Cytomegalovirus Infection in Human Cortical Organoids Induces Unique Developmental and Viral Substates

Human Cytomegalovirus (HCMV) is a ubiquitous {beta}-herpesvirus with exposure resulting in a life-long infection. Severe HCMV disease can occur in conditions of insufficient immunity, and this includes congenital infection. Infection of the developing brain can cause severe birth defects including microcephaly and sensorineural hearing loss. Also, increasing evidence suggests the contribution of HCMV infection in adult CNS diseases. Studying HCMV replication and its impact on CNS is restricted by host tropism and anatomy. In these studies, we define the impact of infection on healthy iPSC-derived cortical organoids using single cell RNA sequencing. We uncovered 16 cell type clusters in uninfected tissues using the Human Neural Organoid Cell Atlas. Following infection by HCMV expressing GFP, several clusters were altered in both GFP-positive and GFP-negative populations, including the likely transition of the Glutamatergic/GABAergic Neuron cluster into a CMV-Infected Cell cluster. Expressions of viral genes varied between cells and cell types, requiring us to introduce a TC (temporal class) indexing score to account for varying stages of infection. This analysis in conjunction with machine learning approaches revealed cells that are likely producing de novo HCMV along with several viral sub-states. Several viral sub-states had significant enrichments of genes involved in epithelial-to-mesenchymal (EMT) transition, including expression of EpCAM. We independently verified HCMV-induction of EpCAM protein expression in cortical organoids and glutamatergic neurons. In summary, our studies reveal unique features of HCMV infection of cortical organoid tissues, including varying viral sub-states and tropisms, and introduced new approaches to analyze these multi-dimensional complex data sets.