A Case Cluster of Aseptic Meningitis Associated With a Newly Identified Recombinant Echovirus 6/Coxsackievirus B1 Enterovirus

Figures

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  Figure 1.. Representative images of cytopathic effects in 3 cell lines. A, Mock-inoculated A549 cells, 5 days postinoculation (dpi). B, A549 cells, 5 dpi with cerebrospinal fluid. C, Mock-inoculated LLC-MK2 cells, 5 dpi. D, LLC-MK2 cells, 5 dpi with nasal swab material. E, Mock-inoculated MRC-5 cells, 10 dpi. F, MRC-5 cells, 10 dpi with nasal swab material. Virus-induced cytopathic effects consisting of rounded cells detaching from the growing surface and floating cells are present in A549 and LLC-MK2 cells inoculated with cerebrospinal fluid and nasal swab material by 5 dpi. By 10 dpi, the mock-inoculated MRC-5 cell monolayer remains intact, whereas most of the MRC-5 cells inoculated with nasal swab material have been destroyed. Images taken at an original magnification of 400×.

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  Figure 2.. Schematics of the coding DNA sequence (CDS) genomic segment, with gene structure and summary of ancestry from the phylogenetic analysis. Coordinates are based on the UF-EPI-1-2024 strain from the initial adult patient.

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  Figure 3.. A and B, Neighbor-joining (NJ) trees inferred from the 2 major nonrecombinant segments of the full genome sequences. Branch lengths are drawn proportional to nucleotide substitutions per site according to the scale at the bottom. Red terminal branches indicate sequences sampled from meninges and/or cerebrospinal fluid (CSF). Arcs indicate strains belonging to well-supported clades (bootstrap >90%). Cox, coxsackievirus; Echo, echovirus; Ent, enterovirus. A, NJ tree inferred from the VP2-VP3-VP1-2A-2B-2C nonrecombinant concatemer. Strains isolated in this study (green branches) are indicated by a green arrow. B, NJ tree inferred from the 3A-3C-3D nonrecombinant concatemer. Strains isolated in this study (brown branches) are indicated by a brown arrow. C and D, Bayesian clock-like phylogenies inferred from the 2 major nonrecombinant segment of the full genome sequences. Each tree includes a subset of sequences most closely related to the strains isolated in this study. Branch lengths are scaled in time according to the scale at the bottom. Red labels indicate sequences sampled from meninges and/or CSF. Horizontal bars represent the 95% highest posterior density interval of estimated…

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  Figure 4.. Similarity plot of the new Florida isolates. The similarity plot was obtained by comparing the new Florida sequences (query) with coxsackievirus and echovirus 6 putative parental strains, according to the legend in the figure. The x-axis indicates genome position; the y-axis is the identity between query and parental strains. The analysis was carried out by using a sliding window of 200 nucleotides with steps of 20 nucleotides. The crossing point indicates the recombination breakpoint.

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  Figure 5.. Recombination and mutation result in the emergence of novel viruses identified in Florida. A, A ribbon diagram depicts the structural prediction of an echovirus 6 polypeptide (2022_FRA_OR840837.1; magenta). B, A ribbon diagram illustrates the structural prediction for a coxsackievirus polypeptide (2023_FRA_OR840844.1; cyan). C, A ribbon diagram shows the predicted structure for a recombinant polypeptide, integrating sequences from echovirus 6 (magenta) and coxsackievirus (cyan; 2024_USA_UF-EP1-3-2024_FL). The residues encoded by the 5′ and 3′ recombinant fragments come from the separate parental strains (magenta for echovirus 6, cyan for coxsackievirus). Positions mutated following recombination are shown as spheres: yellow for carbon, blue for nitrogen, red for oxygen.

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  Figure 6.. Recombination and mutation in coxsackievirus are predicted to affect subunit assembly. AlphaFold 3 predictions indicated that the echovirus 2C polypeptide forms hexamers, whereas the coxsackievirus 2C polypeptide forms heptamers. The recombinant mutated viruses identified in Florida express 2C polypeptides predicted to form hexamers and heptamers. A and B, Side views of the echovirus hexamer and coxsackievirus heptamer, respectively. C and D, Side views of the recombinant mutated virus predicted to form a hexameric and heptameric arrangement, respectively, highlighting that the amino-terminal portion of the recombinant virus is derived from echovirus 6 (magenta) while the carboxy-terminal portion originates from coxsackievirus (cyan). E and F, Top views of the echovirus hexamer and coxsackievirus heptamer, respectively. G, Top view of the recombinant mutated virus predicted to form a hexameric arrangement. H, Top view of the recombinant mutated virus predicted to form a heptameric arrangement.