Astrovirus encephalitis in boy with X-linked agammaglobulinemia

Abstract

Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.

Figure 1

Histologic findings from brain of 15-year-old boy with X-linked agammaglobulinemia and astrovirus encephalitis. A) Frontal cortex with cortical thinning (double-headed arrow) and vacuolation (arrows) (Luxol fast blue stain with periodic acid–Schiff method [LFB/PAS], original magnification ×10). B) Frontal cortex with vacuolation (arrow) and rare residual neurons (arrowheads) (LFB/PAS, original magnification ×50). C) Marked astrogliosis in the frontal cortex (glial fibrillary acidic protein [GFAP] immunostain, original magnification ×100). D) White matter with marked astrogliosis (GFAP immunostain, original magnification ×100). E) Penetrating artery with abundant CD3+ T-cells in the perivascular space and adjacent brain parenchyma (CD3⁺ immunostain, original magnification ×25). F) CD3⁺ T-cells as part of microglial nodules (CD3⁺ immunostain, original magnification ×100). G) White matter in the internal capsule showing myelin clumps (arrowheads) and vacuolation (arrow) (LFB/PAS, original magnification ×100). H) Loss of axons in the internal capsule (Bielschowsky nerve fiber silver stain, original magnification ×100). I) Internal capsule with marked microgliosis (CD68⁺ immunostain, original magnification ×40). J) Microcalcifications (arrow) in the globus pallidus (LFB/PAS, original magnification ×100). All paraffin sections were counterstained with hematoxylin.

Figure 2

Schematic genome organization of human astrovirus Puget Sound (HAstV-PS). Arrows represent the 3 open reading frames of the 6,584-nt single-strand, positive-sense genome. Bars above the schematic indicate the 12 contiguous fragments (contigs A–L) generated through unbiased high-throughput sequencing. PCR primers for amplification across sequence gaps were designed based on the unbiased high-throughput sequencing data, and the draft genome was resequenced by overlapping PCR products that covered the entire genome except for terminal sequences. Genomic termini were characterized with 5′ and 3′ rapid amplification of cDNA ends kits (Invitrogen, Carlsbad, CA, USA). Arrowheads indicate primer locations. PRO, protease; POL, polymerase; CA, capsid; (A)_n, poly-A tail.

Figure 3

Phylogenetic analysis of full-length capsid protein sequences showing the relationship between human astrovirus Puget Sound (HAstV-PS) identified in brain of 15-year-old boy with X-linked agammaglobulinemia and encephalitis and other astroviruses. GenBank accession numbers in parentheses: MLB1 (FJ22245), VA1 (FJ973620), HAstV-1 (AB000295), HAstV-2 (L06802), HAstV-3 (DQ630763), HAstV-4 (AB025803), HAstV-5 (U15136), HAstV-6 (Z46658), HAstV-7 (Y08632), HAstV-8 (Z66541), MAstV (AY179509), OAstV (NC_002469), BatAstV (FJ571074), FAstV (AF056197), ChAstV (EU650331), PAstV (Y15938), ANV 1 (AB033998), ANV 2 (AB046864), TAstV 1 (Y15936), and TAstV 2 (AY769615). Bayesian posterior probability values >75% are shown at respective nodes. FAstV, feline astrovirus; ChAstV, cheetah astrovirus; PAstV, porcine astrovirus; OAstV, ovine astrovirus; MAstV, mink astrovirus; ANV, avian nephritis virus; TAstV, turkey astrovirus. Scale bar indicates number of amino acid substitutions per site.

Figure 4

Immunofluorescence and immunohistochemical analyses with human astrovirus Puget Sound capsid antibodies. A) Indirect double immunofluorescence–stained, formalin-fixed, paraffin-embedded tissue sections from 15-year-old boy with X-linked agammaglobulinemia and astrovirus encephalitis and a control with astrogliosis not caused by astrovirus infection. The sections were stained for the astrocyte marker glial fibrillary acidic protein (GFAP, green) and for viral capsid protein (rabbit serum 1:1,000, red). Viral capsid protein is present in hypertropic astrocytes throughout the subcortical white matter and cortex; astrocytes have swollen cell bodies with intense GFAP immunostaining. Blue signal (DAPI) indicates nuclear counterstaining. Original magnification ×100. B) Immunohistochemical localization of viral antigen in a frontal cortex biopsy specimen. Immunoalkaline phosphatase stain with viral capsid antibodies (rabbit serum 1:1,000) and naphthol-fast red with hematoxylin counterstain. Original magnification ×158.