The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes.

An essential aspect of positive-sense RNA virus replication is anchoring the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. This study provides evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. We identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs through mutational analysis of the N-terminal domain in replicon and reverse genetics systems. In addition, we demonstrate the association of key components of the astrovirus replication complex: double-stranded RNA, RNA-dependent RNA polymerase, protease, and N-terminal protein. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies.

Genetic diversity of astroviruses detected in wild aquatic birds in Hong Kong.

Wild waterfowl serve as a reservoir of some astroviruses. Fecal samples from wild waterfowl collected at Hong Kong's Marshes were tested using pan-astrovirus reverse transcription-PCR. Positive samples underwent subsequent host identification using DNA barcoding. Based on deduced partial sequences, noteworthy samples from three astrovirus groups (mammalian, avian and unclassified astroviruses) were further analyzed by next-generation sequencing. One sample of Avastrovirus 4 clade, MP22-196, had a nearly complete genome identified. The results of ORF2 phylogenetic analysis and genetic distance analysis indicate that Avastrovirus 4 is classified as a distinct subclade within Avastrovirus. MP22-196 has typical astrovirus genome characteristics. The unique characteristics and potential differences of this genome, compared to other avian astrovirus sequences, involve the identification of a modified sgRNA sequence situated near the ORF2 start codon, which precedes the ORF1b stop codon. Additionally, the 3' UTR of MP22-196 is shorter than other avian astroviruses. This study expands our understanding of the Avastrovirus 4 clade.

Astrovirus induced nonpurulent encephalomyelitis in sheep: First report from Türkiye by high-throughput sequencing.

BACKGROUND: This study presents the case of non-purulent encephalomyelitis associated with astrovirus infection in a sheep from Eastern Anatolia, Türkiye. METHODS: A necropsy was performed on a sheep showing nervous signs. Afterwards, brain tissue samples were taken and examined with histopathological, immunohistochemical and molecular techniques. RESULTS: Neuropathologic changes included neuronal degeneration, diffuse gliosis, multifocal perivascular cuffing, neuronophagy and neuronal necrosis in the cerebrum, the cerebellum and the cervical spinal cord. Aerobic and anaerobic bacterial culture, selective culture for Listeria monocytogenes, and PCR analysis for rabies virus, tick-borne encephalitis virus, Türkiye encephalitis virus, small ruminant lentiviruses and border disease virus were negative. However, the presence of astrovirus RNA in cerebral, cerebellar and spinal cord samples was demonstrated by a pan-astrovirus RT-PCR. Immunohistochemical examinations revealed astrovirus antigens within the neuronal cytoplasm. High-throughput sequencing techniques identified the causative agent as a member of the genotype species Mamastrovirus 13 but representing a distinct genetic lineage with similarity to ovine astrovirus 1 in the open-reading frames (ORF)1ab region and muskox astrovirus in the ORF2 region. CONCLUSION: This report provides evidence that astroviruses are potentially encephalitis-causing pathogens in ovine populations in Türkiye, featuring an astrovirus strain distinct from those previously identified in sheep.

Mouse and human immune responses share neutralization epitopes of HAstV-VA1.

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.

Astroviruses in terrestrial Malagasy mammals.

Small terrestrial mammals are major hosts of infectious agents responsible for zoonotic diseases. Astroviruses (AstVs)-the cause of non-bacterial gastroenteritis mainly affecting young children-have been detected in a wide array of mammalian and avian host species. However, understanding the factors that influence AstV infection within and across hosts is limited. Here, we investigated the impact of land use changes on AstVs in terrestrial small mammals in rural northeastern Madagascar. We sampled 515 small mammals, representing seven endemic and four introduced species. Twenty-two positive samples were identified, all but one of which were found in the introduced species Mus musculus and Rattus rattus (family Muridae), with a positivity rate of 7.7% (6/78) and 5.6% (15/266), respectively. The non-introduced rodent case was from an endemic shrew-tenrec (family Tenrecidae). We found the highest positivity rate of AstVs infection in brushy regrowth (17.5%, 7/40) as compared to flooded rice fields (4.60%, 8/174), secondary forest (4.1%, 3/74), agroforest (3.6%, 1/28), village (2.61%, 3/115), and semi-intact forest (0%, 0/84). A phylogenetic analysis revealed an association between AstVs and their rodent host species. None of the viruses were phylogenetically related to AstVs previously described in Malagasy bats. This study supports AstV circulation in synanthropic animals in agricultural habitats of Madagascar and highlights the need to assess the spillover risk to human populations in rural areas.

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Alteration of gut microbiome in goslings infected with goose astrovirus.

