RESUMEN

This report aims to describe the identification of porcine astrovirus 3 (PAstV3) RNA in the central nervous system (CNS) of weaned pigs with clinical signs of neurological disease associated with polioencephalomyelitis in southeastern Brazil. Three, 20 -35 days-old piglets that died after clinical manifestations of a neurological syndrome were submitted to post-mortem evaluations. Tissue samples were examined by histopathology, bacteriology, and molecular assays (RT-PCR, nested-PCR, RT-qPCR, and Sanger sequencing) to detect the primary infectious disease agents associated with neurological disease in pigs. The principal neuropathological alterations occurred in the grey matter of the spinal cord and brainstem resulting in nonsuppurative poliomyelitis and rhombencephalitis. PAstV3 RNA was detected in the CNS samples of all piglets with histopathological evidence of disease and was confirmed by nucleotide sequencing. Nucleic acids from pathogens commonly associated with neurological diseases in pigs, such as porcine teschovirus, porcine sapelovirus, porcine enterovirus G, atypical porcine pestivirus, senecavirus A, and encephalomyocarditis virus was not detected by molecular assays in the three piglets. This is the first report of PAstV3 in piglets with neurological disease and lesions consistent with polioencephalomyelitis in Brazil. This report highlights the importance of monitoring health events that could compromise pig farming productivity and animal welfare.

Asunto(s)

Encefalomielitis , Mamastrovirus , ARN Viral , Enfermedades de los Porcinos , Animales , Porcinos , Brasil , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/patología , ARN Viral/genética , Mamastrovirus/aislamiento & purificación , Mamastrovirus/genética , Encefalomielitis/veterinaria , Encefalomielitis/virología , Encefalomielitis/patología , Infecciones por Astroviridae/veterinaria , Infecciones por Astroviridae/virología , Infecciones por Astroviridae/patología , Filogenia , Sistema Nervioso Central/virología , Sistema Nervioso Central/patología , Médula Espinal/patología , Médula Espinal/virología

RESUMEN

Astroviruses are highly divergent and infect a wide variety of animal hosts. In 2009, a genetically divergent human astrovirus (HAstV) strain VA1 was first identified in an outbreak of acute gastroenteritis. This strain has also been associated with fatal central nervous system disease. In this work, we report the isolation of three high-affinity neutralizing monoclonal antibodies (Nt-MAbs) targeting the capsid spike domain of HAstV-VA1. These antibodies (7C8, 2A2, 3D8) were used to select individual HAstV-VA1 mutants resistant to their neutralizing activity and a HAstV-VA1 triple mutant that escapes neutralization from all three Nt-MAbs. Sequencing of the virus genome capsid region revealed escape mutations that map to the surface of the capsid spike domain, define three potentially independent neutralization epitopes, and help delineate four antigenic sites in human astroviruses. Notably, two of the escape mutations were found to be present in the spike sequence of the HAstV-VA1-PS strain isolated from an immunodeficient patient with encephalitis, suggesting that those mutations arose as a result of the immune pressure generated by the patient's immunotherapy. In agreement with this observation, human serum samples exhibiting strong neutralization activity against wild-type HAstV-VA1 had a 2.6-fold reduction in neutralization titer when evaluated against the triple-escape HAstV-VA1 mutant, suggesting that both mouse and human antibody responses target shared neutralization epitopes. The isolated Nt-MAbs reported in this work will help to characterize the functional domains of the virus during cell entry and have the potential for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1. IMPORTANCE: Human astroviruses (HAstVs) have been historically associated with acute gastroenteritis. However, the genetically divergent HAstV-VA1 strain has been associated with central nervous system disease. In this work high-affinity neutralizing monoclonal antibodies directed to HAstV-VA1 were isolated and characterized. The proposed binding sites for these antibodies and for neutralizing antibodies against classical HAstVs suggest that there are at least four neutralization sites on the capsid spike of astroviruses. Our data show that natural infection with human astrovirus VA1 elicits a robust humoral immune response that targets the same antigenic sites recognized by the mouse monoclonal antibodies and strongly suggests the emergence of a variant HAstV-VA1 virus in an immunodeficient patient with prolonged astrovirus infection. The isolated Nt-MAb reported in this work will help to define the functional sites of the virus involved in cell entry and hold promise for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1.

