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 enteric viruses responsible of gastroenteritis in urban and rural Burkina Faso.

BACKGROUND: Viral gastrointestinal infections remain a major public health concern in developing countries. In Burkina Faso, there are very limited updated data on the circulating viruses and their genetic diversity. OBJECTIVES: This study investigates the detection rates and characteristics of rotavirus A (RVA), norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) in patients of all ages with acute gastrointestinal infection in urban and rural areas. STUDY DESIGN & METHODS: From 2018 to 2021, stool samples from 1,295 patients with acute gastroenteritis were collected and screened for RVA, NoV, SaV and HAstV. Genotyping and phylogenetic analyses were performed on a subset of samples. RESULTS: At least one virus was detected in 34.1% of samples. NoV and SaV were predominant with detection rates of respectively 10.5 and 8.8%. We identified rare genotypes of NoV GII, RVA and HAstV, recombinant HAstV strains and a potential zoonotic RVA transmission event. CONCLUSIONS: We give an up-to-date epidemiological picture of enteric viruses in Burkina Faso, showing a decrease in prevalence but a high diversity of circulating strains. However, viral gastroenteritis remains a public health burden, particularly in pediatric settings. Our data advocate for the implementation of routine viral surveillance and updated management algorithms for diarrheal disease.

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.

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.

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.

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.

PreK-12 school and citywide wastewater monitoring of the enteric viruses astrovirus, rotavirus, and sapovirus.

Wastewater monitoring is an efficient and effective way to surveil for various pathogens in communities. This is especially beneficial in areas of high transmission, such as preK-12 schools, where infections may otherwise go unreported. In this work, we apply wastewater disease surveillance using school and community wastewater from across Houston, Texas to monitor three major enteric viruses: astrovirus, sapovirus genogroup GI, and group A rotavirus. We present the results of a 10-week study that included the analysis of 164 wastewater samples for astrovirus, rotavirus, and sapovirus in 10 preK-12 schools, 6 wastewater treatment plants, and 2 lift stations using newly designed RT-ddPCR assays. We show that the RT-ddPCR assays were able to detect astrovirus, rotavirus, and sapovirus in school, lift station, and wastewater treatment plant (WWTP) wastewater, and that a positive detection of a virus in a school sample was paired with a positive detection of the same virus at a downstream lift station or wastewater treatment plant over 97 % of the time. Additionally, we show how wastewater detections of rotavirus in schools and WWTPs were significantly associated with citywide viral intestinal infections. School wastewater can play a role in the monitoring of enteric viruses and in the detection of outbreaks, potentially allowing public health officials to quickly implement mitigation strategies to prevent viral spread into surrounding communities.

Structure and antigenicity of the divergent human astrovirus VA1 capsid spike.

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

Classic human astrovirus 4, 8, MLB-3, and likely new genotype 5 sublineage in stool samples of children in Nigeria.

Human astrovirus (HAstV) is a nonenveloped RNA virus and has been implicated in acute gastroenteritis among children and elderly. However, there exists a substantial dearth of information on HAstV strains circulating in Nigeria. Viral-like particles were purified from archived 254 stool samples of children with acute flaccid paralysis between January and December 2020 from five states in Nigeria, using the NetoVIR protocol. Extracted viral RNA and DNA were subjected to a reverse transcription step and subsequent random polymerase chain reaction amplification. Library preparation and Illumina sequencing were performed. Using the virome paired-end reads pipeline, raw reads were processed into genomic contigs. Phylogenetic and pairwise identity analysis of the recovered HAstV genomes was performed. Six near-complete genome sequences of HAstV were identified and classified as HAstV4 (n = 1), HAstV5 (n = 1), HAstV8 (n = 1), and MLB-3 (n = 3). The HAstV5 belonged to a yet unclassified sublineage, which we tentatively named HAstV-5d. Phylogenetic analysis of open reading frames 1a, 1b, and 2 suggested recombination events inside the MAstV1 species. Furthermore, phylogenetic analysis implied a geographic linkage between the HAstV5 strain from this study with two strains from Cameroon across all the genomic regions. We report for the first time the circulation of HAstV genotypes 4, 8, and MLB-3 in Nigeria and present data suggestive for the existence of a new sublineage of HAstV5. To further understand the burden, diversity, and evolution of HAstV, increased research interest as well as robust HAstV surveillance in Nigeria is essential.

[Analysis of the complete genome characterization of 11 human astrovirus strains in Shandong Province].

Objective: To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province. Methods: Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software. Results: A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 (n=6), MLB1 (n=6), MLB2 (n=1), and VA2 (n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion: The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Environmental Surveillance of Human Astroviruses in Jinan City of China, 2020-2021.

