preliminary examination of a subset of whole cross sections of pups revealed abnormalities observed in the cortex of ZIKV Ankle2 -/+ mouse pups but not in mock infected animals (Fig C). Taken together, this novel model enabled us to identify at least one putative target (ANKLE2) of congenital ZIKV-associated brain abnormalities which may lead to clinical microcephaly. Expansion of a Drosophila-based screen of viral proteins may identify other viral targets resulting in congenital malformations and provide opportunities for future pharmacologic interventions to prevent these malformations.