Asunto(s)

Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Humanos , Animales , Anticuerpos Neutralizantes/inmunología , Ratones , Epítopos/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Mamastrovirus/inmunología , Mamastrovirus/genética , Mutación , Infecciones por Astroviridae/inmunología , Infecciones por Astroviridae/virología , Pruebas de Neutralización

RESUMEN

AIMS: To estimate the risk of human rotavirus (RV) and astrovirus (HAstV) infections for swimmers and fishers at Las Cañas beach, Uruguay. METHODS AND RESULTS: Surface water samples were collected monthly for 1 year. The dose-response models used were ß-Poisson and 1 F1 hypergeometric for RV and HAstV, respectively. The probabilities of infection were calculated using a kernel density estimate to fitting the data and then sampling from this distribution (Monte Carlo simulation). The probability of RV infection for fishers was between 0 and 65% and for swimmers was between 0 and 50% (<18 years old) and between 0 and 38% (>18 years old). For HAstV, the probability of infection for fishers was between 0% and 45% and for swimmers was between 0 and 38% (<18 years old) and between 0 and 18% (>18 years old). CONCLUSIONS: This study suggests that fishers are at higher risk of infection for both viruses compared with swimmers mainly due to higher viral frequency and concentration at the site for fishing activities.

Asunto(s)

Infecciones por Astroviridae , Mamastrovirus , Rotavirus , Humanos , Adolescente , Rotavirus/genética , Mamastrovirus/genética , Natación , Uruguay/epidemiología , Caza , Heces

RESUMEN

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

Asunto(s)

Infecciones por Astroviridae , Vesículas Extracelulares , Gastroenteritis , Mamastrovirus , Anticuerpos Neutralizantes , Infecciones por Astroviridae/inmunología , Infecciones por Astroviridae/virología , Vesículas Extracelulares/virología , Gastroenteritis/virología , Humanos , Mamastrovirus/inmunología

RESUMEN

Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.

Asunto(s)

Infecciones por Astroviridae , Proteínas de la Cápside , Mamastrovirus , Precursores de Proteínas , Infecciones por Astroviridae/virología , Células CACO-2 , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Humanos , Espacio Intracelular/virología , Mamastrovirus/fisiología , Precursores de Proteínas/metabolismo , Tripsina/metabolismo

RESUMEN

Astroviruses are common pathogens of the human gastrointestinal tract, but they have been recently identified from cases of fatal meningoencephalitis. Astrovirus VA1 is the most frequently detected astrovirus genotype from cases of human encephalitis, but the prevalence of neutralizing antibodies to VA1 in human sera is unknown. We developed a focus reduction neutralization assay (FRNT) for VA1 and measured the seroprevalence of neutralizing antibodies from two cohorts of adult and pediatric serum samples: (i) an age-stratified cohort from St. Louis, MO, collected from 2007 to 2008 and (ii) a cohort from the Peruvian Amazonian River Basin collected in the late 1990s. In the St. Louis cohort, the lowest seropositivity rate was in children 1 year of age (6.9%), rising to 63.3% by ages 9 to 12, and 76.3% of adults ≥20 years were positive. The Peruvian Amazon cohort showed similar seropositivity rates across all ages, with individuals under age 20 having a rate of 75%, while 78.2% of adults ≥20 years were seropositive. In addition, we also identified the presence neutralizing antibodies to VA1 from commercial lots of intravenous immunoglobulin (IVIG). Our results demonstrate that a majority of humans are exposed to VA1 by adulthood, with the majority of infections occurring between 2 and 9 years of age. In addition, our results indicate that VA1 has been circulating in two geographically and socioeconomically divergent study cohorts over the past 20 years. Nonetheless, a significant proportion of the human population lacks neutralizing immunity and remains at risk for acute infection. IMPORTANCE Astroviruses are human pathogens with emerging disease associations, including the recent recognition of their capacity to cause meningoencephalitis. Astrovirus VA1 is the most commonly identified astrovirus genotype from cases of human encephalitis, but it is unknown what percentage of the human population has neutralizing antibodies to VA1. We found that 76.3 to 78.2% of adult humans ≥20 years of age in two geographically and socioeconomically distinct cohorts are seropositive for VA1, with the majority of infections occurring between 2 and 9 years of age. These results demonstrate that VA1 has been circulating in human populations over the past 2 decades and that most humans develop neutralizing antibodies against this virus by adulthood. However, a subset of humans lack evidence of neutralizing antibodies and are at risk for diseases caused by VA1, including encephalitis.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Astroviridae/epidemiología , Mamastrovirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Recién Nacido , Modelos Logísticos , Masculino , Mamastrovirus/genética , Persona de Mediana Edad , Missouri/epidemiología , Perú/epidemiología , ARN Viral/genética , Estudios Seroepidemiológicos , Adulto Joven

