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Electron micrographs of HAstV-8-infected Caco-2 cells. Cells were harvested at 24 hpi and processed for electron microscopy, as described in Materials and Methods. Panels A, C, and E represent three different cells, and panels B, D, and F represent enlargements of the corresponding areas. Astrovirus particles were observed in clusters (VP) and in isolated (circled) forms. Particles that look partially assembled inside or at the edges of vesicles (V) induced during infection are marked with arrows. Bars, 200 nm.
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VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on t...
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... in infected cells. To characterize virus particles that could represent intermediates in morphogenesis, infected cells were analyzed by electron microscopy. Large groups of viral particles (VP in Fig. 6) surrounding “O-ring” structures (V [for vesicles] in Fig. 6) were clearly observed. These O-ring structures, which were not observed in mock-infected cells (not shown), probably correspond to the double-membrane vesicles previously reported by Guix et al. (9, 10), which were also reported to be in close association with virus agglomerates. More dispersed HAstV-8 particles were also detected in the cells’ cytoplasm (circled, Fig. 6C). Additional virus-like particles, which could represent subviral particles at different stages of assembly, were observed inside and at the edges of O-ring structures (Fig. 6; more clearly observable in panels C, D, and F and denoted with arrows). Micrographs shown in Fig. 6 show viral particles with distinct features distributed in different locations inside the cell. To localize the structural proteins in infected cells and on membrane-enriched fractions of iodixanol gradients, gold-la- beled antibodies to VP90 (anti-E4) and to VP70/VP90 (anti- Yuc8) were used for IEM analysis. As expected, anti-Yuc8 antibodies detected the virus clusters, confirming that the structural proteins (VP70 and/or VP90) were present in these particles (Fig. 7A and B). To find out whether VP90 was present in such viral clusters, anti-E4 antibodies were used for IEM in Z-VAD-FMK-treated infected cells (Fig. 7E and F). VP90 was indeed detected in the particle aggregates (Fig. 7E), and of particular interest, the anti-E4 antibodies also inter- acted with material located in the interior of the vesicles (Fig. 7F), where virus-like particles are observed (Fig. 6B to D and F). Anti-Yuc8 antibodies also detected antigen at the edges of these vesicles (Fig. 7C and D). When the membrane-enriched fractions from the iodixanol gradients were analyzed by IEM with anti-E4 antibodies, specific to VP90, antigen was mainly localized at the periphery of vesicles (Fig. 8). Some of these vesicles also had associated small subviral particles, similar to those observed in the vesicles present in infected cells (Fig. 6, compare with enlarged images in Fig. 8). The protein recognized in these fractions most likely represents m VP90, while c VP90 likely forms part of the viral clusters observed in the cell. Thus, structural proteins of astrovirus were localized in virus particles but also in close association with vesicles, where apparent immature subviral particles were observed. The association of m VP90 with vesicle membranes, together with the presence of putative intermediate particles in these structures, suggests that this protein, but not c VP90, represents the form of VP90 that first assembles into ...
