Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

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.

Figure 1. Summary of mNGS diagnostics: improvement over traditional clinical testing

(A) Comparison of mNGS and qRT-PCR detection of EV-A71, E-30 and CVB in CSF, NP, and fecal samples. Statistics performed using the Fisher exact test, with orange and blue p-values corresponding with mNGS and qRT-PCR results, respectively. (B) Comparison of detection levels for each different experiment, with rpM representing mNGS and Ct values for qRT-PCR. Triangles denote samples identified by mNGS but not qRT-PCR. Statistics performed using a Mann-Whitney test. (C) Heatmap of each individual subject with each body site represented. Boxes with 2 colors represent codetections of different viral species. (D) Comparison of mNGS detection rates to qRT-PCR. Statistics performed using the McNemar test. CVB = Coxsackievirus B; E-30 = echovirus 30; EV-A71 = enterovirus A71; mNGS = metagenomic next-generation sequencing; NP = nasopharyngeal; qRT-PCR = quantitative reverse transcription polymerase chain reaction.

Figure 2. Phylogenetic and Genomic Analysis of EV-A71

(A) Phylogenetic tree of full-length viral genomes for enterovirus A71, echovirus 30, coxsackievirus B, and rhinovirus isolated from CSF (L), stool (F), and NP (N) compared with the German neuroinvasive strain (KX139462.1). Rhinovirus obtained from subjects acts as the root. The number refers to the subject. (B) Confirmation of clinical VP1 testing that the Catalonian EV-A71 viral protein 1 (VP1) gene is most closely related to a neuroinfectious German strain. Blue = neuroinvasive EV-A71, Orange = HFMD EV-A71. (C) Phylogenetic tree of 545 EV genomes from every species highlighting the relatedness between the EV strains discovered in this outbreak. (D) Protein alignment of the VP1 gene highlighting the S241P mutation found exclusively in the CSF of subject 16. Scale bars indicate nucleotide substitution rate per position. EV-A71 = enterovirus A71; F = fecal; NP = nasopharyngeal.

Figure 3. VirScan Identifies immunodominant enterovirus antigens and improves the detection of CSF enterovirus in encephalomyelitis/brainstem encephalitis

(A) VirScan identified 136 unique, enriched viral antigens with taxonomies linking them to genus Enterovirus. We mapped 123 of these 136 peptides with BLASTP to a reference EV-A71 genome (Genbank Accession AXK59213.1), as described previously (coding genes in light blue, non-coding genes in orange). Mapping revealed the relative locations of the EV antigens identified in the Catalonia cases (graphed blue shading) across the EV genome, which appeared remarkably conserved, as seen previously in pediatric acute flaccid myelitis (graphed light red shading with overlap appearing gray). (B) Violin plot revealing enrichment for EV antigens by VirScan and EV VP1 antigen by ELISA in Catalonia cases, regardless of whether an EV had been previously identified by mNGS or qRT-PCR (p = ns for both comparisons). In both groups, EV detection was significantly greater than in the pediatric OND controls (p < 0.001 for all comparisons as indicated). The Mann-Whitney test was used, with Bonferroni correction for VirScan. (C) Differences in virus detection for subjects with encephalomyelitis/brainstem encephalitis or encephalitis/meningitis. The detection of CSF EV by mNGS or qRT-PCR was low (0/10). (D) VirScan or ELISA improved EV detection in encephalomyelitis/brainstem encephalitis (0/6 vs 5/6, p = 0.07 by the McNemar test). CSF = cerebral spinal fluid; EV = enterovirus; mNGS = metagenomic next-generation sequencing; NP = nasopharyngeal; OND = other neurologic disease.