RESUMEN

This study combined conventional epidemiology of human astroviruses. From 2010 to 2016, 232 stool samples from children under 5 years of age were screened using NGS and conventional RT-PCR followed by genetic analysis in order to investigate the genotypic diversity of classical human astrovirus (HAstV) circulating in Tocantins State, Brazil. HAstV was detected in 16 cases (6.9%). Seven specimens (43.7%; 7/16) were positive according RT-PCR and next-generation sequencing (NGS) to investigate the molecular to both NGS and RT-PCR. NGS and RT-PCR individually revealed six (37.5%; 6/16) and three (18.8%; 3/16) additional positive samples, respectively. Sequencing of the HAstV-positive samples revealed HAstV-1a (9/16), HAstV-4c (3/16), and HAstV-5c (4/16) lineages.

Asunto(s)

Infecciones por Astroviridae/virología , Gastroenteritis/virología , Mamastrovirus/genética , Infecciones por Astroviridae/epidemiología , Brasil/epidemiología , Preescolar , Heces/virología , Femenino , Gastroenteritis/epidemiología , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Mamastrovirus/aislamiento & purificación , Filogenia , Población Rural

Asunto(s)

Infecciones por Astroviridae/diagnóstico , Infecciones por Astroviridae/virología , Mamastrovirus/clasificación , Mamastrovirus/genética , Brasil , Niño , Preescolar , Heces/virología , Femenino , Gastroenteritis/virología , Humanos , Lactante , Masculino , Mamastrovirus/aislamiento & purificación , ARN Viral/genética

RESUMEN

ABSTRACT Human astrovirus (HAstV) 1-8 and highly divergent HAstVMLB1−3 genotypes have been detected in children both with and without acute gastroenteritis (AGE). One hundred and seventy fecal samples from children (≤5 years old) living in the Amazon region were evaluated for the presence of HAstV1-8, HAstV MLB1−3 and HAstVVA1−3, using an usual RT-PCR protocol and a new protocol with specific primers designed to detect HAstVMLB1−3. HAstVMLB1 and HAstV MLB2, as well as the HAstV3 and 5 genotypes were detected. HAstVMLB1−2 genotype was detected for the first time in Brazil at a frequency of 3.5% (6/170).

Asunto(s)

Niño , Humanos , Lactante , Mamastrovirus , Infecciones por Astroviridae , Gastroenteritis , Filogenia , Mamastrovirus/genética , Brasil , Infecciones por Astroviridae/diagnóstico , Infecciones por Astroviridae/epidemiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Heces , Gastroenteritis/diagnóstico , Genotipo

RESUMEN

Human astrovirus (HAstV) 1-8 and highly divergent HAstVMLB1-3 genotypes have been detected in children both with and without acute gastroenteritis (AGE). One hundred and seventy fecal samples from children (≤5 years old) living in the Amazon region were evaluated for the presence of HAstV1-8, HAstV MLB1-3 and HAstVVA1-3, using an usual RT-PCR protocol and a new protocol with specific primers designed to detect HAstVMLB1-3. HAstVMLB1 and HAstV MLB2, as well as the HAstV3 and 5 genotypes were detected. HAstVMLB1-2 genotype was detected for the first time in Brazil at a frequency of 3.5% (6/170).