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... in infected cells. To characterize virus particles that could represent intermediates in morphogenesis, infected cells were analyzed by electron microscopy. Large groups of viral particles (VP in Fig. 6) surrounding “O-ring” structures (V [for vesicles] in Fig. 6) were clearly observed. These O-ring structures, which were not observed in mock-infected cells (not shown), probably correspond to the double-membrane vesicles previously reported by Guix et al. (9, 10), which were also reported to be in close association with virus agglomerates. More dispersed HAstV-8 particles were also detected in the cells’ cytoplasm (circled, Fig. 6C). Additional virus-like particles, which could represent subviral particles at different stages of assembly, were observed inside and at the edges of O-ring structures (Fig. 6; more clearly observable in panels C, D, and F and denoted with arrows). Micrographs shown in Fig. 6 show viral particles with distinct features distributed in different locations inside the cell. To localize the structural proteins in infected cells and on membrane-enriched fractions of iodixanol gradients, gold-la- beled antibodies to VP90 (anti-E4) and to VP70/VP90 (anti- Yuc8) were used for IEM analysis. As expected, anti-Yuc8 antibodies detected the virus clusters, confirming that the structural proteins (VP70 and/or VP90) were present in these particles (Fig. 7A and B). To find out whether VP90 was present in such viral clusters, anti-E4 antibodies were used for IEM in Z-VAD-FMK-treated infected cells (Fig. 7E and F). VP90 was indeed detected in the particle aggregates (Fig. 7E), and of particular interest, the anti-E4 antibodies also inter- acted with material located in the interior of the vesicles (Fig. 7F), where virus-like particles are observed (Fig. 6B to D and F). Anti-Yuc8 antibodies also detected antigen at the edges of these vesicles (Fig. 7C and D). When the membrane-enriched fractions from the iodixanol gradients were analyzed by IEM with anti-E4 antibodies, specific to VP90, antigen was mainly localized at the periphery of vesicles (Fig. 8). Some of these vesicles also had associated small subviral particles, similar to those observed in the vesicles present in infected cells (Fig. 6, compare with enlarged images in Fig. 8). The protein recognized in these fractions most likely represents m VP90, while c VP90 likely forms part of the viral clusters observed in the cell. Thus, structural proteins of astrovirus were localized in virus particles but also in close association with vesicles, where apparent immature subviral particles were observed. The association of m VP90 with vesicle membranes, together with the presence of putative intermediate particles in these structures, suggests that this protein, but not c VP90, represents the form of VP90 that first assembles into ...
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... in infected cells. To characterize virus particles that could represent intermediates in morphogenesis, infected cells were analyzed by electron microscopy. Large groups of viral particles (VP in Fig. 6) surrounding “O-ring” structures (V [for vesicles] in Fig. 6) were clearly observed. These O-ring structures, which were not observed in mock-infected cells (not shown), probably correspond to the double-membrane vesicles previously reported by Guix et al. (9, 10), which were also reported to be in close association with virus agglomerates. More dispersed HAstV-8 particles were also detected in the cells’ cytoplasm (circled, Fig. 6C). Additional virus-like particles, which could represent subviral particles at different stages of assembly, were observed inside and at the edges of O-ring structures (Fig. 6; more clearly observable in panels C, D, and F and denoted with arrows). Micrographs shown in Fig. 6 show viral particles with distinct features distributed in different locations inside the cell. To localize the structural proteins in infected cells and on membrane-enriched fractions of iodixanol gradients, gold-la- beled antibodies to VP90 (anti-E4) and to VP70/VP90 (anti- Yuc8) were used for IEM analysis. As expected, anti-Yuc8 antibodies detected the virus clusters, confirming that the structural proteins (VP70 and/or VP90) were present in these particles (Fig. 7A and B). To find out whether VP90 was present in such viral clusters, anti-E4 antibodies were used for IEM in Z-VAD-FMK-treated infected cells (Fig. 7E and F). VP90 was indeed detected in the particle aggregates (Fig. 7E), and of particular interest, the anti-E4 antibodies also inter- acted with material located in the interior of the vesicles (Fig. 7F), where virus-like particles are observed (Fig. 6B to D and F). Anti-Yuc8 antibodies also detected antigen at the edges of these vesicles (Fig. 7C and D). When the membrane-enriched fractions from the iodixanol gradients were analyzed by IEM with anti-E4 antibodies, specific to VP90, antigen was mainly localized at the periphery of vesicles (Fig. 8). Some of these vesicles also had associated small subviral particles, similar to those observed in the vesicles present in infected cells (Fig. 6, compare with enlarged images in Fig. 8). The protein recognized in these fractions most likely represents m VP90, while c VP90 likely forms part of the viral clusters observed in the cell. Thus, structural proteins of astrovirus were localized in virus particles but also in close association with vesicles, where apparent immature subviral particles were observed. The association of m VP90 with vesicle membranes, together with the presence of putative intermediate particles in these structures, suggests that this protein, but not c VP90, represents the form of VP90 that first assembles into ...
