Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing

Abstract

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

Keywords: VA/HMO astrovirus clade; deafness; encephalitis; next-generation sequencing; pathogen discovery.

Figure 1.

A, Magnetic resonance imaging of the brain. Coronal (left) and axial (right) views show T2-weighted abnormalities in the thalamus and midbrain (white arrows). B, Histological and immunohistochemical studies of brain biopsy tissue. A brain biopsy from the right frontal lobe showed diffuse striking reactive gliosis in the neuroparenchyma highlighted by immunostaining for glibal fibrillary acidic protein (1). Diffuse scattering of small numbers of reactive T lymphocytes immunopositive for CD3 (2, black arrows) is seen. Immunostaining for CD8, a microglial marker, revealed marked microglial activation within the cortex (3) and white matter (4). Microglial cell processes were often seen in close contact with neurons (5, black arrows). No viral inclusions were observed. C, A metagenomic next-generation sequencing assay for infectious disease diagnosis. Using the sequence-based ultra-rapid pathogen identification (SURPI) pipeline [3], >381 million reads generated from the patient's brain biopsy sample were analyzed in approximately 5 hours for potential pathogens. D, Genome recovery and pairwise comparative analysis of human astrovirus (HAstV) VA1/HMO-C-UK1. Mapping of 1612 Astroviridae reads identified by SURPI resulted in recovery of >94.9% of the 6586 base pair (bp) genome (coverage plot; shown mapped to the genome of HAstV-VA1/HMO-C-UK1), with sequence confirmation using polymerase chain reaction (PCR) (blue lines). Pairwise identity plots revealed high (>95%) nucleotide identity shared between the patient's astrovirus (AstV) strain (HAstV-VA1/HMO-C-UK1) and other VA-1/HMO-C viruses. E, In situ hybridization of brain tissue from the affected patient. Nucleic acid hybridization using probes to the AstV capsid gene (red) and the GADPH control (blue) reveals selective labeling of infected neurons (yellow, nuclei stained with DAPI). The estimated proportion of cells staining positive for AstV was approximately 0.6%. F, Phylogenetic tree of human and encephalitic mammalian AstVs. VA-1/HMO-C viruses in the VA/HMO clade associated with encephalitis in humans (HAstV-VA1/HMO-C-UK1 and HAstV-SG) are more closely related to encephalitic AstVs in other mammals (MAstV and BAstV) than to classic HAstVs 1–8 or AstVs in the MLB clade. Mammalian AstVs linked to encephalitis are highlighted in red text. Abbreviations: BAStV, bovine astrovirus; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMOAst-C, astrovirus HMO-C; MAstV, mink astrovirus; ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; SURPI, sequence-based ultra-rapid pathogen identification.