Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes.

Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm3, whereas this density has a very low viral titer in the prototypic Fermon strain. IMPORTANCE Despite the strong causal link between enterovirus D68 (EV-D68) and acute flaccid myelitis (AFM), it remains unclear how EV-D68 gains entry into the central nervous system and what receptors enable it to infect motor neurons. We show that EV-D68 particles can adhere to exosomes, placing EV-D68 among a handful of other picornaviruses which are known to interact with extracellular vesicles. Uptake and infection of permissive cells by virally contaminated exosomes would have major implications in the search for the EV-D68 receptor, as well as providing a possible route for viral entry into motor neurons. This work identifies a novel cellular entry route for EV-D68 and may facilitate the identification of genetic risk factors for development of AFM.

A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Structure and inhibition of EV-D68, a virus that causes respiratory illness in children.

Enterovirus D68 (EV-D68) is a member of Picornaviridae and is a causative agent of recent outbreaks of respiratory illness in children in the United States. We report here the crystal structures of EV-D68 and its complex with pleconaril, a capsid-binding compound that had been developed as an anti-rhinovirus drug. The hydrophobic drug-binding pocket in viral protein 1 contained density that is consistent with a fatty acid of about 10 carbon atoms. This density could be displaced by pleconaril. We also showed that pleconaril inhibits EV-D68 at a half-maximal effective concentration of 430 nanomolar and might, therefore, be a possible drug candidate to alleviate EV-D68 outbreaks.

[Expression and Preliminary Research on the Soluble Domain of EV-D68 3A Protein].

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.

Enterovirus 70 receptor utilization is controlled by capsid residues that also regulate host range and cytopathogenicity.

Enterovirus type 70, an etiologic agent of acute hemorrhagic conjunctivitis, may bind different cellular receptors depending on cell type. To understand how EV70-receptor interaction is controlled, we studied two variants of the virus with distinct receptor utilization. EV70-Rmk, derived by passage in rhesus monkey kidney cells, replicates poorly in HeLa cells and does not cause cytopathic effects. Decay accelerating factor (DAF) is not a cell receptor for EV70-Rmk. Passage of EV70-Rmk in HeLa cells lead to isolation of EV70-Dne, which does not replicate in rhesus monkey kidney cells but grows to high titers in HeLa cells and causes cytopathic effects. DAF is sufficient for cell entry of EV70-Dne. EV70-Rmk replicates in human eye and brain-derived cell lines, whereas the Dne strain replicates only in HeLa cells and in conjunctiva-derived 15C4 cells. The two EV70 strains differ by five amino acid changes in the viral capsid. Single substitution of four of the five EV70-Rmk amino acids with the residue from EV70-Dne leads to lytic replication in HeLa cells. Conversely, substitution of any of the five EV70-Dne amino acids with the EV70-Rmk amino acid does not alter replication in HeLa cells. Three of these capsid amino acids are predicted to be located in the canyon encircling the fivefold axis of symmetry, one amino acid is found at the fivefold axis of symmetry, and one is located the interior of the capsid. The five EV70 residues define a region of the capsid that controls viral host range, DAF utilization, and cytopathogenicity.

Human rhinovirus 87 identified as human enterovirus 68 by VP4-based molecular diagnosis.

Human rhinoviruses (HRVs) are the major cause of respiratory infections. We developed a diagnostic method for HRVs based on the reverse-transcription polymerase chain reaction (RT-PCR) and VP4-based phylogenetic analysis. A set of primers used in the RT-PCR of human enteroviruses (EVs) appeared to be capable of amplifying all prototype strains of HRVs, each of which generated a 530-bp fragment. The single exception was HRV-87, which generated a 650-bp fragment, as observed in human EVs. The VP4 nucleotide sequence of HRV-87 showed more than 97% nucleotide identity with human EV-68, and formed a monophyletic cluster along with the prototype strain of EV-68 in the human EV-D cluster. HRV-87 showed the second highest homology (76.8%) with EV-70, another member of the human EV-D, in a sample of 66 human EVs and 12 HRVs. Therefore, HRV-87 should be reclassified into the cluster containing human EV-68.