Filoviruses Can Efficiently Infect Human Neuron-Like Cells Without Genetic Adaptation

Abstract

Recent large-scale Ebola outbreaks, combined with improved follow-up of survivors, has permitted the observation of common long-term neurological sequelae in patients that have survived Ebola virus infection. To date there have been few studies into neurological infections by Ebola or related filoviruses, however, recent studies have isolated infectious virus from patients cerebrospinal fluid months after being discharged from the treatment facility. In order to determine whether different filoviruses were capable of infecting human neurons, the human neuroblastoma cell lines, SH-SY5Y and M17, were chemically-differentiated into more neuron-like cells using established protocols. The neuron-like profiles of the differentiated cells were confirmed by the determination of expression of a range of neuron-specific markers. Zaire ebolavirus, Reston ebolavirus, and Marburg virus were serially-passaged in both cell lines to determine permissiveness of the cells, as well as permit the acquisition of adaptive mutations in the viral genomes. Whilst Marburg virus grew to high titres in both cell lines, Zaire ebolavirus only grew in SH-SY5Y cells, and Reston ebolavirus rapidly died out in both cell lines. Whole-genome sequencing of the passaged viruses revealed two consensus-level non-coding mutations in the SH-SY5Y-passaged Marburg virus. Viral growth kinetics were determined for pre- and post-passaging Zaire ebolavirus and Marburg virus in both human neuronal cell lines, as well as the human hepatocyte cell line, Huh7. Growth kinetics were similar for both the pre- and post-passaged viruses, suggesting that adaptive mutations were not required for efficient growth in these cells. This study is the first to demonstrate that filoviruses are capable of infecting human neuron-like cells in a species-specific manner. Marburg virus-infected cells remained alive up to Day 21 post-infection, suggesting that long-term neurological sequelae following filovirus infection may be a result of direct neuronal infection, and that infection of neurons might contribute to viral persistence in survivors. Author SummaryFiloviruses, including Ebola and Marburg viruses, have been traditionally considered "haemorrhagic fever" viruses, with infections causing bleeding and frequently death. Recent large-scale outbreaks in Africa have challenged these assumptions due to a significant number of patients reporting neurological symptoms sometimes months after infection. In many of these patients, virus was present at detectable levels in the fluid surrounding the brain. There has been significant debate about the ability of Ebola and Marburg viruses to infect and grow in human neurons (brain cells), and evidence has been lacking due to the lack of feasibility in taking brain samples. Our study demonstrates that both Zaire ebolavirus and Marburg virus are capable of infecting cells derived from human brains without needing to change, and without killing the cells. Reston ebolavirus, a related virus that appears not to cause disease in humans, was not able to grow efficiently in these cells. Our findings show that these viruses might be capable of living in the brains of survivors for long periods of time, similar to previous observations in the eye and testes. In addition, the response of the body to these infected cells might account for the neurological symptoms described by patients.