Human astroviruses (HAstVs) are a significant etiological agent of acute gastroenteritis in children. In order to investigate the circulation of HAstVs during the COVID-19 pandemic, a 2-year environmental surveillance was conducted in Jinan between 2020 and 2021. A total of 24 sewage samples were collected and concentrated. Real-time PCR indicated a positive rate of 83.3%, 79.2% (19/24), and 62.5% for classic, MLB, and VA types of HAstV in sewage samples, respectively, with genomic copies ranging from 6.4 × 103 to 3.7 × 107, 3.2 × 104 to 2.2 × 106, and 1.2 × 104 to 1.6 × 107 l-1. Next-generation sequencing (NGS) analysis on complete ORF2 amplicons from each sewage concentrate revealed the presence of 11 HAstV types, including HAstV-1, -2, -4, -5, MLB1, and VA1 to VA6, as well as non-human animal astroviruses. The most abundant HAstV types were HAstV-1, -4, and -5, which accounted for 70.3%, 12.6%, and 9.1% of total HAstV reads, respectively. Phylogenetic analysis revealed that the sequences obtained in this study were segregated into multiple transmission lineages, yet exhibited less genetic divergence among themselves than with foreign strains. These findings provide insight into the genotype diversity and genetic characterization of HAstVs during the COVID-19 pandemic, and highlight the effectiveness of utilizing NGS approaches to investigate sewage HAstVs.

Analysis of the complete genome characterization of 11 human astrovirus strains in Shandong Province / 中华预防医学杂志

Objective: To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province. Methods: Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software. Results: A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 (n=6), MLB1 (n=6), MLB2 (n=1), and VA2 (n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion: The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Analysis of the complete genome characterization of 11 human astrovirus strains in Shandong Province / 中华预防医学杂志

Objective: To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province. Methods: Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software. Results: A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 (n=6), MLB1 (n=6), MLB2 (n=1), and VA2 (n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion: The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Prevalence and Characterization of Gastroenteritis Viruses among Hospitalized Children during a Pilot Rotavirus Vaccine Introduction in Vietnam.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) are the most common viral causes of gastroenteritis in children worldwide. From 2016 to 2021, we conducted a cross-sectional descriptive study to determine the prevalence of these viruses in hospitalized children under five years old in Nam Dinh and Thua Thien Hue provinces in Vietnam during the pilot introduction of the RV vaccine, Rotavin-M1 (POLYVAC, Hanoi, Vietnam). We randomly selected 2317/6718 (34%) acute diarrheal samples from children <5 years of age enrolled at seven sentinel hospitals from December 2016 to May 2021; this period included one year surveillance pre-vaccination from December 2016 to November 2017. An ELISA kit (Premier Rotaclone®, Meridian Bioscience, Inc., Cincinnati, OH, USA) was used to detect RV, and two multiplex real-time RT-PCR assays were used for the detection of NoV, SaV and HAstV. The prevalence of RV (single infection) was reduced from 41.6% to 22.7% (p < 0.0001) between pre- and post-vaccination periods, while the single NoV infection prevalence more than doubled from 8.8% to 21.8% (p < 0.0001). The SaV and HAstV prevalences slightly increased from 1.9% to 3.4% (p = 0.03) and 2.1% to 3.3% (p = 0.09), respectively, during the same period. Viral co-infections decreased from 7.2% to 6.0% (p = 0.24), mainly due to a reduction in RV infection. Among the genotypeable samples, NoV GII.4, SaV GI.1, and HAstV-1 were the dominant types, representing 57.3%, 32.1%, and 55.0% among the individual viral groups, respectively. As the prevalence of RV decreases following the national RV vaccine introduction in Vietnam, other viral pathogens account for a larger proportion of the remaining diarrhea burden and require continuing close monitoring.

Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

Human astroviruses (HAstV) are etiologic agents of acute gastroenteritis that most often afflict young children and elderly adults. Most studies of HAstV have focused on epidemiology. In this study, we collected 10 stool samples from a diarrhea outbreak from a diarrhea sentinel surveillance hospital in Beijing. Samples were evaluated immediately using parallel multiplex RT-qPCR and nanopore sequencing, and were then amplified by designed primers and Sanger sequencing to obtain whole genome sequences. Six isolates were categorized as HAstV-5 and subjected to whole genome analysis to characterize their genetic variation and evolution. Full genome analysis revealed low genetic variation (99.38-100% identity) among isolates. Phylogenetic analysis showed that all isolates were closely related to domestic strains Yu/1-CHN and 2013/Fuzhou/85. The recombination breakpoint of the six isolates was located at 2741 bp in the overlap region of ORF1a and ORF1b, similar to those of Yu/1-CHN and 2013/Fuzhou/85. Overall, our study highlights the combined use of RT-qPCR and sequencing as an important tool in rapid diagnosis and acquisition of whole genome sequences of HAstV.

