Contemporary Enterovirus D68 strains show enhanced replication and translation at 37°C

Abstract

Enterovirus D68 (EV-D68) emerged in 2014 as an important pathogen linked to severe lower respiratory disease and acute flaccid myelitis outbreaks. Historically associated with mild common-cold-like symptoms, clusters of severe disease attributed to EV-D68 appeared during a series of outbreaks in 2014, 2016, and 2018. Previous studies of historic EV-D68 strains demonstrated attenuated replication at temperatures of the lower respiratory tract (37°C), when compared to the upper respiratory tract (32°C). By testing a panel of historic and contemporary EV-D68 strains at 32°C and 37°C, we demonstrate that contemporary strains of EV-D68 undergo little to no attenuation at increased temperatures. Contemporary strains produced higher levels of viral proteins at 32°C and 37°C than historic strains, although both strains infected similar numbers of cells and had comparable amounts of replication complexes. IRES activity assays with dual-luciferase reporter plasmids demonstrated enhanced translation in recent EV-D68 strains mapped to regions of variability in the 5' UTR found only in contemporary strains. Using an infectious clone system, we demonstrate that the translation advantage dictated by the 5' UTR does not solely mediate temperature sensitivity. The strain-dependent effects of temperature on the EV-D68 life cycle gives insight into the susceptibility of the lower respiratory system to contemporary strains. IMPORTANCEEnterovirus-D68 (EV-D68) emerged in 2014 as a causative agent of biannual severe pediatric respiratory disease and acute flaccid myelitis (AFM). We show that recent EV-D68 viruses have gained the ability to replicate at 37⁰C. Enhanced virus protein translation seemed to correlate with enhanced virus replication at 37⁰C but other genetic factors are also contributing to this phenotype. An enhanced ability to replicate at core body temperature may have allowed EV-D68 to penetrate both lower in the respiratory tract and into the central nervous system, explaining the recent surge in severe disease associated with virus infection.

Enterovirus D68 molecular and cellular biology and pathogenesis

“…EV-D68 is therefore more phenotypically similar to rhinoviruses, and accordingly, it is transmitted by respiratory droplets and produces a predominantly respiratory syndrome in most patients. While the prototypical strains of EV-D68 show attenuated growth at higher temperatures, multiple strains isolated in 2014 and later replicate with equal efficiency at 32 °C or 37 °C ( 41 ). These changes may contribute to the apparent increased propensity for newer EV-D68 strains to establish systemic infection and neuroinvasion.…”

“…Another notable divergence of contemporary strains from the historical prototypes is contraction of the spacer region in the 5′-UTR, located between the IRES and the open reading frame. This change may be associated with increased activity of the IRES ( 41 , 63 ). The 5′-UTR is a well-established determinant of virulence in other enteroviruses including poliovirus ( 64 ), and therefore, the evolution of the EV-D68 5′-UTR may contribute to increased virulence.…”

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism

“…However, both contemporary and pre-outbreak isolates of EV-D68 can replicate in neuronal cell lines as well in primary neurons and astrocytes derived from human pluripotent stem cells (hPSCs) indicating that this contraction of the spacer might not be responsible for increased virulence ( 46 ). Another possibility is the ability of contemporary strains to replicate ( in vitro on cell lines) at equal efficiency at 32°C or 37°C, while the replication of prototype strains such as the EV-D68 Fermon strain (isolated from a respiratory sample in 1962) is attenuated at higher temperatures ( 47 ). It would be worthwhile to verify these findings using complex 3D CNS models, such as motor neuron or spinal cord models.…”

Enterovirus D: A Small but Versatile Species

“…Severe complications arose from respiratory illness to a polio-like disease, described as AFM [ 160 ]. The recently characterized strains of EV-D68 from the 2014 outbreak seem to be less susceptible to attenuation at 37 °C, can infect neural tissues independently of SA, and can utilize the neural receptor ICAM-5 (abundantly found in the telencephalon and also identified as a receptor for prototypic strains) [ 123 , 161 , 162 , 163 ]. Infection via intramuscular injection in neonatal mice using several contemporary strains of this virus results in limb paralysis and interestingly the severity of the paralysis is age-dependent [ 164 ].…”