Granular cytoplasmic inclusions in astrocytes and microglial activation in the fetal brain of pigtail macaques in response to maternal viral infection

The fetal origins of neuropsychiatric disorders are poorly understood but have been linked to viral or inflammatory injury of the developing brain. The fetal white matter is particularly susceptible to injury as myelination, axonal growth, and deep white matter tracts become established. We have used the pigtail macaque (Macaca nemestrina) to study the maternal and fetal effects of influenza A virus (FLUAV) and Zika virus (ZIKV) infection during pregnancy, in cohorts with different time intervals between inoculation and delivery. We observed a striking histopathological alteration in a subset of astrocytes which contained granular cytoplasmic inclusions ("inclusion cells", ICs) within a specific region of the deep cerebral white matter in the fetal brains from specific FLUAV and ZIKV cohorts. Immunohistochemical and ultrastructural characteristics of ICs indicated that they are astrocytes (GFAP+) undergoing autophagocytosis (p62+) with activated lysosomes (LAMP1+, LAMP2+) and reactive changes in neighboring microglia. There was also a positive correlation between the number of ICs and LAMP1 or LAMP2 immunoreactivity in the fetal brain (LAMP1: rho 0.66; LAMP2: rho 0.54, p < 0.001 for both). Interestingly, ICs were significantly more prevalent in the 5-day FLUAV cohort and the 21-day intermediate ZIKV cohort than in controls (p < 0.005 and p = 0.04, respectively), but this relationship was not apparent in the ZIKV cohort with a shorter (2-3 days) or longer (months) time course. Virologic and immunologic assays indicated that the appearance of these cells was not linked with fetal brain infection. ICs were not observed in a macaque model of perinatal hypoxic ischemic encephalopathy. These alterations in fetal white matter are pathologically abnormal and may represent a transient neuropathologic finding that signifies a subtle brain injury in the fetus after maternal viral infection.