RESUMO
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.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Humanos , Animais , Anticorpos Neutralizantes/imunologia , Camundongos , Epitopos/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Mamastrovirus/imunologia , Mamastrovirus/genética , Mutação , Infecções por Astroviridae/imunologia , Infecções por Astroviridae/virologia , Testes de NeutralizaçãoRESUMO
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.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Astroviridae/epidemiologia , Mamastrovirus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Masculino , Mamastrovirus/genética , Pessoa de Meia-Idade , Missouri/epidemiologia , Peru/epidemiologia , RNA Viral/genética , Estudos Soroepidemiológicos , Adulto JovemRESUMO
The causes of the broad spectrum of severity in COVID-19 are unknown. A protective effect through humoral immunity from previous infections by viruses of the SARS-CoV-2 family could explain a mild form of this disease. This study aimed to address whether the presence of antibodies against human seasonal coronaviruses (HCoVs) could prevent severe manifestations of COVID-19. A cross-sectional study was carried out in 165 participants. The presence of pre-existent antibodies against the seasonal HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63 were detected. From all of the seasonal HCoVs studied, it was only found that being seropositive to HCoV-229E presented an association (p = 0.012) with developing mild clinical symptoms of COVID-19 or being asymptomatic. Multinomial regression analysis showed that being seropositive to HCoV-229E is associated with mild or moderate clinical symptoms for COVID-19. Statistical analysis also showed that being female is associated with being asymptomatic for SARS-CoV-2 infection or developing mild COVID-19. A subgroup analysis taking only seropositive to HCoV-229E revealed that females are more likely to develop asymptomatic SARS-CoV-2 infection (OR = 27.242, 95% CI 2.092-354.706, p = 0.012). Our results suggest that previous infections by HCoV-229E could prevent more serious clinical manifestations of COVID-19, but these are not the only variables that influence this event.
Assuntos
COVID-19 , Coronavirus Humano 229E , Anticorpos Antivirais , Estudos Transversais , Feminino , Humanos , SARS-CoV-2RESUMO
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.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Antígenos Virais/imunologia , Infecções por Astroviridae/imunologia , Mamastrovirus/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Infecções por Astroviridae/virologia , Células CACO-2 , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Variação Genética , Humanos , Imunização , Mamastrovirus/genética , Camundongos , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.
Assuntos
Mamastrovirus/fisiologia , Internalização do Vírus , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Infecções por Astroviridae/genética , Infecções por Astroviridae/metabolismo , Infecções por Astroviridae/virologia , Linhagem Celular , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Inativação Gênica , Humanos , Mamastrovirus/efeitos dos fármacos , Mutação , Ligação Viral , Liberação de Vírus , Replicação Viral/efeitos dos fármacos , Desenvelopamento do Vírus , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
In this work we evaluated the ability of rotavirus strains with different receptor requirements to infect the apical and basolateral surfaces of polarized MDCKII cells. We used neuraminidase (NA)-sensitive (RRV and TFR-1) and neuraminidase-resistant (Wa and UK) viruses that differ in their use of integrins. Regardless of their receptor requirements, all virus strains tested were found to efficiently infect cells from both membrane surface domains, with preference for the basolateral domain, since: (i) disruption of tight junctions of polarized cell monolayers by calcium chelation led to a reversible increase of rotavirus infectivity, (ii) the viruses infected preferentially the cells located at the borders of microcolonies of polarized cells, and (iii) in cells grown on a permeable support all four virus strains were able to start the infection by either plasma membrane domain. Preferential infection (5-11-fold more efficiently) of the basolateral surface correlated with the neuraminidase resistance of the virus strains, but not with their requirement for integrins, which in MDCKII cells seem to be used by all four viruses. The infection of both cell surface domains by RRV was found to depend on the presence of terminal sialic acids, since its infectivity was reduced by neuraminidase treatment of the cells and it was also blocked by incubation of the virus with glycophorin A. The efficient infection through the basolateral membrane surface of polarized cells might be relevant for the pathogenesis of rotavirus, especially given the recent reports of antigenemia and extraintestinal spread of the virus in children and animal models.
