Multimodal Investigation of the Etiology for Acute Flaccid Myelitis Points to Enterovirus as a Source

Description:

Acute Flaccid Myelitis (AFM) is an acute paralysis resembling polio that since at least 2012 has displayed seasonal spikes in occurrence across the United States, typically in children. Enterovirus (EV), specifically EV-D68, has been implicated as the causal organism, but doubts persist because EV infection is common, and EV RNA is rarely detected in spinal fluid and is not detected in non-sterile sites in 50% of children with AFM. In this study, spinal fluid (CSF) samples from 42 children with AFM- and 54 non-AFM-afflicted children were incubated with virus particle-encoding peptides representing all known vertebrate virus and arbovirus genomes. Antibodies in the CSF could then bind their targets and be collected for identification by deep sequencing. Separately, sequencing of spinal fluid RNA, both in an unbiased survey and enriching for EV RNAs, was performed. This extensive, pan-viral antibody-dependent search found antibodies to enterovirus in 29 of 42 patients compared to 4 of 58 control subjects. When antibody testing was combined with traditional methods to directly detect EV in the CSF or a non-sterile site such as the mouth, 36 of 42 patients with AFM had evidence of EV. Thus, the study strongly supports the hypothesis that AFM is due to EV infection and not to other viruses. This is an important advance that will require testing in a larger patient population on the way to developing an anti-EV therapeutic to prevent AFM.

Full abstract, to be presented at the American Neurological Association 2019 Annual Meeting (October 13-15 in St. Louis):

Multimodal Investigation of the Etiology for Acute Flaccid Myelitis

Ryan Schubert, MD¹, Prashanth Ramachandran, MBBS¹, Isobel Hawes, BA¹, Emily Cawford, PhD², Akshaya Ramesh, PhD¹, Brian O' Donovan, PhD¹, Cristina Tato, PhD², Amy Lyden, BS², Hannah A. Sample, BS¹, Kelsey Zorn, MHS¹, John Pak, PhD², Wesley Wu, PhD², Carly Cheung, BS², Riley Bove, MD¹, Stephen Hauser, MD¹, Sean Pittock, MD³, Divyanshu Dubey, MD³, Mark Gorman, MD⁴, Leslie Benson, MD⁴, Terry Ng, PhD⁵, Rachel Marine, PhD⁵, W. Allan Nix, BS⁵, Jennifer L. Konopka-Anstadt, PhD⁵, M. Steven Oberste, PhD⁵, Joseph DeRisi, PhD², Michael R. Wilson, MD¹. ¹UCSF, San Francisco, CA, USA, ²CZ Biohub, San Francisco, CA, USA, ³Mayo Clinic, Rochester, MN, USA, ⁴Boston Children’s Hospital, Boston, MA, USA, ⁵Centers for Disease Control and Prevention, Atlanta, GA, USA.

Objective: We deployed a pan-viral serologic assay using programmable phage display in combination with metagenomic next-generation sequencing (mNGS) on cerebrospinal fluid (CSF) from patients with acute flaccid myelitis (AFM). Background: Since 2014, there has been a biennial spike in pediatric AFM across the United States. Epidemiologic evidence suggests enterovirus D68 (EV-D68) and other EVs are a potential etiology. However, uncertainty persists about the role of EVs in AFM because EV RNA is rarely detected in CSF or other sterile sites and is not detected in any non-sterile site specimen of more than half of children with AFM. Design/Methods: CSF samples from children with AFM (n=29) and unmatched pediatric cases of other neurologic diseases (OND) not known to be caused by EV (n=71) were incubated with T7 bacteriophage expressing 481,966 sixty-two amino acid peptides with a fourteen amino acid overlap tiled across all known vertebrate virus and arbovirus genomes. Antibody-bound phage were deep sequenced to quantify enriched peptides with normalized counts expressed as reads per hundred thousand (rpK). Separately, mNGS of CSF RNA, both unbiased and with targeted enrichment for EV, was performed with 500 million paired-end reads/sample. Results: On the pan-viral phage display assay, 21/29 AFM CSF samples specifically enriched EV peptides above 2,500 rpK (median rpK 5,641; IQR 2,024-18,526) compared with 7/71 OND (median rpK 335; IQR 53-1,066). mNGS did not detect additional EV RNA in CSF. Conclusions: Despite the rare detection of EV RNA in the CNS of patients with AFM, our pan-viral serologic assay identified CSF antibodies to the EV genus in most patients (21/29) with AFM compared to a minority of pediatric OND (7/71). Follow-up studies are underway to validate our findings in larger cohorts and to provide EV species-level epitope resolution. These results support the hypothesis that EVs, not other viruses, are associated with AFM.

All abstracts from ANA2019 will be available under embargo starting October 4. Contact Katherine Pflaumer ([email protected]) for full meeting abstracts, and for call-in information for the ANA2019 Media Roundtable (Oct. 15, 11 a.m. US Central).