Neurotropism of Enterovirus D68 Isolates Is Independent of Sialic Acid and Is Not a Recently Acquired Phenotype

Rosenfeld, Amy; Warren, Audrey L.; Racaniello, Vincent R.

doi:10.1128/mbio.02370-19

Cited by 47 publications

(37 citation statements)

References 83 publications

(129 reference statements)

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“…Heparan sulfate is abundantly expressed on RD cells ( 32 ) and associated with increased virus attachment, infection efficiency, and virus replication in cells in vitro . The ability of EV-D68 to replicate in mouse neuroblastoma cells and human induced pluripotent stem cell motor neurons were independent of sialic acid recognition, although the usage of heparan sulfate was not investigated ( 30 , 33 ). In our study, unfortunately, viruses from earlier passages without E271K in VP1 were not available and an infectious clone of the clinical isolate B2/947 with the original VP1 amino acid E271 could not be recovered ( 21 ), which made a direct comparison of viruses with and without E271K in VP1 not possible.…”

Section: Discussionmentioning

confidence: 99%

“…However, since 2016, subclade B3 and to a lesser extent subclade D1 became predominant, and both were associated with neurological complications ( 16 , 27 – 29 ). In addition, recent studies have shown that multiple isolates from different clades are able to infect both neuronal cells and neuroblastoma cell lines ( 30 ) and that non-subclade B1 and B3 isolates were able to cause paralysis in mice after intracranial inoculation. Interestingly, isolates from the same clade differed in their ability to cause paralysis in mice ( 31 ).…”

Section: Introductionmentioning

confidence: 99%

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Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Ayudhya

Meijer

Bauer

et al. 2020

mSphere

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Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro. Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates. IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Ayudhya

Meijer

Bauer

et al. 2020

mSphere

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“…However, sialylated glycans, which are not required for the infection of neurons or astrocytes, are able to support a conformational intermediate of EV-D68, and bovine serum albumin can render some enteroviruses (e.g., echovirus 1) more permeable to small molecules. These intermediate conformations were less stable than those of the untreated viruses and were described as "primed" for transition to A-particles (32)(33)(34).…”

Section: Discussionmentioning

confidence: 92%

Albumin Enhances the Rate at Which Coxsackievirus B3 Strain 28 Converts to A-Particles

Carson

Cole

2020

J Virol

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Three strains of coxsackievirus B3 (CVB3) differ by single mutations in capsid protein VP1 or VP3 and also differ in stability at 37°C in tissue culture medium. Among these strains, the CVB3/28 parent strain has been found to be uniquely sensitive to a component in fetal bovine serum (FBS) identified as serum albumin. In cell culture medium, serum increased the rate of CVB3/28 conversion to noninfectious particles at least 2-fold. The effect showed a saturable dose response. Rates of conversion to noninfectious virus with high concentrations of soluble coxsackievirus and adenovirus receptor (sCAR) were similar with and without FBS, but FBS amplified the catalytic effect of 100 nM sCAR nearly 3-fold. Such effects in other systems are due to nonessential activating cofactors. IMPORTANCE A factor other than the virus receptor expressed by target cells has been found to accelerate the loss of an enterovirus (CVB3/28) infectious titer, with little effect on nearly identical mutant strains. The destabilizing factor in fetal bovine serum, identified as albumin, does not interfere with the catalytic activity of soluble receptor at saturating receptor concentrations and amplifies the catalytic activity of the soluble receptor at a concentration that otherwise produces about one-third the saturated receptor-catalyzed rate of virus decay. This finding evidences the possibility that other virus-“priming” ligands may also be nonessential activating cofactors that serve to accelerate receptor-catalyzed viral eclipse.

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“…One theory for the apparent sudden emergence of EV-D68 and AFM in 2014 is a change in the virus, resulting in alterations in tissue tropism, neurovirulence, or other key pathogenic properties, and some data support this theory (42). However, studies have not identified a clear viral genetic signal that consistently correlates with neurologic disease, and data from other investigators indicate that neurotropism is not a recently acquired phenotype (44). Studies of EV-D68 tropism and replication in cell culture models, including those of neural or respiratory origin, might identify more subtle evolutionary changes that influence pathogenesis.…”

Section: Online Reportmentioning

confidence: 99%

Enterovirus D68–Associated Acute Flaccid Myelitis, United States, 2020

Kidd¹,

Lopez²,

Konopka-Anstadt³

et al. 2020

Emerg. Infect. Dis.

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A FM is a syndrome characterized by the acute onset of flaccid limb weakness and lesions in the gray matter of the spinal cord visible on magnetic resonance imaging; the lesions represent damage to the lower motor neurons in the anterior horns. This feature distinguishes AFM from other disorders associated with acute flaccid limb weakness or paralysis, such as disorders of peripheral nerves (e.g., Guillain-Barré syndrome) or neuromuscular transmission (e.g., myasthenia gravis or botulism). AFM can be caused by multiple infectious and noninfectious etiologies. Poliovirus, nonpolio enteroviruses, and flaviviruses are all known to cause AFM in a subset of persons who are infected (1-4). In addition, noninfectious etiologies such as neuroinflammatory conditions or spinal vascular disease can result in a clinical and radiographic picture that overlaps with that of AFM caused by infection (5-7). Although the

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Neurotropism of Enterovirus D68 Isolates Is Independent of Sialic Acid and Is Not a Recently Acquired Phenotype

Cited by 47 publications

References 83 publications

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Albumin Enhances the Rate at Which Coxsackievirus B3 Strain 28 Converts to A-Particles

Enterovirus D68–Associated Acute Flaccid Myelitis, United States, 2020

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