138 these data suggested that ZikV causes a symptomatic but transient meningoencephalitis in 139 neonatal mice.
140 Increased levels of fluid in CNS and reduced cerebellum volume in convalescent mice. 141 While ZikV-infected mice older than 30 days showed no obvious tremors, ataxia or seizures, the 142 extensive tissue disruption during acute disease suggested that the transient infection could 143 result in permanent loss of cerebral mass or calcifications paralleling the lesions described for 144 exposed human offspring. To evaluate whether the infection led to permanent structural 145 damage, we performed magnetic resonance imaging (MRI) of infected mice at 60 dpi ("young"), 146 4-6 ("adult"), and 12-14 ("aged") months post-infection (mpi). As shown in Figure 2 , at 60 dpi 147 the overall volume of the brain was smaller compared to controls, consistent with recently 148 published findings (19) . Of note, as the mice age and catch-up in body weight (Supplementary 149 Figure 3 ), the differences in total brain volume relative to age-matched controls became less 150 evident suggesting that the tissue repairs over time (Figure 2B ). However, convalescent 151 animals showed increased percentage fluid (as indicated by white) in the CNS, especially in and 152 around cerebellum and a reduction in cerebellar volume that was evident at all the time points. 153 This fluid increase appears to be associated with expansion of the 4 th ventricle and/or the 154 thickness of the pia layers surrounding the cerebellum but may also reflect changes to the 155 microstructural composition of the tissue. Therefore, although animals that survived neonatal 156 ZikV infection with PRVABC059 do not exhibit overt microcephaly, they do have reduced 157 cerebellar volume and increased cerebellar fluid. Importantly, multiple studies have shown 158 that defects in cerebellum can affect not only balance and coordination, but language, emotion, 159 social behavior and working memory as well (25, 26). 161 Despite the reduction in cerebellar volume, the behavior of convalescent mice in their home 162 cage environment was grossly indistinguishable from age-matched control mice. To examine 163 more closely whether the early infection resulted in functional sequelae, we tested locomotor 164 activity, as well as social, emotional and learning behavior of convalescent mice in three age 165 groups: "young adult" (60 dpi), "adults" (4-6 months post infection (mpi)) and "aged" (> 1-year 166 post infection (ypi)) mice. All animals were exposed to the same battery of 5 tests, under the 167 same conditions and in the same environment, by a blinded researcher. The rotarod test, 168 which examines motor function and coordination ( Figure 3A ) showed a significant reduction in 169 latency to fall among ZikV-infected mice for all ages compared to age-matched controls, 170 revealing an impairment in motor coordination and endurance (U = 253; p < 0.0001; Figure 3 ). 171 Assessment of motility and exploratory behavior using the Open Field Test (OFT) showed that 172 total horizontal motor activity and velocity were increased in ZikV-infected mice regardless of 173 age, suggesting a hyperactive phenotype (U = 409, p = 0.005; Figure 3B ). The increased mobility 174 was more pronounced in younger mice (p<0.005), but evident at all stages (p<0.05).
175 Interestingly, while convalescent mice covered more distance in the OFT, they did not spend Figure 6A ). Further, CD45 + immune cells were found in proximity to and within these foci, 226 which were surrounded by GFAP + astrocytes and rounded Iba-1 + activated microglia (Figure 5 227 and supplementary Figure 6A) . Surprisingly, similar apparently active foci of infection could be 320 Further, we show that while the most severe tissue damage appears to occur during acute 321 infection, the brain parenchyma in convalescent mice harbors low levels of replicating virus 322 localized in small foci with active apoptotic processes and cellular infiltrates that are 323 surrounded by activated astrocytes and microglia. The lesions are associated with a pattern of 324 gene expression that indicates a continuous inflammatory milieu as late as a year post-325 infection. Together, these data suggest that subjects exposed to Zika early in development may 326 not only experience long-term sequelae associated with the tissue damage that occurs during 327 acute infection, but also harbor live replicating virus in their CNS that leads to continuous local 328 inflammation and may worsen their long-term prognosis.
329 The most severe cases of CZS can include severe malformations of the brain (microcephaly, 330 simplified gyral pattern, cerebellar hypoplasia, calcifications, ventriculomegaly), eyes 331 (microphthalmia), and limbs and are diagnosed in 5-10% of children exposed in utero early in 332 pregnancy. Further, there is mounting evidence that some infants exposed to the virus who do 560 Each group underwent 1 test per day. For adult and aged groups, there was a six-week delay 561 between NOR and EPM tests due to equipment availability. All mice were tested blindly so the 562 tester did not know the age or prior infection status of the mice. 