Asunto(s)

Infecciones por Astroviridae , Gastroenteritis , Mamastrovirus , Infecciones por Astroviridae/diagnóstico , Infecciones por Astroviridae/epidemiología , Brasil , Niño , Heces , Gastroenteritis/diagnóstico , Genotipo , Humanos , Lactante , Mamastrovirus/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Classical human astroviruses (HAstV) are agents of nonbacterial acute gastroenteritis (AGE), being predominant among children. There are only a few studies reporting HAstV loads in samples from patients with AGE, data are even scarcer regarding asymptomatic patients. The aim of this study was to evaluate the occurrence and estimate the viral load of HAstV and to perform molecular characterization of positive samples obtained from children, up to 6 years old, with and without AGE. One fecal sample was obtained from each of the 250 children enrolled in the study, from May 2014 to April 2015. Real-time reverse transcription-polymerase chain reaction (RT-qPCR TaqMan) was performed, followed by a conventional RT-PCR directed to ORF2, region C, of the positive samples. Then, these amplicons were sequenced and a phylogenetic analysis was performed to determine the HAstV-1 lineages. A global positivity index of 3.2% (8 of 250) was observed for HAstV with a similar frequency (50%) in both symptomatic and asymptomatic group. Viral loads ranged from 2.8 × 105 to 1.6 × 1011 genome copy/mL Four samples were characterized as HAstV-1, lineage 1a and two as HAstV-4, lineage 4c. Our findings show similar HAstV positivity rates for children with and without AGE, providing evidence of HAstV-1a and HAstV-4c lineage cocirculation in the Central West region of Brazil. Data contributes to the molecular epidemiology of these agents in the region.

Asunto(s)

Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/virología , Infecciones Asintomáticas/epidemiología , Mamastrovirus/genética , Brasil/epidemiología , Preescolar , Heces/virología , Genoma Viral , Genotipo , Humanos , Lactante , Mamastrovirus/clasificación , Mamastrovirus/aislamiento & purificación , Filogenia , Carga Viral

RESUMEN

Although human astroviruses (HAstVs) are important agents of gastroenteritis in young children, the studies aimed at characterizing their biology have been limited, in particular regarding their cell entry process. It has been shown that HAstV serotype 8 enters human cells by a classical clathrin-mediated endocytosis pathway; however, the cell receptor or other cell entry factors that may be relevant for an efficient viral infection are unknown. In this work we used a far-Western blotting approach to identify cellular proteins that interact with the recombinant capsid spike proteins of HAstV serotypes 1, 2, and 8, synthesized in Escherichia coli. We identified the 72 kDa protein disulfide isomerase A4 (PDIA4) as a binding partner for HAstV-1 and -8 spikes, but not for the HAstV-2 spike. In agreement with this observation, the PDI inhibitor 16F16 strongly blocked infection by HAstV serotypes 1 and 8, but not serotype 2. RNA interference of PDIA4 expression selectively blocked HAstV-8 infectivity. We also showed that the PDI activity does not affect virus binding or internalization but is required for uncoating of the viral genome.

Asunto(s)

Infecciones por Astroviridae/metabolismo , Infecciones por Astroviridae/virología , Interacciones Huésped-Patógeno , Mamastrovirus/fisiología , Proteína Disulfuro Isomerasas/metabolismo , Desencapsidación Viral , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Células Cultivadas , Humanos , Mamastrovirus/efectos de los fármacos , Unión Proteica , Internalización del Virus

RESUMEN

Metagenomics is helping to expand the known diversity of viruses, especially of those with poorly studied hosts in remote areas. The Neotropical region harbors a considerable diversity of avian species that may play a role as both host and short-distance vectors of unknown viruses. Viral metagenomics of cloacal swabs from 50 Neotropical birds collected in French Guiana revealed the presence of four complete astrovirus genomes. They constitute an early diverging novel monophyletic clade within the Avastrovirus phylogeny, representing a putative new astrovirus species (provisionally designated as Avastrovirus 5) according to the International Committee on Taxonomy of Viruses (ICTV) classification criteria. Their genomic organization shares some characteristics with Avastrovirus but also with Mamastrovirus. The pan-astrovirus RT-PCR analysis of the cloacal samples of 406 wild Neotropical birds showed a community-level prevalence of 4.9% (5.1% in passerines, the highest described so far in this order of birds). By screening birds of a remote region, we expanded the known host range of astroviruses to the avian families Cardinalidae, Conopophagidae, Furnariidae, Thamnophilidae, Turdidae and Tyrannidae. Our results provide important first insights into the unexplored viral communities, the ecology, epidemiology and features of host-pathogen interactions that shape the evolution of avastroviruses in a remote Neotropical rainforest.