Goose astrovirus (GoAstV) is an emerging avian pathogen that induces gout in goslings with a mortality of up to 50%. Organ damage caused by GoAstV infection was considered the cause of gout, but it is still unclear whether other factors are involved. Human and murine studies have linked the gut microbiome-derived urate and gout, thus we hypothesized that gut microbiome may also play an important role in gout induced by GoAstV infection. This study tested the pathogenicity of our isolated GoAstV genotype 2 strain on goslings, while the appearance of clinical signs, histopathological changes, viral distribution and the blood level of cytokines were monitored for 18 d postinfection (dpi). The dynamics in the gut microbiome were profiled by 16S sequencing and then correlated with GoAstV infection. Results showed that this study successfully developed an experimental infection model for studying the pathogenicity of the GoAstV infection which induces typical symptoms of gout. GoAstV infection significantly altered the gut microbiome of goslings with the enrichment of potential proinflammatory bacteria and depletion of beneficial bacteria that can produce short-chain fatty acids. More importantly, the microbial pathway involved in urate production was significantly increased in goslings infected with GoAstV, suggesting that gut microbiome-derived urate may also contribute to the gout symptoms. Overall, this study demonstrated the role of gut microbiome in the pathogenesis of GoAstV infection, highlighting the potential of gut microbiome-based therapeutics against gout symptoms.

Goose IFIT5 positively regulates goose astrovirus replication in GEF cells.

Interferon-induced protein with tetratricopeptide repeats (IFITs), a family of proteins strongly induced by type I interferon (IFN-I), are deeply involved in many cellular and viral processes. IFIT5, the sole protein in this family found in birds, also plays a crucial role in regulating virus infection. In this study, goose IFIT5 (gIFIT5) was first cloned from peripheral blood lymphocyte (PBL) and phylogenetic analysis showed that it was highly homologous with duck IFIT5 (dIFIT5), sharing 94.6% identity in amino acid sequence. Subsequently, the expression kinetics of gIFIT5 during goose astrovirus (GAstV) infection and the regulatory effect of gIFIT5 on GAstV proliferation were evaluated. Results showed that the mRNA and protein expression level of gIFIT5 was greatly induced by GAstV infection, especially at 12 hpi. Importantly, gIFIT5 could conversely promote GAstV replication in GEF cells. Virus titers in gIFIT5 overexpression group were significantly higher than those in control group at 12 and 24 hpi. Western blot and quantitative real-time PCR (qRT-PCR) further demonstrated that the production of viral cap protein was significantly facilitated in gIFIT5-transfected group. Collectively, GAstV facilitates self-replication via promoting gIFIT5 expression.

The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Molecular characterization of human astrovirus infection in children under 5 years of age with acute gastroenteritis in Tehran, Iran, 2021-2022: co-infection with rotavirus.

Human astroviruses (HAstVs) are considered important causative pathogens of acute gastroenteritis (AGE) in children under 5 years of age worldwide, along with group A rotavirus (RVA), norovirus (NoV), and enteric adenovirus (EAdV). The present study was aimed to both detect HAstV and its co-infections and investigate genetic analysis of circulating HAstV and co-infected virus in hospitalized children under 5 years of age with AGE in Iran. Accordingly, a sum of 200 stool specimens were screened by PCR for HAstV during 2021-2022. The HAstV was found in 0.5% of 200 specimens (n = 1) while was co-infected with RVA. The genetic and phylogenetic analysis indicated HAstV1 genotype, which clustered with viruses from lineage 1b, which has not been previously reported in Iran. The detected RVA strain belonged to G1 lineage II/P[8]-lineage III, which has been reported previously in Iran as the most common strain. The further genetic analysis of RVA VP6 and NSP4 demonstrated an atypical genotype pattern G1P[8]-I1-E2, as a mono-reassortant of a Wa-like genogroup, which appeared to be reassorted with the NSP4 gene of E2 genotype of the G2P[4] DS-1 genogroup. Although the clinical outcomes of the AGE-causing viruses co-infection is not yet entirely clear, it seems that future studies will be helpful to merge clinical and epidemiological data of co-infecting viruses for a more accurate medical and clinical relevance in symptomatic children.

Investigation of caliciviruses and astroviruses in Gabonese rodents: A possible influence of national and international trade on the spread of enteric viruses.

