Recovery Infectious Enterovirus 71 by Bac-to-Bac Expression System in vitro and in vivo.

Enterovirus 71 (EV71) is one of the most important etiological agents for hand-foot-mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system in vitro. After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the Vp1 gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 in vivo. Thus, we established a new approach to rescue EV71 based on a baculovirus expression system in vitro and in vivo, which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.

Design of Small Nanoparticles Decorated with Amphiphilic Ligands: Self-Preservation Effect and Translocation into a Plasma Membrane.

Design of nanoparticles (NPs) for biomedical applications requires a thorough understanding of cascades of nano-bio interactions at different interfaces. Here, we take into account the cascading effect of NP functionalization on interactions with target cell membranes by determining coatings of biomolecules in biological media. Cell culture experiments show that NPs with more hydrophobic surfaces are heavily ingested by cells in both the A549 and HEK293 cell lines. However, before reaching the target cell, both the identity and amount of recruited biomolecules can be influenced by the pristine NPs' hydrophobicity. Dissipative particle dynamics (DPD) simulations show that hydrophobic NPs acquire coatings of more biomolecules, which may conceal the properties of the as-engineered NPs and impact the targeting specificity. Based on these results, we propose an amphiphilic ligand coating on NPs. DPD simulations reveal the design principle, following which the amphiphilic ligands first curl in solvent to reduce the surface hydrophobicity, thus suppressing the assemblage of biomolecules. Upon attaching to the membrane, the curled ligands extend and rearrange to gain contacts with lipid tails, thus dragging NPs into the membrane for translocation. Three NP-membrane interaction states are identified that are found to depend on the NP size and membrane surface tension. These results can provide useful guidelines to fabricate ligand-coated NPs for practical use in targeted drug delivery, and motivate further studies of nano-bio-interactions with more consideration of cascading effects.