York, S. B. | Search bioRxiv

bioRxiv2022

MagPEG: a complete extracellular vesicle isolation/analysis solution

Current extracellular vesicle (EV) isolation methods depend on large expensive equipment like ultracentrifuges and are laborious and time consuming. There is also currently no method available for high throughput isolation to meet clinical demands. Here, we present a method that combines our previous published ExtraPEG method and magnetic beads. Western blot and nanoparticle tracking analysis (NTA) of the purified EVs revealed higher or equivalent recovery and purity with this method compared to ExtraPEG or size exclusion chromatography (SEC) methods. With this newly developed workflow and automated liquid handling instrument, we have successfully isolated up to 96 EV samples from 5 {micro}L pre-cleared serum in 45 minutes. Moreover, DNA / small RNA / protein purification and profiling steps could be seamlessly integrated into the isolation workflow. To profile EV protein markers, EVs were lysed from the binding step and covalently bound to the surface of the beads. TotalSeq or ELISA antibody can be applied with under a standard protocol. With this extended protocol, researchers can easily complete EV isolation and protein profiling experiment within 8 hours. Taken together, we provide a high throughput method for EV isolation and molecular analyses that may be used for sensitive biomarker detection from biological fluids.

cell biology↗

bioRxiv2021

Zika virus hijacks extracellular vesicle tetraspanin pathways for cell-to-cell transmission

Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the progression of neurological conditions and both the spread and pathogenesis of infectious diseases. It has recently been demonstrated that EVs from Hepatitis C virus (HCV) infected individuals and cells contained replicative-competent viral RNA that was capable of infecting hepatocytes. Being a member of the same viral family, it is likely the Zika virus also hijacks EV pathways to package viral components and secrete vesicles that are infectious and potentially less immunogenic. As EVs have been shown to cross blood-brain and placental barriers, it is possible that Zika virus could usurp normal EV biology to gain access to the brain or developing fetus. Here, we demonstrate that Zika virus infected cells secrete distinct EV sub-populations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with varying sizes and density compared to those released from non-infected cells. We also show that the EV enriched tetraspanin CD63 regulates the release of EVs, and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika infection. ImportanceZika virus is a re-emerging infectious disease that spread rapidly across the Caribbean and South America. Infection of pregnant women during the first trimester has been linked to microcephaly, a neurological condition where babies are born with smaller heads due to abnormal brain development. Babies born with microcephaly can develop convulsions and suffer disabilities as they age. Despite the significance of Zika virus, little is known about how the virus infects the fetus or causes disease. Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells that are present in all biological fluids. EVs carry signaling factors, proteins and microRNAs that mediate intercellular communication. EVs have been shown to be a means by which some viruses can alter cellular environments and cross previously unpassable cellular barriers. Thus gaining a greater understanding of how Zika affects EV cargo may aid in the development of better diagnostics, targeted therapeutics and prophylactic treatments.

microbiology↗