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.

Goose Nephritic Astrovirus Infection of Goslings Induces Lymphocyte Apoptosis, Reticular Fiber Destruction, and CD8 T-Cell Depletion in Spleen Tissue.

The emergence of a novel goose nephritic astrovirus (GNAstV) has caused economic losses to the Chinese goose industry. High viral load is found in the spleen of goslings infected with GNAstV, but pathological injuries to the spleen due to GNAstV are largely unknown. In this study, 50 two-day-old goslings were infected orally with GNAstV, and 50 goslings were treated with PBS as control. Spleens were collected at different times following infection to assess damage. GNAstV infection caused visceral gout and urate deposition in joints, and resulted in 16% mortality. GNAstV was found in the lymphocytes and macrophages within the spleen. Lymphocyte loss, especially around the white pulp, and destruction and decline in the number of reticular fibers was observed in GNAstV-infected goslings. Moreover, in GNAstV-infected goslings, ultrahistopathological examination found that splenic lymphocytes exhibited condensed chromatin and apoptotic bodies, and reticular cells displayed damage to plasma membrane integrity and swollen mitochondria. Furthermore, TUNEL staining confirmed apoptosis of lymphocytes, and the mRNA levels of Fas and FasL were significantly increased in the GNAstV-infected goslings. In addition, GNAstV infection reduced the number and protein expression of CD8. In conclusion, GNAstV infection causes lymphocyte depletion, reticular cell necrosis, reticular fiber destruction, lymphocyte apoptosis, and reduction in CD8 levels, which contribute to spleen injury.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3.

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5' and 3' regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

Chicken Astrovirus (CAstV) Molecular Studies Reveal Evidence of Multiple Past Recombination Events in Sequences Originated from Clinical Samples of White Chick Syndrome (WCS) in Western Canada.

In this study, we aimed to molecularly characterize 14 whole genome sequences of chicken astrovirus (CAstV) isolated from samples obtained from white chick syndrome (WCS) outbreaks in Western Canada during the period of 2014-2019. Genome sequence comparisons showed all these sequences correspond to the novel Biv group from which no confirmed representatives were published in GenBank. Molecular recombination analyses using recombination detection software (i.e., RDP5 and SimPlot) and phylogenetic analyses suggest multiple past recombination events in open reading frame (ORF)1a, ORF1b, and ORF2. Our findings suggest that recombination events and the accumulation of point mutations may have contributed to the substantial genetic variation observed in CAstV and evidenced by the current seven antigenic sub-clusters hitherto described. This is the first paper that describes recombination events in CAstV following analysis of complete CAstV sequences originated in Canada.

Isolation and characterization of a duck-origin goose astrovirus in China.

In 2019, a new type of infectious disease characterized with haemorrhage and swellings of kidneys, occurred on commercial duck farms in Shandong province, China. Our systematic investigation led to the isolation of an astrovirus, designated AstV-SDTA strain and was isolated from a diseased duckling using LMH cells. Similar clinical symptoms were reproduced by experimental infection using the AstV-SDTA strain. The complete genome sequencing characterization of AstV-SDTA was conducted using next-generation sequencing (NGS) technique on Illumina HiSeq platform, and used polymerase chain reaction method to verify the NGS results for the obtained whole sequences. Phylogenetic analysis revealed that AstV-SDTA strain belongs to a novel goose astrovirus (GoAstV) branch of avian astroviruses, and the nucleotide homology based on the complete genome sequences among AstV-SDTA and other GoAstV strains deposited in Genbank was 97.2-98.8%. Taken together, these results suggest that the cross-species transmission of novel GoAstV between domestic waterfowl is possible. Further surveillance of novel GoAstV in poultry are needed in order to gain a better understanding of both the molecular and evolutionary characteristics of novel GoAstV.

Diversity of Astroviruses Circulating in Humans, Bats, and Wild Birds in Egypt.

Astroviruses belong to Astroviridae family which includes two main genera: Mamastroviruses that infect mammals, and Avastroviruses that infect avian hosts. Bats and wild birds are considered among the natural reservoirs for astroviruses. Infections in humans are associated with severe gastroenteritis, especially among children. We conducted surveillance for astroviruses in bats, wild birds, and humans in Egypt. Our results indicated relatively high prevalence of astroviruses in those hosts. Phylogenetic analysis revealed diversity of these viruses within hosts. Detected human viruses showed similarity with classic and variant human astroviruses, as well as similarity with animal-origin viruses. Viruses in bats were dispersed, with similarities to other bat viruses as well as other mammalian, including human, viruses. Wild bird viruses varied and were related to other avastroviruses, as well as human astroviruses. Our results indicate that astroviruses are common in bats, wild birds, and humans in Egypt, with a wide gene pool. Potential cross-species transmission may be occurring but should be verified by further surveillance and molecular studies.