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... in infected cells. To characterize virus particles that could represent intermediates in morphogenesis, infected cells were analyzed by electron microscopy. Large groups of viral particles (VP in Fig. 6) surrounding “O-ring” structures (V [for vesicles] in Fig. 6) were clearly observed. These O-ring structures, which were not observed in mock-infected cells (not shown), probably correspond to the double-membrane vesicles previously reported by Guix et al. (9, 10), which were also reported to be in close association with virus agglomerates. More dispersed HAstV-8 particles were also detected in the cells’ cytoplasm (circled, Fig. 6C). Additional virus-like particles, which could represent subviral particles at different stages of assembly, were observed inside and at the edges of O-ring structures (Fig. 6; more clearly observable in panels C, D, and F and denoted with arrows). Micrographs shown in Fig. 6 show viral particles with distinct features distributed in different locations inside the cell. To localize the structural proteins in infected cells and on membrane-enriched fractions of iodixanol gradients, gold-la- beled antibodies to VP90 (anti-E4) and to VP70/VP90 (anti- Yuc8) were used for IEM analysis. As expected, anti-Yuc8 antibodies detected the virus clusters, confirming that the structural proteins (VP70 and/or VP90) were present in these particles (Fig. 7A and B). To find out whether VP90 was present in such viral clusters, anti-E4 antibodies were used for IEM in Z-VAD-FMK-treated infected cells (Fig. 7E and F). VP90 was indeed detected in the particle aggregates (Fig. 7E), and of particular interest, the anti-E4 antibodies also inter- acted with material located in the interior of the vesicles (Fig. 7F), where virus-like particles are observed (Fig. 6B to D and F). Anti-Yuc8 antibodies also detected antigen at the edges of these vesicles (Fig. 7C and D). When the membrane-enriched fractions from the iodixanol gradients were analyzed by IEM with anti-E4 antibodies, specific to VP90, antigen was mainly localized at the periphery of vesicles (Fig. 8). Some of these vesicles also had associated small subviral particles, similar to those observed in the vesicles present in infected cells (Fig. 6, compare with enlarged images in Fig. 8). The protein recognized in these fractions most likely represents m VP90, while c VP90 likely forms part of the viral clusters observed in the cell. Thus, structural proteins of astrovirus were localized in virus particles but also in close association with vesicles, where apparent immature subviral particles were observed. The association of m VP90 with vesicle membranes, together with the presence of putative intermediate particles in these structures, suggests that this protein, but not c VP90, represents the form of VP90 that first assembles into ...
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... in infected cells. To characterize virus particles that could represent intermediates in morphogenesis, infected cells were analyzed by electron microscopy. Large groups of viral particles (VP in Fig. 6) surrounding “O-ring” structures (V [for vesicles] in Fig. 6) were clearly observed. These O-ring structures, which were not observed in mock-infected cells (not shown), probably correspond to the double-membrane vesicles previously reported by Guix et al. (9, 10), which were also reported to be in close association with virus agglomerates. More dispersed HAstV-8 particles were also detected in the cells’ cytoplasm (circled, Fig. 6C). Additional virus-like particles, which could represent subviral particles at different stages of assembly, were observed inside and at the edges of O-ring structures (Fig. 6; more clearly observable in panels C, D, and F and denoted with arrows). Micrographs shown in Fig. 6 show viral particles with distinct features distributed in different locations inside the cell. To localize the structural proteins in infected cells and on membrane-enriched fractions of iodixanol gradients, gold-la- beled antibodies to VP90 (anti-E4) and to VP70/VP90 (anti- Yuc8) were used for IEM analysis. As expected, anti-Yuc8 antibodies detected the virus clusters, confirming that the structural proteins (VP70 and/or VP90) were present in these particles (Fig. 7A and B). To find out whether VP90 was present in such viral clusters, anti-E4 antibodies were used for IEM in Z-VAD-FMK-treated infected cells (Fig. 7E and F). VP90 was indeed detected in the particle aggregates (Fig. 7E), and of particular interest, the anti-E4 antibodies also inter- acted with material located in the interior of the vesicles (Fig. 7F), where virus-like particles are observed (Fig. 6B to D and F). Anti-Yuc8 antibodies also detected antigen at the edges of these vesicles (Fig. 7C and D). When the membrane-enriched fractions from the iodixanol gradients were analyzed by IEM with anti-E4 antibodies, specific to VP90, antigen was mainly localized at the periphery of vesicles (Fig. 8). Some of these vesicles also had associated small subviral particles, similar to those observed in the vesicles present in infected cells (Fig. 6, compare with enlarged images in Fig. 8). The protein recognized in these fractions most likely represents m VP90, while c VP90 likely forms part of the viral clusters observed in the cell. Thus, structural proteins of astrovirus were localized in virus particles but also in close association with vesicles, where apparent immature subviral particles were observed. The association of m VP90 with vesicle membranes, together with the presence of putative intermediate particles in these structures, suggests that this protein, but not c VP90, represents the form of VP90 that first assembles into ...
