Recent Clinical Isolates of Enterovirus D68 Have Increased Replication and Induce Enhanced Epithelial Immune Response Compared to the Prototype Fermon Strain

Abstract

In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared the viral binding and replication of eight recent EV-D68 clinical isolates collected both before and during the 2014 outbreak and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We selected pairs of closely related isolates from the same phylogenetic clade that were associated with severe vs. asymptomatic infections. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater binding (2-3 logs) and virus progeny yields (2-4 logs) but a similar level of replication (1.5-2 log increase in viral RNA from 2 h to 24 h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1.5-2-log higher virus progeny yields than Fermon due to increased replication. Interestingly, no significant differences in replication were identified between the pairs of genetically close recent EV-D68 clinical isolates despite the observed differences in associated disease severity. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and the Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by an increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates; however, host factors are likely the main determinants of illness severity.

Keywords: RNA-seq; enterovirus D68; respiratory illness; viral replication.

Figure 1

The phylogenetic tree of EV-D68 clinical isolates selected for cloning. Full-length genome sequences were analyzed. The numbers next to each tree branch represent bootstrap values showing the percentage of replicate trees that the viral strain clustered together. The branch lengths represent the evolutionary distance in units of base pair differences per viral sequence, with the scale bar denoting difference. Severity of symptoms designations: As—asymptomatic; Ex—exacerbation; M—mild; S—severe; numbers following the designations indicate symptom score where available. Filled and empty circles of the same color show symptomatic and asymptomatic infections, respectively.

Figure 2

Viral binding and replication in HeLa cells. (A) Cell-associated input virus (2 h p.i.) and progeny yields (24 h p.i.) were determined by RT-qPCR and (B) viral replication was calculated and compared for each isolate. The first letters in isolate designations represent the phylogenetic clade, and the following letters and numbers indicate the clinical isolate ID. The * represents a difference (p < 0.05) in the clinical isolates vs. Fermon, + represents a difference (p < 0.05) in isolate 7787 vs. other isolates, and # represents a difference (p < 0.05) in isolates C2386, C2204, and C7720 vs. other isolates. Data are means and SEM from five independent experiments. The viruses are color-coded according to clade, with darker color indicating severe illness and lighter color indicating asymptomatic infection.

Figure 3

Time course of infection with isolate C7788 and Fermon in differentiated human primary BEC-ALI cultures. The * represents difference (p < 0.05) between Fermon and B1-7788 isolate. Data are means and SEM from five independent experiments.

Figure 4

Viral binding and replication in differentiated human primary BEC-ALI. (A) Cell-associated input virus (2 h p.i.) and progeny yields (24 h p.i.) were determined by RT-qPCR and (B) viral replication was calculated and compared for each isolate. The first letters in isolate designations represent the phylogenetic clade, and the following letters and numbers indicate the clinical isolate ID. The * represents a difference (p < 0.05) between Fermon and the recent clinical isolates. Data are means and SEM from five independent experiments. The viruses are color-coded according to clade with darker color indicating severe illness and lighter color indicating asymptomatic infection.

Figure 5

RNA-seq volcano plot identifying differentially expressed genes in BEC-ALI cultures after infection with recent EV-D68 isolates vs. Fermon. The x-axis represents the log₂ values of the fold change observed for each mRNA transcript, and the y-axis represents the log₁₀ of the p-values of the significance tests between replicates for each transcript. The orange circles represent genes that had an FDR of >0.2, circles in red are genes with an FDR between 0.05–0.2, and the circles in burgundy are genes with an FDR of <0.05. The labeled genes (n = 6) had an FDR of <0.03 and a fold change >2.