Caliciviruses (Caliciviridae) and astroviruses (Astroviridae) are among the leading cause of non-bacterial foodborne disease and gastroenteritis in human. These non-enveloped RNA viruses infect a wide range of vertebrate species including rodents. Rodents are among the most important hosts of infectious diseases globally and are responsible for over 80 zoonotic pathogens that affect humans. Therefore, screening pathogens in rodents will be is necessary to prevent cross-species transmission to prevent zoonotic outbreaks. In the present study, we screened caliciviruses and astroviruses in order to describe their diversity and whether they harbor strains that can infect humans. RNA was then extracted from intestine samples of 245 rodents and retrotranscribed in cDNA to screen caliciviruses and astroviruses by PCRs. All the samples tested negative for caliciviruses and while astroviruses were detected in 18 (7.3%) samples of Rattus rattus species. Phylogenetic analyses based on the RdRp gene showed that all the sequences belonged to Mamastrovirus genus in which they were genetically related to R. rattus related AstVs previously detected in Gabon or in Rattus spp. AstV from Kenya and Asia. These findings suggested that transportation such as land and railway, as well national and international trade, are likely to facilitate spread of AstVs by the dissemination of rodents.

Primary goose kidney tubular epithelial cells for goose astrovirus genotype 2 infection: establishment and RNA sequencing analysis.

Goose astrovirus genotype 2 (GAstV-2) mainly causes gout in goslings; therefore, it is a major pathogen threatening to goose flocks. However, the mechanisms underlying host-GAstV-2 interactions remain unclear because host cells suitable for GAstV-2 replication have been unavailable. We previously noted that GAstV-2 is primarily located in goose renal epithelial cells, where it causes kidney damage. Therefore, here, we derived goose primary renal tubular epithelial (RTE) cells (GRTE cells) from the kidneys of goose embryos after collagenase I digestion. After culture in Dulbecco's modified Eagle medium/Nutrient mixture F-12 with 10% fetal bovine serum (FBS), the isolated cells had polygonal with roadstone-like morphology; they were identified to be epithelial cells based on the presence of cytokeratin 18 expression detected through immunofluorescence assay (IFA). GAstV-2 infection in GRTE cells led to no obvious cytopathic effects; the maximum amounts of infectious virions were observed 48 h post infection through IFA and quantitative PCR. Next, RNA-seq was performed to identify and map post-GAstV-2 infection differentially expressed genes. The downregulated pathways were mainly related to metabolism, including tryptophan metabolism, drug metabolism by cytochrome P450, xenobiotic metabolism by cytochrome P450, retinol metabolism, butanoate metabolism, starch and sucrose metabolism, ascorbate and aldarate metabolism, and drug metabolism by other enzymes and peroxisome. In contrast, the upregulated pathways were mostly related to the host cell defense and proliferation, including extracellular matrix-receptor interaction, complement and coagulation cascades, phagosome, PI3K-Akt signaling pathway, human T-lymphotropic virus 1 infection, lysosome, and tumor necrosis factor signaling pathway. In conclusion, we developed a GRTE cell line for GAstV-2 replication and analyzed the potential host-GAstV-2 interactions through RNA-seq; our results may aid in further investigating the pathogenic mechanisms underlying GAstV-2 infection and provide strategies for its prevention and control.

Isolation, Identification, and Pathogenicity of a Goose Astrovirus Genotype 1 Strain in Goslings in China.

Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3-98.4%; ORF1b: 92.3-99.1%; ORF2: 95.8-98.8%) and amino acid sequences (ORF1a: 99.4-99.5%; ORF1b: 98.2-98.8%; ORF2: 97.0-99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese.

Impact of duck astrovirus on susceptibility to infection across duck ages.

An outbreak of duck astrovirus (DAstV) has occurred in duck farming regions of China, causing substantial economic setbacks in the duck industry. This investigation aimed to examine the variations in DAstV pathogenicity among ducks at different age intervals. Infections were induced in ducks at distinct age groups (1, 7, 14, 21, and 28 d) utilizing the DAstv-1-GDB-2022 strain. The results indicate increased pathogenicity of the DAstv-1-GDB-2022 strain in ducklings aged 21 to 28 d, manifesting as liver and kidney enlargement, severe bleeding, and potential fatalities. Conversely, ducklings aged 1 and 14 d displayed milder symptoms postinfection. Notably, viral shedding continued in ducks of diverse age groups even 21 d postinfection (Dpi). Moreover, DAstV replicates in various tissues, predominantly affecting the liver. Immunohistochemical tests using rabbit anti-DAstV antibodies revealed robust positive signals in both the liver and kidneys, which correlated with the clinical symptom severity observed through macroscopic and microscopic examinations. Serum biochemical assays and indirect ELISA demonstrated a consistent response to DAstV infection across different age groups, with older ducklings exhibiting increased sensitivity. In conclusion, this study successfully replicated clinical symptoms similar to those of natural DAstV infection using the DAstv-1-GDB-2022 strain. Importantly, we systematically delineated the differences in susceptibility to DAstV among ducks at various ages, laying the foundation for further research into the pathogenic mechanisms of DAstV and potential vaccine development.