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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 ini...
Citations
... Their genomes are about 6.1 to 7.9 kb long and are organized in the following order: 5′ untranslated region (UTR), open reading frames (ORF1a, ORF1b, ORF2), 3′UTR and a poly (A) tail . ORF1a and ORF1b encode non-structural polyproteins (nsp1a and nsp1ab), ORF2 encoding capsid protein, which is a highly variable region of the genome and the main protein inducing host immune response in the immune response process (Méndez et al., 2007;Jamnikar-Ciglenecki et al., 2020). The International Committee on Taxonomy of Viruses (ICTV) depending on the host infected by astroviruses classified them as two viral genera, including Mamastrovirus (mammal astrovirus, MAstV) and Astroviruses (avian astrovirus, AAstV) Among them, MAstV mainly causes gastroenteritis and encephalitis in mammals, while AAstV mainly causes nephritis and enteritis in chickens, hepatitis in ducks, gout and nephritis in goslings (Chu et al., 2012;Zhang et al., 2018). ...
Waterfowl astroviruses are mainly duck astroviruses and goose astroviruses, of which duck astroviruses (DAstV-3, -4), goose astroviruses (GoAstV-1, -2) are the four new waterfowl 21 astroviruses in recent years, which can lead to enteritis, viral hepatitis, gout and reduce the growth performance of waterfowl, affecting the healthy development of the waterfowl farming industry. Since no targeted drugs or vaccines on the market, studies on the epidemiology of the virus are necessary for vaccine development. In this study, we collected 1546 waterfowl samples from 13 provinces in China for epidemiological investigation. The results showed that 260 samples (16.8%) were positive. Four species of astrovirus were detected in 13 provinces except Fujian province. Among the four sites tested, the highest positive rates were found in farms and slaughterhouses. Cross-host and mixed infection were observed in four species of waterfowl astroviruses. The whole genome of 17 isolates was sequenced and compared with published sequences. Genetic evolution and homology analysis showed that the isolated strains had high similarity to their reference sequences. To assess the pathogenicity of GoAstV, 7-day-old goslings were inoculated with GoAstV-1 and GoAstV-2 by the intramuscular route, and infected geese showed similar clinical signs, such as anorexia, depression, and weight loss. Organ damage was seen after infection, with histopathological changes in the heart, liver, spleen, kidney, and intestine, and higher viral loads in throat and anal swabs. These findings increase our understanding of the pathogenicity of GoAstV-1 and GoAstV-2 in goslings and provide more references for future research.
... Nonstructural proteins alone from other viruses such as HCV and SARS-CoV-2 are sufficient for induction of DMV formation (40)(41)(42). Little is known about the function of astrovirus nonstructural proteins, and it is likely that they play a role in DMV formation (43,44). ...
Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients.
IMPORTANCE
These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections.
... It is hypothesized that this assembly was a result of the heterologous expression system. Previous studies have shown that the acidic domain (AD) orients the C-terminal region of the HAstV VP90 to cellular membranes (29). Without mammalian membranes present during expression, the protein may form non-icosahe dral assemblies. ...
... Our finding that HAstV8 VP90 can be isolated in large quantities from E. coli allows us to study HAstV assembly and maturation mediated by host proteases (caspases, trypsin, etc.) in vitro. The observation of rod-shaped protein assemblies not seen in cell-culture derived virions supports earlier hypotheses that eukaryotic host membranes and/or viral nucleic acids are required for proper virus assembly (29). The liposome disruption assay used in this study provides a simple system for a thorough investigation of the membrane penetration activity mediated by HAstV capsids. ...