Assuntos
Receptores Virais/fisiologia , Rotavirus/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Cães , Integrinas/metabolismo , Neuraminidase/metabolismo , Receptores Virais/química , Ácidos Siálicos/metabolismoRESUMO
Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) surrounded by three protein layers. The virus contains an RNA-dependent RNA polymerase that synthesizes RNA transcripts corresponding to all segments of the viral genome. These transcripts direct the synthesis of the viral proteins and also serve as templates for the synthesis of the complementary strand to form the dsRNA genome. In this work, we analyzed the kinetics of transcription and replication of the viral genome throughout the replication cycle of the virus using quantitative reverse transcription-PCR. The role of the proteins that form double-layered particles ([DLPs] VP1, VP2, VP3, and VP6) in replication and transcription of the viral genome was analyzed by silencing their expression in rotavirus-infected cells. All of them were shown to be essential for the replication of the dsRNA genome since in their absence there was little synthesis of viral mRNA and dsRNA. The characterization of the kinetics of RNA transcription and replication of the viral genome under conditions where these proteins were silenced provided direct evidence for a second round of transcription during the replication of the virus. Interestingly, despite the decrease in mRNA accumulation when any of the four proteins was silenced, the synthesis of viral proteins decreased when VP2 and VP6 were knocked down, whereas the absence of VP1 and VP3 did not have a severe impact on viral protein synthesis. Characterization of viral particle assembly in the absence of VP1 and VP3 showed that while the formation of triple-layered particles and DLPs was decreased, the amount of assembled lower-density particles, often referred to as empty particles, was not different from the amount in control-infected cells, suggesting that viral particles can assemble in the absence of either VP1 or VP3.
Assuntos
RNA Viral/biossíntese , Rotavirus/fisiologia , Transcrição Gênica , Replicação Viral , Técnicas de Silenciamento de Genes/métodos , Cinética , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Estruturais Virais/antagonistas & inibidores , Proteínas Estruturais Virais/metabolismo , Montagem de VírusRESUMO
Rotavirus entry is a complex multistep process that depends on the trypsin cleavage of the virus spike protein VP4 into polypeptides VP5 and VP8 and on the interaction of these polypeptides and of VP7, the second viral surface protein, with several cell surface molecules, including integrin alphavbeta3. We characterized the effect of the trypsin cleavage of VP4 on the binding to MA104 cells of the sialic acid-dependent virus strain RRV and its sialic acid-independent variant, nar3. We found that, although the trypsin treatment did not affect the attachment of these viruses to the cell surface, their binding was qualitatively different. In contrast to the trypsin-treated viruses, which initially bound to the cell surface through VP4, the non-trypsin-treated variant nar3 bound to the cell through VP7. Amino acid sequence comparison of the surface proteins of rotavirus and hantavirus, both of which interact with integrin alphavbeta3 in an RGD-independent manner, identified a region shared by rotavirus VP7 and hantavirus G1G2 protein in which six of nine amino acids are identical. This region, which is highly conserved among the VP7 proteins of different rotavirus strains, mediates the binding of rotaviruses to integrin alphavbeta3 and probably represents a novel binding motif for this integrin.
Assuntos
Antígenos Virais/química , Proteínas do Capsídeo/química , Regulação Viral da Expressão Gênica , Integrina alfaVbeta3/metabolismo , Rotavirus/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Virais/metabolismo , Sítios de Ligação , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Macaca mulatta/virologia , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/metabolismo , Rotavirus/genética , Tripsina/metabolismoRESUMO
RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.
Assuntos
Regulação Viral da Expressão Gênica , Interferência de RNA , RNA Interferente Pequeno , Rotavirus/efeitos dos fármacos , Rotavirus/genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/efeitos dos fármacos , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , RNA Viral/metabolismoRESUMO
Rotavirus infection seems to be a multistep process in which the viruses are required to interact with several cell surface molecules to enter the cell. The virus spike protein VP4, which is cleaved by trypsin into two subunits, VP5 and VP8, is involved in some of these interactions. We have previously shown that the neuraminidase-sensitive rotavirus strain RRV initially attaches to a sialic acid-containing cell molecule through the VP8 subunit of VP4 and subsequently interacts with integrin alpha2beta1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin alphavbeta3 and the heat shock cognate protein Hsc70. In this work, we have shown that rotavirus RRV and its neuraminidase-resistant variant nar3 interact with Hsc70 through a VP5 domain located between amino acids 642 and 658 of the protein. This conclusion is based on the observation that a recombinant protein comprising the 300 carboxy-terminal amino acids of VP5 binds specifically to Hsc70 and a synthetic peptide containing amino acids 642 to 658 competes with the binding of the RRV and nar3 viruses to the heat shock protein. The VP5 peptide also competed with the binding to Hsc70 of the recombinant VP5 protein, and an antibody to Hsc70 reduced the binding of the recombinant protein to the surface of MA104 cells. The fact that the synthetic peptide blocks the infectivity of rotaviruses RRV and nar3 but not their binding to cells indicates that the interaction of VP5 with Hsc70 most probably occurs at a postattachment step during the virus entry process.