Molecular characterization of a virulent goose astrovirus genotype-2 with high mortality in vitro and in vivo.

Goose astrovirus (GAstV) is a newly identified viral pathogen threatening waterfowl, exhibiting a high prevalence across various regions in China. Notably, the Guanghan District of Deyang City, situated in Sichuan Province, has faced a outbreak of GAstV, resulting in significant mortality among goslings due to the induction of gout-like symptoms. In our research, we successfully isolated a GAstV strain known as GAstV SCG3. This strain exhibits efficient replication capabilities, proving virulent in goslings and goose embryos. Our study delved into the characteristics of GAstV SCG3 both in vitro and in vivo. Additionally, we examined tissue phagocytosis and the distribution of GAstV SCG3 in deceased goslings using H&E staining and IHC techniques. According to the classification established by the ICTV, GAstV SCG3 falls under the category of GAstV genotype-2. Notably, it demonstrates the highest homology with the published AHAU5 sequences, reaching an impressive 98%. Furthermore, our findings revealed that GAstV SCG3 exhibits efficient proliferation exclusively in goose embryos and in LMH cells, while not manifesting in seven other types of avian and mammalian cells. Significantly, the mortality of GAstV on goslings and goose embryos are 93.1 and 80%, respectively. Moreover, the viral load in the livers of infected goslings surpasses that in the kidneys when compared with the attenuated strain GAstV SCG2. The mortality of GAstV is usually between 20% and 50%, our study marks the first report of a virulent GAstV strain with such a high mortality.

Bovine astrovirus and its role in lymphocytic encephalitis in cattle in Ontario, Canada, 1988-2019.

Astroviruses have been found in cattle and other species with encephalitis. Our objective was to determine the frequency of neurotropic bovine astrovirus (BoAstV) in cases of encephalitis in cattle ≥ 4-mo-old. Of 56 cases of idiopathic lymphocytic encephalitis examined retrospectively (1988-2019), fixed brain from 11 cases (19%) tested positive by semi-quantitative RT-PCR for BoAstV CH13/NeuroS1. None of the control cases tested positive, including 32 with other forms of encephalitis and 40 with no neurologic disease. Most astrovirus-positive cases were 1-2-y-old, with a range of 7 mo to 7 y, and affected both beef and dairy breeds with wide geographic distribution. BoAstV-positive cases had acute onset of neurologic signs of 12 h to 7 d before death or euthanasia. Affected cattle had lymphocytic inflammation throughout the brain including cerebrum, thalamus, midbrain, cerebellum, medulla oblongata, and spinal cord, and affecting gray and white matter. Further PCR testing identified a possible cause in 9 of the 45 (20%) remaining idiopathic cases of lymphocytic encephalitis, including eastern equine encephalitis virus, Listeria monocytogenes, bovine viral diarrhea virus, bovine alphaherpesvirus 1, and ovine gammaherpesvirus 2 (malignant catarrhal fever); we found no cases of infection by West Nile virus, rabies virus, or Chlamydia spp. No cause was identified in 36 of 56 (64%) cases of lymphocytic encephalitis. We frequently identified neurotropic BoAstV in cases of lymphocytic encephalitis that had no previously identified cause. Neurotropic BoAstV infections had gone undetected for decades, but the frequency of BoAstV infections has not increased among contemporary cases.

The first evidence of shaking mink syndrome-astrovirus associated encephalitis in farmed minks, China.

A novel neurological disorder, shaking mink syndrome (SMS), emerged in Denmark and Sweden in 2000. SMS has seldom been reported in China, but the causative agent has not been detected in the country. SMS outbreaks occurred in multiple provinces in 2020. A total of 44 brain samples from minks associated with SMS were collected from Heilongjiang, Liaoning and Shandong provinces of which 28 samples (63.3%) were SMS-astrovirus (SMS-AstV)-positive by reverse transcription PCR. Histopathological examination revealed non-suppurative encephalitis in three minks. Moreover, the complete coding region sequences (CDSs, 6559 bp) of a sample collected from a 2-month-old mink (termed SMS-AstV-H1, GSA accession No. SAMC816786) were amplified by PCR and Sanger sequencing. The complete CDS and open reading frame 2 sequences of SMS-AstV-H1 were 94.3% and 96.4% identical to an SMS-AstV strain (GenBank accession number: GU985458). Phylogenetically, SMS-AstV-H1 was closely related to an SMS-AstV strain (GU985458). Based on the above results, we describe SMS-AstV-associated encephalitis in farmed minks in China. Future studies need to focus on epidemiology, virus isolation and potential interspecies transmission of SMS-AstV.

The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

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.

The Capsid Precursor Protein of Astrovirus VA1 Is Proteolytically Processed Intracellularly.

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.