Exosomes cloak the virion to transmit Enterovirus 71 non-lytically

Abstract

Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection. In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM). It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the "non-enveloped" EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis.Abbreviation: EV71: enterovirus 71; EXO: exosome; RD: rhabdomyosarcoma; TEM: transmission electron microscope; HFMD: hand, foot, and mouth disease; HIV: immunodeficiency virus; HCV: hepatitis C virus; HTLV: Human T-cell lymphotropic virus; HAV: hepatitis A virus; MOI: multiplicity of infection; EVs: extracellular vesicles; VP1: viral capsid protein 1; NTA: nanoparticle tracking analysis; CNS: central nervous system.

Keywords: Density; EV71; EXO-EV71; exosomes; non-lytically.

Figure 1.

Enterovirus 71 Exit Cells Non-lytically In Vitro. (a) viral RNA was detected in culture supernatant after infected 1–9 h by qRT-PCR assay (mean±SD; three independent experiments). (b) infected cells incubated with SYTOX^TM Blue Nucleic Acid Stain to monitor plasma membrane intactness. c, quantification of fluorescence intensity in Figure 1(b) (mean±SD; three independent experiments; *p ≤ 0.05). Statistical analysis were performed using unpaired two-tailed student’s t-test. D, single-step growth curve of RD cells infected with EV71 at MOI of 1. Supernatants were collected from infected cells at the indicated time points, and virus titers were calculated by standard TCID₅₀ assay.

Figure 2.

EV71 virions could be wrapped within exosomes in vitro. (a) buoyant density of EV71 particles released by RD cells in iodixanol gradients. Exosome-like vesicles wrapping EV71 particles (EXO-EV71) gathered in 1.10–1.12g/mL (fraction 7–8), non-enveloped EV71 particles gathered in 1.18–1.26g/mL (fraction 4–5). (b) TEM images of EXO-EV71 (a-c, fraction 7–8 in A) and non-enveloped EV71 (d, fraction 4–5 in A). c, distribution of the number of virion-like particles contained within individual exosomes. d, distribution of EXO-EV71 size measured by nanoparticle NTA. e, immunoblots of EV71 capsid proteins (VP1) and exosomes positive marker (CD63 and TSG101) and negative marker (CANX and Albumin) in lysate of EV71-infected cells, EXO-EV71 and EV71 particles. f, immunoblot analysis of CD63, TSG101 and VP1 in EXO-EV71 exposed to proteinase K with or without detergent (0.1% Triton X-100, used to ensure degradation of both surface and internal components).

Figure 3.

Exosomes Containing Viral RNA (EXO-EV71-RNA) Existed in Patients’ Plasma. (a) viral RNA could be detected in patients’ plasma and in the exosomes of the plasma. (b) viral RNA was positive in patients’ stools.