Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.Human astroviruses (HAstV) have been found to be a frequent cause of gastroenteritis among young children worldwide (5,7,8,15). The virions are formed by a nonenveloped protein capsid and a positive-stranded RNA genome of approximately 7 kb (9, 22). The RNA genome has three open reading frames (ORFs) (ORF1a, -1b, and -2), each encoding at least one polyprotein. The ORF1a contains viral serine protease and nuclear localization signal motifs, whereas the ORF1b has an RNA-dependent RNA polymerase motif (9,22). The products of ORF1a and ORF1b are synthesized from the genomic RNA as two polyproteins, with the latter being produced as a polyprotein 1a-1b (approximately 160 kDa) through a frameshift translational mechanism (12)(13)(14). It is believed that the products of these ORFs, processed to smaller polypeptides by the viral protease, are involved in the viral RNA replication. ORF2, of approximately 780 amino acid residues, depending on the strain (21), codes for the structural virus polypeptides (18). The structural polyprotein is translated from a polyadenylated subgenomic RNA produced at high levels during infection, which is 3Ј-colinear with the genomic RNA (17). Based on the homology among strains belonging to different serotypes, at least two domains in the product of this ORF have been predicted (16,21). The first domain includes amino acid residues 1 to 415, and it is highly conserved among all the human serotypes and some viruses from animal origin; the second domain (amino acid 416 to the end) is highly divergent among human serotypes (10,16,21,23). Neutralizing epitopes have been mapped to the second domain (3, 20); therefore, it is likely that this hypervariable region is exposed on the viral particle. It is known that astrovirus in...
The prevalence and type diversity of human astroviruses (HAstV) in children with symptomatic and asymptomatic infections were determined in five localities of Mexico. HAstV were detected in 4.6 (24 of 522) and 2.6% (11 of 428) of children with and without diarrhea, respectively. Genotyping of the detected strains showed that at least seven (types 1 to 4 and 6 to 8) of the eight known HAstV types circulated in Mexico between October 1994 and March 1995. HAstV types 1 and 3 were the most prevalent in children with diarrhea, although they were not found in all localities studied. HAstV type 8 was found in Mexico City, Monterrey, and Mérida; in the last it was as prevalent (40%) as type 1 viruses, indicating that this astrovirus type is more common than previously recognized. A correlation between the HAstV infecting type and the presence or absence of diarrheic symptoms was not observed. Enteric adenoviruses were also studied, and they were found to be present in 2.3 (12 of 522) and 1.4% (6 of 428) of symptomatic and asymptomatic children, respectively.
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
In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.
The relative contribution of the rotavirus surface proteins, VP4 and VP7, to the induction of homotypic as well as heterotypic neutralizing antibodies (NtAbs) in natural infections was studied. The NtAb titers of paired sera from 70 infants with serologically defined primary rotavirus infections were determined with a panel of rotavirus reassortants having one surface protein from a human rotavirus (serotypes G1 to G4 for VP7 and P1A and P1B for VP4) and the other surface protein from a heterologous animal rotavirus strain. A subset of 37 children were evaluated for epitope-specific antibodies to the two proteins by an epitope-blocking assay. The infants were found to seroconvert more frequently to VP4 than to VP7 by both methods, although the titers of the seroconverters were higher to VP7 than to VP4. Both proteins induced homotypic as well as heterotypic NtAbs. G1 VP7 frequently induced a response to both G1 and G3 VP7s, while G3 VP7 and P1A VP4 induced mostly homotypic responses.
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