Serological and metagenomic interrogation of cerebrospinal fluid implicates enteroviruses in pediatric acute flaccid myelitis

Background: Since 2014, the United States has experienced a biennial spike in pediatric acute flaccid myelitis(AFM). Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF) and only inconsistently identified from the respiratory tract, serum, or stool. Methods: We interrogated CSF from children with AFM (n=42) and pediatric controls with other neurologic diseases (OND) (n=58). Samples were incubated with T7 bacteriophageexpressing 481,966 sixty-two amino acid peptides with a fourteen amino acid overlap tiled across all known vertebrate virus and arbovirusgenomes. Antibody-bound phage were deep sequencedto quantify enriched peptides with normalized counts expressed as reads per hundred thousand (rpK). EV antibody findings were confirmed with ELISA using whole viral protein 1 (VP1) from contemporary enterovirus(EV) A71 and D68 strains. Separately, metagenomic next-generation sequencing (mNGS) of CSF RNA, both unbiased and with targeted enrichment for EVs, was performed. Results: The most significantly enriched viral family by CSF pan-viral phage displayin AFM versus OND controls was Picornaviridae(mean rpK 11,266 versus mean rpK 950, p-adjusted < 0.001, Wilcoxon signed-rank testwith Bonferroni adjustment). Enriched Picornaviridaepeptides belonged almost entirely to the genus Enterovirus. The mean EV VP1 ELISA signal in AFM (mean OD 0.51) was significantly higher than OND controls (mean OD 0.08, p-value < 0.001, Mann-Whitney test). mNGS did not detect additional enterovirusRNA in CSF. Conclusion: Despite the rare detection of EV RNA in the CNS of patients with AFM, our pan-viral serologic assay identified high levels of CSF EV antibodies in AFM CSF compared to CSF from OND controls. These results provide further evidence for a causal role of non-polio enteroviruses in AFM.