Entry and egress of human astroviruses.

Astroviruses encapsidate a positive-sense, single-stranded RNA genome into ∼30nm icosahedral particles that infect a wide range of mammalian and avian species, but their biology is not well understood. Human astroviruses (HAstV) are divided into three clades: classical HAstV serotypes 1-8, and novel or non-classical HAstV of the MLB and VA clades. These viruses are part of two genogroups and phylogenetically cluster with other mammalian astroviruses, highlighting their zoonotic potential. HAstV are a highly prevalent cause of nonbacterial gastroenteritis, primarily in children, the elderly and immunocompromised. Additionally, asymptomatic infections and extraintestinal disease (e.g., encephalitis), are also observed, mostly in immunocompetent or immunocompromised individuals, respectively. While these viruses are highly prevalent, no approved vaccines or antivirals are available to prevent or treat infections. This is in large part due to their understudied nature and the limited understanding of even very basic features of their life cycle and pathogenesis at the cellular and organismal level. This review will summarize molecular features of human astrovirus biology, pathogenesis, and tropism, and then focus on two stages of the viral life cycle, namely entry and egress, since these are proven targets for therapeutic interventions. We will further highlight gaps in knowledge in hopes of stimulating future research into these understudied viruses.

Coinfection of porcine astrovirus and other porcine viruses in diarrheic pigs in Haryana, India.

In this study, 306 rectal swabs from diarrheal pigs of various ages (0-3 weeks, 3-6 weeks, and >6 weeks) were collected from 54 piggery units in different climatic zones in Haryana state, India. These samples were tested for the presence of porcine astrovirus (PAstV), porcine rotavirus group A (PRV-A), and classical swine fever virus (CSFV) by reverse transcription polymerase chain reaction (RT-PCR), and porcine circovirus 2 (PCV-2) by polymerase chain reaction (PCR). Out of the 306 samples tested, 153 (50%), 108 (35.3%), 32 (10.6%), and three (0.9%) tested positive for PAstV, PCV-2, PRV-A, and CSFV, respectively. A single infection was detected in 135 samples, while mixed infections were found in 77 samples: 70 with two viruses and seven samples with more than two. PAstV was detected most frequently (55.31%) in pigs aged 3-6 weeks. PCV-2 was more predominant in pigs aged 0-3 weeks (36.53%), whereas PRV-A was more common in pigs aged 3-6 weeks (11.3%). CSFV was observed in the age group of 0-3 weeks (1.92%). Phylogenetic analysis revealed the circulation of lineages 2 and 4 of PAstV in this region. Thus, it can be concluded that one or more than one virus is circulating in piggery units in Haryana, India.

Astrovirus replication is dependent on induction of double-membrane vesicles through a PI3K-dependent, LC3-independent pathway.

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.

Genetic characteristics of classical astroviruses in Shenzhen, China, 2016-2019.

Human astrovirus (HAstV) is a single-stranded, positive-sense RNA virus and is the leading cause of viral gastroenteritis. However, despite its prevalence, astroviruses still remain one of the least studied enteroviruses. In this study, we sequenced 11 classical astrovirus strains from clinical samples collected in Shenzhen, China from 2016 to 2019, analyzed their genetic characteristics, and deposited them into GenBank. We conducted phylogenetic analysis using IQ-TREE software, with references to astrovirus sequences worldwide. The phylogeographic analysis was performed using the Bayesian Evolutionary Analysis Sampling Trees program, through Bayesian Markov Chain Monte Carlo sampling. We also conducted recombination analysis with the Recombination Detection Program. The newly sequenced strains were categorized as HAstV genotype 1, which is the predominant genotype in Shenzhen. Phylogeographic reconstruction indicated that HAstV-1 may have migrated from the United States to China, followed by frequent transmission between China and Japan. The recombination analysis revealed recombination events within and across genotypes, and identified a recombination-prone region that produced relatively uniform recombination breakpoints and fragment lengths. The genetic analysis of HAstV strains in Shenzhen addresses the current lack of astrovirus data in the region of Shenzhen and provides key insights to the evolution and transmission of astroviruses worldwide. These findings highlight the importance of improving surveillance of astroviruses.