Chapter 51 : Astrovirus

(A) The six- and five-point star-like morphology of astrovirus can be observed in fecal samples by negative staining and EM. (Reprinted from reference 1 with permission.) (B) Paracrystalline arrays of human astrovirus particles observed by transmission EM in infected Caco-2 cells; virus clusters (V) are usually localized at the periphery of nuclei (N). (C) Three-dimensional electron cryomicroscopy density maps of immature and mature HAstV. Mature virions only display 30 of 90 spikes after proteolytic cleavage. (Reprinted from reference 13 with permission.)

Astrovirus genome organization and synthesis of the nonstructural proteins. (A) The genome of astrovirus (shown here for HAstV-8) is organized into three ORFs: 1a, 1b, and 2. A fourth hypothetical ORFx is shown. The genomic RNA is a positive-sense RNA of around 6.8 kilobases that has a VPg protein covalently bound to its 5’-end and a polyA tail at the 3’-end. (B) Three nonstructural proteins have been positively identified: the viral protease, a VPg protein, and the RNA-dependent RNA polymerase (RdRp). In addition, other conserved structural motifs in the nsp1a polyprotein have also been identified: a putative helicase domain (Hel), a coiled-coil (cc) domain, several transmembrane domains (tm), and a hypervariable region (hvr). The RdRp is synthesized as part of a long polyprotein called nsp1ab, that results from translation of ORF1a and ORF1b as a single polypeptide, through a ribosomal frame shift mechanism directed by highly conserved cis-acting sequences (the heptanucleotide AAAAAAC and one pseudoknot) localized in the overlap region between ORFs 1a and 1b ( 26 ). The intermediate protease (viral and cellular) cleavage products of polyproteins nsp1a and nsp1b, as well as the final produced polypeptides, are not shown here, but can be consulted for HAstV-8 in other publications ( 28 , 40 ). The cellular protease (red arrow head) and viral protease (black arrow heads) cleavage sites are indicated.

Astrovirus genome replication and transcription and synthesis of the structural proteins. (A) The genome of astrovirus (shown here for HAstV-8) is used as template to synthesize the negative-sense antigenomic RNA (agRNA), which, in turn, serves as template to synthesize the full-length positive sense genomic (gRNA) and subgenomic (sgRNA) RNAs. The sgRNA is about 2.4 kilobases and has a polyA tail at the 3’-end and likely also has a VPg protein covalently bound at its 5’-end. (B) The sgRNA is translated into the structural polyprotein precursor VP90, which contains an N-terminal conserved domain and a C-terminal hypervariable domain. The conserved domain has a basic amino acid region that is thought to interact with the viral RNA and a second region that has been predicted to be the shell of the viral capsid. The hypervariable domain conforms the viral spikes, and contains an acidic region towards the carboxy terminus of VP90. The processing pathway of this precursor protein has been described in detail ( 3 ). Briefly, VP90 is assembled into particles, and the acidic region at its carboxy terminus is processed to yield a protein of 70 kilodaltons (VP70) ( 43 ). This processing is carried out by caspases, cellular proteases activated during virus infection that are involved in cell death by apoptosis. The caspase cleavages correlate with, and are required for, the release of virions from the cells ( 43 , 44 ). The released virions formed by VP70 are poorly infectious, and full activation of their infectivity requires that VP70 is cleaved by extracellular trypsin. Processing of the VP70-containing particles by this enzyme is sequential, producing final protein products of 34, 27, and 25 kilodaltons through at least eight intermediary polypeptides ( 3 ). The black arrow head represents a site for caspase cleavage and the red arrow heads, sites for trypsin cleavage. The intermediate caspases and trypsin cleavage products are not shown but can be found in other publications ( 28 , 40 ).

(A) Photomicrograph of a jejunal biopsy specimen from a bone marrow transplant recipient with astrovirus infection demonstrating villus blunting, nonspecific alterations in surface epithelial cells and a mixed lamina propria inflammatory infiltrate but without the presence of viral inclusion bodies (original magnification, x100). Also shown are photomicrographs of duodenal (B) and jejunal (C) biopsy samples from a bone marrow transplant recipient with astrovirus infection immunostained with anti-astrovirus antibody and demonstrating progressively extensive staining of surface epithelial cells, most commonly near the villus tips (original magnifications, x40 and x100, respectively). (D) Electron micrographs of a jejunal enterocyte demonstrating cytoplasmic paracrystalline viral arrays of astrovirus (original magnifications, x32,000 and x100,000 [inset]). (Reprinted from reference 68 with permission.)

Detection of the capsid protein of HAstV VA-1 in neuronal tissue from immunosuppressed individuals. (A) Immunohistochemistry with an antibody against astrovirus shows extensive staining of cell bodies and processes in the neurophil. Some of the astrovirus-positive cells have the morphology of pyramidal neurons (indicated by an arrow; scale bar, 50 µm). (B) Electron microscopy of the same patient in (A) showed a rare focus of crystalline material but no viral particles (scale bar, 2 µm). (C) Indirect immunofluorescence of brain tissue sections from a 15-year-old boy with X-linked agammaglobulinemia and astrovirus encephalitis (patient), and a control patient with astrogliosis not caused by astrovirus infection (control). The sections were stained for the astrocyte marker glial fibrillary acidic protein (GFAP, green) and for viral capsid protein (red); the blue color (DAPI) indicates nuclear counterstaining. Original magnification, ×100. (Adapted from references 103 (A and B) and 102, with permission.)