The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus–host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro .
IMPORTANCE
Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.
... Of all the stages in the life cycle of HAstV, cellular egress or exit is the least characterized stage. Even though HAstV egress mechanism has not been fully characterized, it has been suggested that HAstV release is a non-lytic process with possible association with membranous structures (Aguilar-Hernández et al.; Méndez et al. 2007) . HAstV8 infection enhances extracellular vesicle (EV) secretion from Caco-2 cells, and it seems HastV8 is associated with released EV (Baez-Navarro et al. 2022a). ...
During infection, viruses are released from infected cells and spread to neighboring
cells either through a lytic pathway (by rupturing cells), a non-lytic pathway (such
as the use of extracellular vesicles), or both pathways. Human astroviruses (HAstV)
are highly prevalent non-enveloped positive sense RNA viruses. They are separated
into three clades, classic-type, MLB-type and VA-type, in humans. They are
identified as the third leading cause of viral gastroenteritis in children, the elderly,
and immunocompromised patients. Yet, little is known about the replication,
transmission, immunity, and epidemiology of these viruses. Thus, I sought to
investigate the possible pathways HAstV utilize to exit infected cells. First, I
investigated the role of cell lysis in HAstV release. To do this, I measured cell death
in HAstV infected Caco2 and Huh7.5 cells WST-1 assay for cell proliferation, and
Annexin V/propidium iodide staining for cell apoptosis using microscopy and flow
cytometry. Interestingly, HAstV-1, MLB-2 and VA-1 did not show significant
cytopathic effect in infected cells despite the several log increases in viral titer as
determined by RT-qPCR. Second, we investigated the role of extracellular vesicles
(EV) in HAstV release. Using pharmaceuticals to modulate EV synthesis, we found
that HAstV release increased when Forskolin and Norepinephrine were used to
enhance EV production in infected Caco2 and Huh7.5 cells. Conversely, inhibition of EV production by GW4869 significantly reduced HAstV release in infected cells.
Additionally, EV isolated from infected cells supports new infection without trypsin
activation. Our data showed an association between EV produced by host cells and
the released HAstV particles. This suggests that human astrovirus egress is non-lytic and may involve the use of EV
... The CP encoded by astrovirus is approximately 90 kD (viral protein [VP] 90) and assembles into immature virus particles in cells [24]. VP90 is cleaved by caspase to produce VP70, which is then cleaved by trypsin to VP34, VP27, and VP25 [25]. These three proteins are distributed on the surface of the virus to form mature virus particles [26,27]. ...
In recent years, an infection in geese caused by goose astrovirus (GAstV) has repeatedly occurred in coastal areas of China and rapidly spread to inland provinces. The infection is characterized by joint and visceral gout and is fatal. The disease has caused huge economic losses to China’s goose industry. GAstV is a nonenveloped, single-stranded, positive-sense RNA virus. As it is a novel virus, there is no specific classification. Here, we review the current understanding of GAstV. The virus structure, isolation, diagnosis and detection, innate immune regulation, and transmission route are discussed. In addition, since GAstV can cause gout in goslings, the possible role of GAstV in gout formation and uric acid metabolism is discussed. We hope that this review will inform researchers to rapidly develop effective methods to prevent and treat this disease.
... One of the less characterized phases of the astrovirus replication cycle, is cell egress. It has been proposed that astrovirus release is a nonlytic process, during which the extracellular virions appear to be associated with membranous structures (9,12). In this regard, it is of interest that the cell exit of different viruses has been associated with extracellular vesicles (EV) (13)(14)(15). ...
... Detergent treatment increases HAstV Yuc8 infectious titer in supernatant from 15 h postinfection. It has been previously observed that a portion of the astrovirus particles produced in infected cells floats to low-density fractions when separated by density centrifugation, suggesting that they interact with membranous structures (12). To characterize the possible association of astrovirus particles with membranes in the cell culture media, we evaluated the kinetics of astrovirus release in Caco-2 cells. ...
