Pan-viral serology implicates enteroviruses in acute flaccid myelitis

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Abstract

Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)1,2,3,4,5,6. Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)2. CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM.

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Fig. 1: EV immunoreactivity in AFM on a pan-viral phage display assay.
Fig. 2: Primary EV antigens identified by pan-viral phage display in AFM.
Fig. 3: Independent validation of pan-viral phage display with purified EV VP1 capsid protein.

Data availability

VirScan and clinical data analyzed in the manuscript have been made available in the online Supplementary material. The non-human sequence reads from the mNGS experiments for each sample were deposited at the National Center for Biotechnology Information Sequence Read Archive (PRJNA557094).

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Acknowledgements

This work is supported by a National Multiple Sclerosis Society—American Brain Foundation Clinician Scientist Development Award (no. FAN-1608-25607 to R.D.S.), the UCSF Biomedical Sciences Graduate Program (to I.A.H.), an American Academy of Neurology Clinical Research Training Scholarship (no. P0534134 to P.S.R.), the University of California, San Francisco (UCSF) Dean’s Office Medical Student Research Program (G.A.S.), UCSF Bioinformatics Graduate Program (B.O.), NIH grant nos. K08NS096117 (to M.R.W.) and K23AI28069 (to K.M.), the Chan Zuckerberg Biohub (J.L.D., C.M.T., J.E.P., E.D.C., W.W., C.K.C., A.L., M.T. and R.S.), an endowment from the Rachleff family (to M.R.W.), and the Sandler and William K. Bowes, Jr. Foundations (M.R.W., K.C.Z., H.A.S., C.Y.C., L.M.K., B.O. and J.L.D.). We thank H. Sandler, W. Bowes, Jr., and D. and A. Rachleff for their encouragement. We thank the patients and their families for their participation in this study.

Author information

A.R. computationally designed the VirScan peptide library. R.D.S., I.A.H. and G.A.S. cloned the VirScan library. R.D.S. and I.A.H. performed the VirScan experiments. R.D.S. and B.O. developed the automated IP protocols and analysis pipeline for VirScan. J.E.P., W.W. and C.K.C. cloned and expressed EV VP1 proteins. R.D.S. performed the ELISA experiments. P.S.R., E.D.C., A.L., C.M.T., M.T. and R.S. performed metagenomic sequencing and FLASH. P.S.R., M.R.W. and E.D.C. analyzed metagenomic and FLASH data. D.B. and L.M.K. helped prepare samples for sequencing. R.D.S., H.A.S., K.C.Z., R.B., S.L.H., A.A.G., B.L.J.-K., K.N., K.S.K., T.C., J.Z.D., H.J.M., C.Y.C., B.B., C.A.G., C.Y., V.C., D.A.W., S.R.D., R.L.M., A.S.L., W.A.N., A.S., M.P.G., L.B., K.M., J.L.K.-A. and M.S.O. identified patients, performed clinical phenotyping and provided patient samples. R.D.S., A.R., T.F.F.N., J.L.D. and M.R.W. analyzed VirScan and ELISA data. R.D.S. and J.L.D. generated the figures. J.L.K.-A. and M.S.O. provided critical expert guidance on the manuscript. R.D.S., J.L.D. and M.R.W. conceived of and wrote the manuscript. All authors discussed the results and contributed critical reviews to the manuscript.

Correspondence to Michael R. Wilson.

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The authors declare no competing interests.

Additional information

Peer review information Alison Farrell and Saheli Sadanand are the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Disclaimer The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention, the National Institutes of Health or the California Department of Public Health.

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Extended data

Extended Data Fig. 1 Flow chart depicting patient enrollment by institution.

mNGS with and without FLASH were performed on CSF samples acquired from the CDC (n=14 AFM, n=4 OND, n=2 EV positive controls) and UCSF AFM Cohort (n=6 AFM). Samples from all institutions were tested by VirScan. Due to limited sample, a subset of those tested by VirScan were tested by confirmatory ELISA.

Extended Data Fig. 2 Comparison of VirScan and ELISA.

A comparison of the total amount of enterovirus signal generated by VirScan (x-axis) to the maximum OD generated by either EV-D68 or EV-A71 signal ELISA (greater of the two values shown) for all samples run (n = 26 AFM + 50 OND). The 95% confidence intervals are shaded in grey.

Extended Data Fig. 3 Geographic distribution of cases and controls.

Geographic comparison of cases (blue) and controls (orange) with average EV signal by ELISA (top), average EV signal by VirScan (middle), and total number (bottom).

Extended Data Fig. 4 Season and year of cases and controls.

Season and year comparison for cases (blue) and controls (orange) with average EV signal by ELISA (top), average EV signal by VirScan (middle), and total number (bottom).

Extended Data Fig. 5 Analysis of effect of year and season on enterovirus signal in the OND controls.

EV VirScan (left, n = 54) and EV VP1 ELISA (right, n = 50) for the OND control cohort by year (top) and season (bottom). Bar graphs depict heights as median values with error bars reflecting the interquartile range. Statistics for year were performed with the Mann-Whitney test and for seasons with the Kruskal-Wallis test.

Extended Data Fig. 6 CSF cell count and IgG concentration in cases and controls do not explain EV signal by VirScan or ELISA.

CSF enterovirus antibodies by VirScan a and ELISA are not correlated with the overall amount of CNS inflammation as measured by the CSF cell count (panel A) or CSF IgG (panel B) in a subset of patients. The 95% confidence intervals for each measurement are shaded in blue (ELISA) or red (Virscan). When comparing the concentration of IgG in a subset of AFM cases and OND controls, there was no difference in CSF IgG concentration (p = ns by Mann-Whitney, mean with errors bars showing standard deviation displayed). Two OND CSF IgG values were reported as < 0.9 mg/dL and were conservatively estimated to be 0. Errors bars represent 95% confidence intervals.

Extended Data Fig. 7 Enterovirus antibody enrichment in AFM cases with VirScan is not a reflection of bias in the input library.

Dotplot demonstrating replicate IPs from a typical sample (left panel) correlate with each other but not with the input library (right panel). Values plotted are log10 of the raw rpK values + 1. The sum of the signal at each point on the axes is expressed as a barplot on the axes of the graph.

Extended Data Fig. 8 Strain calling by ELISA versus VirScan.

ELISA and VirScan data from subjects with EV-A71 or EV-D68 detected by RT-PCR in either CSF, stool or respiratory fluid. Top panels with strain-specific VP1 ELISA data from EV-A71 (n = 8, red) and EV-D68 (n = 3, blue) patients show cross reactivity. Bottom panels show VirScan data from known EV-A71 (n = 9, red) and EV-D68 (n = 7, blue) patients. EV-A and EV-D signals were generated by summing the total rpK generated against EV-A and EV-D derived peptides within a sample.

Extended Data Fig. 9 Enterovirus species per-subject heatmap.

VirScan enterovirus signal in each subject demonstrating enrichment for a cross-reactive EV signal in the AFM subjects (left) as compared with OND subjects (right). Signal represents the log2 of the subject’s EV rpK value divided by the mean rpK value in the OND subjects for each EV species. To increase clarity, values below 3 are not shown.

Supplementary information

Supplementary Information

Supplementary Methods and Table 7

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Supplementary Tables 1–6, 8 and 9

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Schubert, R.D., Hawes, I.A., Ramachandran, P.S. et al. Pan-viral serology implicates enteroviruses in acute flaccid myelitis. Nat Med 25, 1748–1752 (2019) doi:10.1038/s41591-019-0613-1

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