Asunto(s)

Astroviridae/genética , Especificidad del Huésped , Passeriformes/virología , Secuencia de Aminoácidos , Animales , Astroviridae/clasificación , Astroviridae/fisiología , Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/patología , Infecciones por Astroviridae/virología , Cloaca/virología , Guyana Francesa/epidemiología , Genoma Viral , Mamastrovirus/genética , Sistemas de Lectura Abierta/genética , Filogenia , Prevalencia , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/clasificación , Proteínas Virales/metabolismo

RESUMEN

Enteric adenovirus (AdV), sapovirus (SaV), and human astrovirus (HAstV) are important pathogens involved in the gastroenteritis etiology. In this study, a total of 219 fecal samples and sera were collected from children hospitalized for acute gastroenteritis (AGE) in two large pediatric hospitals in Belém, from March 2012 to April 2015. The samples were analyzed by polymerase chain reaction (PCR) for AdV and HAstV (astrovirus) detection, and Nested-PCR and qPCR for SaV detection. AdV was detected in 50.2% (110/219) of the cases, with 42.7% (47/110) being sequenced and classified as: species F (63.9% - 30/47), A (4.2% - 2/47), B (6.4% - 3/47), C (17.1% - 8/47), D (4.2% - 2/47), and E (4.2% - 2/47). Of the 110 AdV-positive feces samples, 80 paired sera presented sufficient amounts and were also tested for this virus, of which 51 (63.7%) showed positive results and 26 (70.3%) pairs (feces plus sera) presented concordant results after sequencing being classified as: species F (21/26; 80.8%), A (1/26; 3.8%), B (1/26; 3.8%), and C (3/26; 11.5%). Overall, HAstV rate in the feces samples was 1.8% (4/219), including both HAstV-1a (2/4; 50%) and HAstV-2c (2/4; 50%). SaV was detected in 4.6% (10/219) of the fecal samples, out of which 50% (5/10) of the positive samples were characterized into the genogroups GI.1 (1), GI.2 (2), and GII.4 (2). These findings highlighted the important contributions of AdV, HAstV, and SaV in the enteric virus spectrum in our region and showed the high genetic diversity of AdV. In addition, it demonstrated for the first time in Brazil, the circulation of AdV in the serum of hospitalized children with AGE.

Asunto(s)

Infecciones por Adenoviridae/epidemiología , Gastroenteritis/virología , Variación Genética , Viremia/epidemiología , Enfermedad Aguda/epidemiología , Adenoviridae/genética , Infecciones por Adenoviridae/virología , Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/virología , Brasil/epidemiología , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Niño , Preescolar , Diarrea/epidemiología , Diarrea/virología , Femenino , Gastroenteritis/epidemiología , Genotipo , Hospitalización , Humanos , Lactante , Recién Nacido , Masculino , Mamastrovirus/genética , Filogenia , Sapovirus/genética

RESUMEN

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

Asunto(s)

Anticuerpos Neutralizantes/aislamiento & purificación , Antígenos Virales/inmunología , Infecciones por Astroviridae/inmunología , Mamastrovirus/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/genética , Infecciones por Astroviridae/virología , Células CACO-2 , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Variación Genética , Humanos , Inmunización , Mamastrovirus/genética , Ratones , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología

RESUMEN

Human astrovirus (HAstV) constitutes a major cause of acute gastroenteritis in children. The viral 5' and 3' untranslated regions (UTR) have been involved in the regulation of several molecular mechanisms. However, in astrovirues have been less characterized. Here, we analyzed the secondary structures of the 5' and 3' UTR of HAstV, as well as their putative target sites that might be recognized by cellular factors. To our knowledge, this is the first bioinformatic analysis that predicts the HAstV 5' UTR secondary structure. The analysis showed that both the UTR sequence and secondary structure are highly conserved in all HAstVs analyzed, suggesting their regulatory role of viral activities. Notably, the UTRs of HAstVs contain putative binding sites for the serine/arginine-rich factors SRSF2, SRSF5, SRSF6, SRSF3, and the multifunctional hnRNPE2 protein. More importantly, putative binding sites for PTB were localized in single-stranded RNA sequences, while hnRNPE2 sites were localized in double-stranded sequence of the HAstV 5' and 3' UTR structures. These analyses suggest that the combination of SRSF proteins, hnRNPE2 and PTB described here could be involved in the maintenance of the secondary structure of the HAstVs, possibly allowing the recruitment of the replication complex that selects and recruits viral RNA replication templates.