... Cell culture and viral propagation. Caco-2 cells were cultured in DMEM supplemented with nonessential amino acids and 15% heat-inactivated FBS, in a 10% CO 2 atmosphere at 37°C (12). MA104 cells were grown in A-DMEM, supplemented with 5% FBS, at 37°C in a 5% CO 2 atmosphere. ...
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 CP is an external structural barrier that not only surrounds the nucleic acids, but also interacts with the host, influencing cell tropism and mediating entry into the cell. Furthermore, it is an antigen that induces an immune response in the host [8]. ...
Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein (length of sequencing product 170 bp) from all samples. The presence of astrovirus RNA was detected in each of the samples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples. Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other countries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe.
... After translation, 180 subunits are assembled into the T = 3 icosahedral capsid and form the VP90 state of Viruses 2021, 13, 821 2 of 11 the virus. In cellular studies, VP90 mostly associates with the membrane fractions [11]. VP90 can dissociate from the membranes and become soluble after cleavage by proteases, specifically caspases from virus-induced apoptosis [11][12][13]. ...
... After translation, 180 subunits are assembled into the T = 3 icosahedral capsid and form the VP90 state of the virus. In cellular studies, VP90 mostly associates with the membrane fractions [11]. VP90 can dissociate from the membranes and become soluble after cleavage by proteases, specifically caspases from virus-induced apoptosis [11][12][13]. ...
... In cellular studies, VP90 mostly associates with the membrane fractions [11]. VP90 can dissociate from the membranes and become soluble after cleavage by proteases, specifically caspases from virus-induced apoptosis [11][12][13]. There are several candidate caspase cleavage sites in the C-terminal acidic domain, and this maturation occurs over a period of 10-20 min after translation [14]. ...
Astroviruses (AstVs) are non-enveloped, positive single-stranded RNA viruses that cause a wide range of inflammatory diseases in mammalian and avian hosts. The T = 3 viral capsid is unique in its ability to infect host cells in a process driven by host proteases. Intercellular protease cleavages allow for viral egress from a cell, while extracellular cleavages allow for the virus to enter a new host cell to initiate infection. High-resolution models of the capsid core indicate a large, exposed region enriched with protease cleavage sites. The virus spike protein allows for binding to target cells and is the major target for naturally occurring and engineered neutralizing antibodies. During maturation, the capsid goes through significant structural changes including the loss of many surface spikes. The capsid interacts with host membranes during the virus life cycle at multiple stages such as assembly, host cell entry and exit. This review will cover recent findings and insights related to the structure of the capsid and its function. Further understanding of the viral capsid structure and maturation process can contribute to new vaccines, gastric therapeutics, and viral engineering applications.
... Star-like structures surrounding the icosahedral capsid were also observed ( Fig. 3A). Ultrathin sections showed that the virions aggregated as crystalline lattices in the cytoplasm of infected cells (Fig. 3B), which is consistent with reports of human AstVs (33,34). Enhancement of PAstV5 replication by coculture with CSFV. ...
Porcine astroviruses are mainly associated with gastroenteritis and neurological diseases in pigs, and five genotypes have been identified (PAstV1-5). However, the clinical manifestations of genotypes other than PAstV1 have not yet been determined because of the failure of in vitro virus isolation. Here, we report a surprising isolation of a PAstV5 strain from a clinical classical swine fever virus (CSFV)-infected tissue sample, which can stably passage in PK-15 cells, and coinfection with CSFV significantly enhanced the replication of PAstV5, possibly through suppression of beta interferon production. Thus, the first isolated PAstV5 strain will be useful for investigating the biological and pathogenic properties of this virus, and the findings obtained in this study provide new insights into defining the interaction mechanism between CSFV and PAstV5.
... Interestingly, our data showed that ORF2 and ORF2-SϩP1ϩP2 could efficiently restrict the viral replication of both GAstV-GD and H9N2 avian influenza virus in vitro. Notably, the acidic domain of ORF2 is thought to be a binding site for cell receptors during astrovirus infection (25). ORF2-SϩP1ϩP2 in this study lacks the acidic domain present in the full-length ORF2. ...
Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo . The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses.
IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.
