Molecular analysis of a serotype 8 human astrovirus genome

Abstract

Human astroviruses are an important cause of gastroenteritis. As part of a molecular epidemiological study carried out in Mexico a human astrovirus isolate, Yuc-8, was adapted to grow in CaCo-2 cells, and its entire genome was sequenced. A 15 amino acid deletion in ORF1a, which has been associated with adaptation of astroviruses to grow in cells other than CaCo-2, was present in Yuc-8. Comparative sequence analysis of the Yuc-8 ORF2 with reported human astrovirus sequences revealed that this isolate belongs to genotype (serotype) 8. Two distinct domains in ORF2 were observed : an amino-terminal domain (residues 1 to 415), with identities higher than 81 % among the strains analysed, and a carboxy-terminal domain (residues 416 to 782) with identities between 36 and 60 %. Two non-superimposable phylogenetic trees were generated by separate analysis of these two domains, suggesting that a differential selective pressure is exerted along the structural polyprotein.Human astroviruses are recognized as an important cause of infantile gastroenteritis around the world . Astrovirus virions are formed by a non-enveloped protein capsid which surrounds a genome consisting of a positive-sense, single-stranded RNA molecule of 6n8 to 7n2 kb in length . The genomic RNA has three open reading frames (ORFs) designated 1a, 1b and 2. ORF1a and ORF1b code apparently for the nonstructural proteins of the virus ; ORF1a contains viral serine protease and nuclear localization signal motifs ; ORF1b contains motifs suggestive of an RNA-dependent RNA polymerase

Wastewater Analysis Indicates that Genetically Diverse Astroviruses, Including Strains Belonging to Novel Clades MLB and VA, Are Circulating within Japanese Populations

“…The assay targeting the 5= end of ORF2 (30) showed improved detection and quantification compared to those of the other assay targeting the 3= end of ORF2 (36). The genetic sequences on the 5= end of the ORF2 region are more conserved than those on the 3= end of the ORF2 region among classic HAstV strains (40). Thus, the concentration of classic HAstVs determined in our previous study using the assay targeting the 3= end of ORF2 was underestimated.…”

C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA

“…An immunoreactive epitope close to the C terminus of nsP1a was partially characterized some years ago (35). In addition, a hypervariable region has been identified close to this epitope by different authors after genetic characterization of HAstV-3 and HAstV-8 (37,41).…”

Protein Products of the Open Reading Frames Encoding Nonstructural Proteins of Human Astrovirus Serotype 8

“…Total RNA from Yuc8-infected cells was extracted with Trizol reagent (GIBCO/BRL) 12 h postinfection (hpi) and used for reverse transcription-PCR to amplify ORF1a and ORF1b. The cDNA fragments obtained were sequenced and compared with the previously reported sequence (18) to confirm their identity. Three nucleotide changes were observed in ORF1a, one conservative and two which resulted in amino acid changes (Leu to His at amino acid positions 3 and 361).…”

“…Cells were cultured in a CO 2 atmosphere at 37°C with minimum essential medium (Eagle's salts) (MEM), supplemented with glutamine, and 10% (BHK-21) or 15% (Caco-2) fetal bovine serum (GIBCO/BRL). The HAstV strain Yuc8, isolated from a Mexican child, was adapted to grow in Caco-2 cells (18). Viral stocks from passages 7 to 15 were used in this work, and they were propagated as described previously (17), except that the virus was treated with 200 g of trypsin/ml for 1 h at 37°C before infection.…”