Enterovirus D68 VP1 and VP3 determine neurotropism in human spinal cord organoids

Abstract

Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68 associated AFM cases waned in the US after 2018 and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesized that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCO). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I/N2D/T98A/E283K or L1P/V148A/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I/N2D/T98A/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018.

Figure 1.

Confirmation of recombinant EV-D68 with VP1 and VP3 mutations. A.) Schema of proposed recombinant viruses with point mutations in either VP1 or VP3. Gray represents rEV-D68 18952, blue – rVP1–4AA-CA (L1I/N2D/T98A/E283K), pink – rVP1–3AA-MO (L1P/V148A/K282R), and yellow – rVP3-I88V-MO. B.) Infectious clones were cloned into pUC19-EV-D68_49131 and were transfected with pCAGGS Y7 at a ratio of 10:1 with the Transit-LT1 transfection reagent (Mirus Bio) into HEK293T cells. Cells were incubated until cytopathic effects were visible and then were collected. Viral stocks were obtained by incubation of transfected lysates on RD cells. Created with Biorender.com. Schematic is modeled from Jones et al. 2024 [6]. C.) RD cells were infected at an MOI 0.01 PFU/cell in biological triplicate with each recombinant virus. Viral titers were determined by TCID₅₀ in RD cells. Black circle – mock, gray triangle – rEV-D68 18952, blue upside-down triangle – rVP1–4AA-CA, pink diamond – rVP1–3AA-MO, yellow hexagon – rVP3-I88V-MO.

Figure 2.

rVP1–4AA-CA and rVP3-I88V-MO result in decreased infection of hSCOs. A.) IF images of SCTi003A hSCOs infected with recombinant viruses. hSCOs were infected with 10⁴ PFU and were fixed in 4% paraformaldehyde at 96 hpi. hSCOs were then stained with DAPI (blue), VP1 (green), and phalloidin (red). B.) MFI of VP1 staining was quantified across five biological replicate images of each recombinant virus across multiple Z stacks and organoids. Quantified values were calculated using Fiji ImageJ. One way ANOVA with multiple comparisons to EV-D68 18952 was performed (ns – no significance, *** p < 0.001, **** p < 0.0001). C.) SCTi003A hSCO were infected with 10⁴ PFU of each recombinant virus in biological triplicate. Supernatants were collected every 24 hours up to 96 hpi. Viral titers were determined by TCID₅₀ in RD cells. A two-way ANOVA with multiple comparisons to EV-D68 18952 was performed (* p < 0.05 ** < 0.005 **** < 0.00005). Black asterisks – compared to Mock, gray asterisks – compared to rFermon, blue asterisks – compared to rVP1–4AA-CA, and yellow asterisks – compared to rVP3-I88V-MO. Black circle – mock, white square – rFermon, gray triangle - rEV-D68 18952, blue upside-down triangle – rVP1–4AA-CA (L1I/N2D/T98A/E283K), pink diamond – rVP1–3AA-MO (L1P/V148A/K282R), and yellow hexagon – rVP3-I88V-MO.

Figure 3.

Combining VP1–4AA-CA and VP3-I88V-MO point mutations results in decreased viral titer compared to rEV-D68 18952. A.) Schema of rVP3-I88V/VP1–4AA-CA recombinant viruses with VP3 I88V and VP1 L1I/N2D/T98A/E283K. Gray represents rEV-D68 18952, and yellow and blue represent rVP3-I88V/VP1–4AA-CA. B.) RD cells were infected at an MOI 0.01 PFU/cell in biological triplicate with rVP3-I88V/VP1–4AA-CA. Viral titers were determined by TCID₅₀ in RD cells. C.) SCTi003A hSCO were infected with 10⁴ PFU of either rEV-D68 18952 (gray triangle) or rVP3-I88V/VP1–4AA-CA (yellow and blue circle) in biological triplicate. Supernatants were collected every 24 hours up to 96 hpi. Viral titers were determined by TCID50 in RD cells. A two-way ANOVA with multiple comparisons to EV-D68 18952 was performed (* p < 0.05 ** < 0.005 **** < 0.00005). D. IF images of SCTi003A hSCOs infected with rEV-D68 18952 or rVP3-I88V/VP1–4AA-CA. hSCOs were infected with 10⁴ PFU and were fixed in 4% paraformaldehyde at 96 hpi. hSCOs were then stained with DAPI (blue), VP1 (green), and phalloidin (red). E.) MFI of VP1 staining was quantified across five biological replicate images of each recombinant virus across multiple Z stacks and organoids. Quantified values were calculated using Fiji ImageJ. An unpaired student t-test was performed (* p < 0.05).

Figure 4.

Frequency of VP1 and VP3 point mutations in clinical isolates that have circulated between 2014 to 2024. A. Schema of VP1 and VP3 recombinant viruses and the effect they have on mice paralysis and on hSCO replication. Gray represents rEV-D68 18952, blue – rVP1–4AA-CA (L1I/N2D/T98A/E283K), pink – rVP1–3AA-MO (L1P/V148A/K282R), and yellow – rVP3-I88V-MO. Black arrow – decreased viral replication in hSCO compared to reference strain (EV-D68 18952), black ‘=‘ – similar rate of viral replication to EV-D68 18952. B. Global EV-D68 sequences from 2014–2024 were obtained from the Bacterial and Viral Bioinformatics Resource Center (n=1,968) and aligned using NextClade. Amino acid substitutions were visualized in an area plot as a percentage of the total sequences analyzed in each year. The identity of each residue in the reference strain, EV-D68 18952, is shown in red, and substitutions are colored according to relative abundance in each year. C. Amino acid types were overlaid onto the substitution frequencies shown in panel B.