SARS‐CoV‐2‐associated neuropathology in non‐human primates

Rutkai, Ibolya; Mayer, Meredith; Hellmers, Linh; Ning, Bo; Huang, Zhen; Monjure, Christopher; Coyne, Carol; Silvestri, Rachel; Golden, Nadia; Hensley, Krystle; Chandler, Kristin; Lehmicke, Gabrielle; Bix, Gregory; Maness, Nicholas; Russell‐Lodrigue, Kasi; Hu, Tony; Roy, Chad; Blair, Robert; Bohm, Rudolf; Doyle‐Meyers, Lara; Rappaport, Jay; Fischer, Tracy

SARS‐CoV‐2 infection impacts multiple organ systems, including the central nervous system (CNS). Multiple reports have described a variety of neurological manifestations associated with infection that may contribute to worsening COVID‐19. The neuropathology of SARS‐CoV‐2 is not well understood, necessitating the development of relevant animal models for investigation. Here, we report marked neuropathology but with limited virus in the CNS of two non‐human primate models (NHPs) of SARS‐CoV‐2 infection. Adult male and female purpose‐bred Rhesus macaques (RMs; n = 4) and wild‐caught African green monkeys (AGMs; n = 4) were inoculated with the 2019‐nCoV/USA‐WA1/2020 strain of SARS‐CoV‐2 via multi‐route mucosal or aerosol challenge. SARS‐CoV‐2 nucleocapsid (SARS‐N) mRNA was detected in nasal swabs within the first week of inoculation, demonstrating infection of all study animals. All animals were euthanized at the study endpoint of 4 weeks post‐inoculation, with the exception of two AGMs that reached humane endpoints at 8‐ and 22‐days post‐challenge. Seven regions of the CNS were investigated for pathology and virus infection. Archival brain tissues from two non‐infected adult female RMs were used as aged‐matched controls. Mild, but chronic, hypoxemia with impaired gas exchange were suggested by SpO(2) values that stayed at or below 95% and elevated blood CO(2) in the majority of the study animals. Neuroinflammation was seen throughout the brain and brainstem but with limited virus detection by immunohistochemistry and RNAscope of fixed tissues and viral RNA detection using a highly sensitive CRISPR‐fluorescent detection system on RNA extracted from sectioned brain lysates. In addition, neuronal injury and death were suggested by pyknotic and karyolytic nuclei and cellular blebbing. Limited myelin vacuolation was revealed in two infected animals through Luxol Fast Blue staining. Neuronal cleaved caspase 3 positivity was seen at a greater frequency in infected animals compared to controls, suggesting increased apoptosis in infection. Microhemorrhages were larger and more frequent among infected NHPs, as compared to controls. Neuroinflammation, neuronal injury and death, and microhemorrhages were seen in animals that did not develop severe respiratory disease and may suggest neuropathology contributes to on‐going symptoms of convalesced patients. Our findings in NHPs are in agreement with human autopsy and neuroimaging studies and demonstrate this is a relevant animal model for investigating neuropathological changes associated with COVID‐19. Our results also suggest that hypoxic‐ischemic events leading to energy failure and neuronal injury, contribute to the neuropathological consequences of COVID‐19. Further studies are warranted to elucidate the mechanisms of SARS‐CoV‐2 neuropathogenesis.