Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients

Abstract

Background: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy.

Methods: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking.

Results: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child.

Conclusions: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Keywords: RNASeq; astrovirus; deep sequencing; encephalopathy; pathogen discovery.

Figure 1.

Phylogenetic relationship of human astrovirus (HAstV)–VA1/HMO-C-UK1(a) () and -UK1(b) () to other astroviruses (AstVs) based on RNA-dependent RNA polymerase nucleotide sequences. indicates other AstV species that have been reported in patients with neurological disease. indicates sequences of animal origin, not human. Scale bar represents the number of base substitutions per site. Abbreviations: BAstV, bat astrovirus; MAstV, mink astrovirus; OAstV, ovine astrovirus; TAstV, turkey astrovirus.

Figure 2.

A, AstV-contig polymerase chain reaction results for all retrospectively tested samples from patient A. Data points at the base of the y-axis indicate astrovirus (HAstV)–VA1/HMO-C-UK1(a) RNA not detected. Cycle threshold (Ct) values represent the cycle number at which amplification was detected and thus have an inverse relationship with viral titer; a smaller Ct value indicates a higher titer. In clinical practice, Ct >38 may be considered equivocal but, in the context of other positive results, is considered a true positive. A difference of 3 Ct values is approximately equivalent to a 10-fold difference in viral load. B, Clinical and diagnostic timeline. Abbreviations: AstV, astrovirus; CSF, cerebrospinal fluid; HAstV, human astrovirus; NPA, nasopharyngeal aspirate.

Figure 3.

The brain biopsy showed extensive neuronal apoptosis in the cerebral cortex as demonstrated by pyknotic and karyorrhectic neurons (A, arrow) (hematoxylin and eosin [H&E], scale bar, 10 µm). There was a brisk microglial reaction demonstrated by CD68 immunohistochemistry with some nodules of microglia (B; scale bar, 100 µm). In contrast, there was no significant lymphocytic reaction (C, CD3 immunohistochemistry; scale bar, 100 µm). There was an extensive astrocytosis (D, glial fibrillary acidic protein [GFAP]; scale bar, 100 µm). Immunohistochemistry with an antibody against astrovirus showed extensive staining of cell bodies and processes in the neuropil (E; scale bar, 50 µm). Some of the positive cells had the morphology of pyramidal neurons (arrow). Electron microscopy showed a rare focus of crystalline material but no viral particles (F; scale bar, 2 µm).