167 COVID-19 Variants and Infection Rates of Neurons, Astrocytes, and Microglia in the Human Brain Demonstrate Direct Viral Replication Within Parenchymal Cells Causing Neurological Symptoms

INTRODUCTION: Neurological symptoms are among the most frequent extrapulmonary complications of COVID-19, affecting more than 30% of patients. To this date, there has not been any direct evidence of viral replication within living human brain tissue. METHODS: Brain specimens were obtained from ten patients undergoing epileptogenic disconnection surgery. Slices were oxygenated in artificial CSF(aCSF), cultured with neuroprotective media, then co-cultured with COVID-19 viral variants Delta, Omicron, Washington. RNA was extracted and sequenced using Next-Generation Sequencing. Differentially expressed genes (DEGs) were identified at p-values <0.05 and log2FC of 1. Gene-ontology analysis was conducted, and normalized enrichment score (NES) was calculated. RESULTS: Quantitative-PCR initially demonstrated robust viral infection and replication within isolated cells from brain slices. PCA was conducted, showing clustering between COVID-19 variants compared to control brain slices. A total of 1743, 564 and 1011 DEGs were identified in the Omicron, Delta and Washington infected tissues, respectively. Interestingly, Gene-Ontology annotation of DEGs showed increased likelihood of transcriptomic correlation with neurological and psychiatric diseases including depression, PTSD, anxiety, suicidality, insomnia, and brain aging, with Delta variant having the highest enrichment score. RNA deconvolution analysis showed significant viral-induced transcriptomic changes on different brain tissue cells including astrocytes, excitatory neurons, and oligodendrocytes, with NES of 1.86, 1.64 and 1.94, respectively (P < 0.001). CONCLUSIONS: For the first time since the COVID-19 pandemic, our unique ex-vivo human brain slice assay has allowed for the ability to capture direct viral replication within the human brain parenchyma, in addition to demonstrating transcriptomics correlation with neurological manifestations such as depression and PTSD, that have become a highly prevalent sequala of this debilitating infection.