Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

Kristoffer E Leon; Ryan D Schubert; Didac Casas-Alba; Isobel A Hawes; Prashanth S Ramachandran; Akshaya Ramesh; John E Pak; Wesley Wu; Carly K Cheung; Emily D Crawford; Lillian M Khan; Cristian Launes; Hannah A Sample; Kelsey C Zorn; Maria Cabrerizo; Ana Valero-Rello; Charles Langelier; Carmen Muñoz-Almagro; Joseph L DeRisi; Michael R Wilson

doi:10.1212/NXI.0000000000000703

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

Neurol Neuroimmunol Neuroinflamm. 2020 Mar 5;7(3):e703. doi: 10.1212/NXI.0000000000000703. Print 2020 May.

Authors

Kristoffer E Leon¹, Ryan D Schubert¹, Didac Casas-Alba¹, Isobel A Hawes¹, Prashanth S Ramachandran¹, Akshaya Ramesh¹, John E Pak¹, Wesley Wu¹, Carly K Cheung¹, Emily D Crawford¹, Lillian M Khan¹, Cristian Launes¹, Hannah A Sample¹, Kelsey C Zorn¹, Maria Cabrerizo¹, Ana Valero-Rello¹, Charles Langelier¹, Carmen Muñoz-Almagro¹, Joseph L DeRisi¹, Michael R Wilson²

Affiliations

¹ From the Medical Scientist Training Program (K.E.L.), University of California, San Francisco; Biomedical Sciences Graduate Program (K.E.L., I.A.H.), University of California, San Francisco; Weill Institute for Neurosciences (R.D.S., I.A.H., P.S.R., A.R., M.R.W.), University of California, San Francisco; Department of Neurology (R.D.S., I.A.H., P.S.R., A.R., M.R.W.), University of California, San Francisco; Institut de Recerca Pediàtrica Hospital Sant Joan de Déu (D.C.-A., C.L., A.V.-R., C.M.-A.), Barcelona, Spain; Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., E.D.C., J.L.D.), San Francisco; Department of Biochemistry and Biophysics (L.M.K., H.A.S., K.C.Z., J.L.D.), University of California, San Francisco; CIBER Epidemiología y Salud Pública (CIBERESP) (C.L., M.C., C.M.-A.), Health Institute Carlos III; Department of Pediatrics (C.L.), Universitat de Barcelona, Barcelona; Enterovirus Unit (M.C.), Spanish National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain; Division of Infectious Diseases (C.L.), Department of Medicine, University of California, San Francisco; and Department of Medicine. Universitat Internacional de Catalunya (C.M.-A.), Barcelona, Spain.
² From the Medical Scientist Training Program (K.E.L.), University of California, San Francisco; Biomedical Sciences Graduate Program (K.E.L., I.A.H.), University of California, San Francisco; Weill Institute for Neurosciences (R.D.S., I.A.H., P.S.R., A.R., M.R.W.), University of California, San Francisco; Department of Neurology (R.D.S., I.A.H., P.S.R., A.R., M.R.W.), University of California, San Francisco; Institut de Recerca Pediàtrica Hospital Sant Joan de Déu (D.C.-A., C.L., A.V.-R., C.M.-A.), Barcelona, Spain; Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., E.D.C., J.L.D.), San Francisco; Department of Biochemistry and Biophysics (L.M.K., H.A.S., K.C.Z., J.L.D.), University of California, San Francisco; CIBER Epidemiología y Salud Pública (CIBERESP) (C.L., M.C., C.M.-A.), Health Institute Carlos III; Department of Pediatrics (C.L.), Universitat de Barcelona, Barcelona; Enterovirus Unit (M.C.), Spanish National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain; Division of Infectious Diseases (C.L.), Department of Medicine, University of California, San Francisco; and Department of Medicine. Universitat Internacional de Catalunya (C.M.-A.), Barcelona, Spain. michael.wilson@ucsf.edu.

Abstract

Objective: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization.

Methods: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA.

Results: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject.

Conclusion: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Brain Stem*
Child, Preschool
Cohort Studies
Encephalitis, Viral / virology*
Enterovirus A, Human / genetics*
Enterovirus A, Human / isolation & purification*
Enterovirus Infections / virology*
Enzyme-Linked Immunosorbent Assay
Female
High-Throughput Nucleotide Sequencing
Humans
Infant
Male
Meningitis, Viral / virology*
Phylogeny
RNA, Viral / blood
RNA, Viral / cerebrospinal fluid
RNA, Viral / metabolism*
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis, RNA

Substances

RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding