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Human astrovirus (HAstV) represents the third most common virus associated with acute diarrhea (AD). This study aimed to estimate the prevalence of HAstV infection in Brazilian children under 5 years of age with AD, investigate the presence of recently described HAstV strains, through extensive laboratory-based surveillance of enteric viral agents in three Brazilian coastal regions between 2005 and 2011. Using reverse transcription-polymerase chain reaction (RT-PCR), the overall HAstV detection rate reached 7.1% (207/2.913) with percentage varying according to the geographic region: 3.9% (36/921) in the northeast, 7.9% in the south (71/903) and 9.2% in the southeast (100/1.089) (p < 0.001). HAstV were detected in cases of all age groups. Detection rates were slightly higher during the spring. Nucleotide sequence analysis of a 320-bp ORF2 fragment revealed that HAstV-1 was the predominant genotype throughout the seven years of the study. The novel AstV-MLB1 was detected in two children with AD from a subset of 200 samples tested, demonstrating the circulation of this virus both the in northeastern and southeastern regions of Brazil. These results provide additional epidemiological and molecular data on HAstV circulation in three Brazilian coastal regions, highlighting its potential to cause infantile AD.

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Os astrovírus humanos (HAstVs) pertencem a família Astroviridae e são associados agastrenterite aguda (GA) em crianças menores de cinco anos, tanto nos paísesdesenvolvidos como naqueles em desenvolvimento, o que os tornam de interesse nocampo da Saúde Pública. A família Astroviridae é dividida em dois gêneros:Avastrovirus e Mamastrovirus. No gênero Mamastrovirus, encontram-se os astrovirusassociados à infecção em mamíferos, tanto humanos como animais. Até 2008, osastrovirus associados a doenças em humanos eram restritos a oito genótipos,conhecidos como HAstV 1-8. A partir de então novos HAstVs foram sendo descritos,associados a doenças em humanos, como os HAstVs MLB1-3 e os HAstVs VA1-4.Opresente estudo consiste no estudo epidemiológicos retrospectivos (1994 a 2011) paradetecção e caracterização molecular de HAstV em amostras de fezes provenientes decrianças com menos de cinco anos de idade com GA, em diferentes regiões do Brasil:Nordeste, Sudeste e Sul. Incluem-se neste trabalho três estudos: 1) Estudo dosHAstV em casos esporádicos de GA ocorridos em crianças menores de cinco anos deidade, em três regiões brasileiras (Nordeste, Sudeste e Sul), durante o período de2005 a 2011, incluindo a pesquisa dos novos HAstV; 2) Estudo dos HAstV em criançascom GA, atendidas na creche Bertha Lutz, FIOCRUZ-RJ, durante o período de janeirode 1994 a dezembro de 2008; 3)...

Human astrovirus (HAstVs), belong to Astroviridae family, and are associatedwith acute gastroenteritis (GA) in children under five years-old, both indeveloped and in developing countries, which makes them of interest in thePublic Health field. The Astroviridae family is divided into two genera:Avastrovirus and Mamastrovirus. Mamastrovirus are the astrovirusesassociated to infection in mammals, both humans and animals. By 2008, theastrovirus associated with human disease were restricted to eight genotypes,known as HAstV 1-8. Since then, new HAstVs have been described, associatedwith human disease, such as HAstVs MLB1-3 and HAstVs VA1-4. The presentstudy is the retrospective epidemiological study (1994 to 2011) for the detectionand molecular characterization of HAstV in stool samples from children underfive years old presenting GA, in different regions of Brazil: Northeast, Southeastand South. Three studies are presented: 1) Study of HAstV in sporadic cases ofGA occurred in children under five years old in three Brazilian regions(Northeast, Southeast and South) from 2005 to 2011, including the descriptionof a new HAstV; 2) Study of HAstV in children with GA, attending the day careBertha Lutz, FIOCRUZ-RJ from January 1994 to December 2008 and 3) Studyof HAstV in children under two years old presenting GA and hospitalized inNiteroi, Rio de Janeiro from April to September 2003. The detection of HAstVwas performed using different protocols for detection and molecularcharacterization such as: Reverse–transcriptase polymerase chain reaction,(RT- PCR), Single step RT -PCR (OneStep RT-PCR) and RealTime RT- PCR.The HAstV detected were characterized by partial sequencing of ORF2 regionof the viral genome. The study 1 demonstrated the HAstV detection frequencyin 7.1 % of samples, and described the first ASTV MLB1 in Brazil. Themolecular characterization identified the circulation genotypes HAstV -1, 2, 3, 4,5, 6 and 8...

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In this work we have shown that astrovirus infection induces apoptosis of Caco-2 cells, since fragmentation of cellular DNA, cleavage of cellular proteins which are substrate of activated caspases, and a change in the mitochondrial transmembrane potential occur upon virus infection. The human astrovirus Yuc8 polyprotein capsid precursor VP90 is initially processed to yield VP70, and we have shown that this processing is trypsin independent and occurs intracellularly through four cleavages at its carboxy-terminal region. We further showed that VP90-VP70 processing is mediated by caspases, since it was blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk), and it was promoted by the apoptosis inducer TNF-related apoptosis-inducing ligand (TRAIL). Although the cell-associated virus produced in the presence of these compounds was not affected, the release of infectious virus to the cell supernatant was drastically reduced in the presence of z-VAD-fmk and increased by TRAIL, indicating that VP90-VP70 cleavage is important for the virus particles to be released from the cell. This is the first report that describes the induction and utilization of caspase activity by a virus to promote processing of the capsid precursor and dissemination of the viral particles.

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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.

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