Asunto(s)

Simulación por Computador , Mamastrovirus/genética , Proteínas/metabolismo , Regiones no Traducidas/genética , Secuencia de Bases , Sitios de Unión , Conformación de Ácido Nucleico

RESUMEN

Viral infections affecting cattle lead to economic losses to the livestock industry worldwide, but little is known about the circulation, pathogenicity and genetic diversity of enteric bovine astrovirus (BoAstV) in America. The aim of this work was to describe the prevalence and genetic diversity of enteric BoAstV in dairy cattle in Uruguay. A total of 457 fecal and 43 intestinal contents from dairy calves were collected between July 2015 and May 2017 and tested by RT-PCR, followed by sequencing and phylogenetic analyses of the polymerase and capsid regions. Twenty-six percent (128/500) of the samples were positive. Three different species within the Mamastrovirus genus were identified, including Mamastrovirus 28, Mamastrovirus 33 (3 samples each) and an unclassified Mamastrovirus species (19 samples). The unclassified species was characterized as a novel Mamastrovirus species. BoAstV circulates in Uruguayan dairy cattle with a high genetic diversity. The eventual clinicopathological significance of enteric BoAstV infection in cattle needs further investigation.

Asunto(s)

Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/virología , Variación Genética , Kobuvirus/clasificación , Mamastrovirus/clasificación , Infecciones por Picornaviridae/veterinaria , Animales , Bovinos , Industria Lechera , Heces/virología , Kobuvirus/patogenicidad , Mamastrovirus/aislamiento & purificación , Filogenia , Infecciones por Picornaviridae/epidemiología , Uruguay/epidemiología

RESUMEN

Astroviruses are a common cause of gastroenteritis in children worldwide and can also cause infection in a range of domestic and wild animal species. Canine astrovirus (formally named as Mamastrovirus 5, MAstV5) has been reported worldwide, and its role as an enteric pathogen is still controversial. Herein, we describe the genomic characterization of a MAstV5 (strain crab-eating fox/2016/BRA) identified in a wild canid (Cerdocyon thous) diagnosed with canine distemper virus (CDV) as causa mortis. The nearly complete genome comprised 6579 nt in length and displayed the archetypal organization of astroviruses. The present report is the first evidence of MAstV5 infection in an animal species other than the dog and highlights a possible natural astrovirus spillover between domestic and wild canids. Moreover, these results show the first evidence of extra-intestinal MAstV5, suggesting a virus systemic spread. This work is expected to contribute to a better understanding of the astroviruses biology and their interactions with the wildlife health.

Asunto(s)

Infecciones por Astroviridae/veterinaria , Canidae , Mamastrovirus/aislamiento & purificación , Animales , Animales Domésticos , Animales Salvajes , Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/transmisión , Infecciones por Astroviridae/virología , Braquiuros , Brasil/epidemiología , Canidae/virología , Cerebelo/patología , Cerebelo/virología , Virus del Moquillo Canino/inmunología , Virus del Moquillo Canino/aislamiento & purificación , Perros/virología , Genoma Viral , Especificidad del Huésped , Inmunohistoquímica/veterinaria , Mamastrovirus/clasificación , Mamastrovirus/genética , Filogenia , Análisis de Secuencia de ADN , Proteínas Virales/química , Proteínas Virales/genética

RESUMEN

Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.(AU)

Asunto(s)

Animales , Perros , Mamastrovirus/clasificación , Mamastrovirus/genética , Mamastrovirus/aislamiento & purificación , Infecciones por Astroviridae/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Brasil

RESUMEN

Abstract Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype 'a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.

Asunto(s)

Animales , Perros , Mamastrovirus/aislamiento & purificación , Mamastrovirus/genética , Infecciones por Astroviridae/veterinaria , Enfermedades de los Perros/virología , Gastroenteritis/veterinaria , Filogenia , Mamastrovirus/clasificación , Sistemas de Lectura Abierta , Infecciones por Astroviridae/virología , Heces/virología , Gastroenteritis/virología , Genotipo