RESUMO
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.
Assuntos
Genoma Viral , Mamastrovirus/química , Mamastrovirus/genética , Sequência de Aminoácidos , Células CACO-2 , Genótipo , Humanos , Mamastrovirus/classificação , Mamastrovirus/crescimento & desenvolvimento , México , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Poliproteínas/química , Poliproteínas/genética , Alinhamento de Sequência , Deleção de Sequência/genética , Homologia de Sequência de Aminoácidos , Sorotipagem , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Glicoproteínas/metabolismo , Mamastrovirus/metabolismo , Sequência de Aminoácidos , Células CACO-2 , Capsídeo/genética , Glicoproteínas/genética , Humanos , Mamastrovirus/classificação , Mamastrovirus/genética , Mamastrovirus/patogenicidade , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sorotipagem , Tripsina , VirulênciaRESUMO
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.