Complete genome sequence and phylogenetic analysis of novel avastroviruses circulating in China from 2016 to 2018.

Avastrovirus-specific antibodies are widely detected in chickens in China. However, there are currently no commercially available vaccines for this group of viruses. To address this issue, we collected 76 tissue samples from Avastrovirus (AAstVs) antibody-positive chickens from farms across eight provinces in China from 2016 to 2018. The samples were then screened for the presence of AAstVs sequences by polymerase chain reaction analysis and a phylogenetic tree was constructed. Specific primers were designed to amplify the whole genome sequences of the viruses from four positive samples, with the genetic characteristics and structures of the resulting genomes then analyzed further. Overall, 42 (55.3 %) of the 76 samples were positive for AAstVs RNA. Phylogenetic analysis along with the ORF1b gene showed that 15 isolates were grouped in AAstV-1 and 27 of them were grouped in AAstV-2. None of the isolates was belonged to AAstV-3. Sequencing and structural analyses revealed that the genomes of the four isolates showed the typical characteristics of AAstVs genomes but were genetically distinct from other AAstVs. The results of this study will contribute to our understanding of the genetic characteristics of AAstVs in China.

Specific detection of the novel goose astrovirus using a TaqMan real-time RT-PCR technology.

Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/µL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV.

Genetic identification of astroviruses in wild boars.

Astroviruses are widely detected in pigs but their detection in wild boars is rather sporadic. In this study, astroviruses were detected in organ homogenates of wild boars by applying nested reverse transcriptase polymerase chain reaction, and the typing was carried out by phylogenetic analysis. Overall, 30/200 (15.0%) homogenates were positive for astroviruses. Genetic typing revealed that of 13 amplicons analyzed, 8 were typed as porcine astrovirus lineage 2 (PAstV-2), 2 as lineage 4 (PAstV-4), 2 identical sequences were grouped with chicken astrovirus, and 1 sequence belonged to a bat astrovirus lineage. This first identification of chicken and bat astroviruses in wild boars indicates interspecies transmission.

Genome analysis of newly emerging goose-origin nephrotic astrovirus in China reveals it belongs to a novel genetically distinct astrovirus.

Since 2017, a new type of goose-origin astrovirus (GoAstV) disease occurred in China. This disease can cause joint swelling of sick geese, and the anatomy shows a clear urate precipitation in the viscera. The rate of death or amputation can reach more than 30%, revealing its severe pathogenicity. One novel goose-origin astrovirus strain, designated as CXZ18, was isolated from diseased geese with a fatal infection characterized by visceral urate deposition. Similar clinical anatomy symptoms were partially reproduced by attacking infection of healthy geese. The CXZ18 has no hemagglutination with chicken erythrocyte, only reproduced in goose embryos, not in SPF chicken or duck embryos. The complete genome-encoded three open reading frames (ORFs) of CXZ18 were 7252 nt in length. BLAST-based homology analysis of viral complete genome showed that CXZ18 has only 53.0%-61.8% with other classic avian astrovirus from various hosts. Further analysis of ORF 1a, ORF 1b, and ORF 2 genes revealed that the isolate was genetically distinct from known astroviruses and belonged to a distinctive branch of avian astroviruses. To conclude, a naturally occurring novel nephrotic astrovirus, distinguished with all previously reported avian astroviruses, was derived from goose.

Genetic characterization of a new astrovirus in goslings suffering from gout.

Since early 2016, the Chinese goose industry has experienced severe outbreaks of gout; however, the etiological factor of the disease is still unclear. Here, we investigated the possible involvement of viral infection in the disease. Using sequence-independent PCR amplification, astrovirus sequences were generated from a gout case. Full-length genomic sequencing and sequence analysis of three goose astrovirus (GoAstV) strains revealed that they belong to a new avastrovirus most closely related to viruses classified within species Avastrovirus 3. The GoAstV was detected in 16/16 gout cases collected from two provinces, supporting a pathogenic role for the new avastrovirus.

White Chick Syndrome Associated with Chicken Astrovirus in Ontario, Canada.

Sixty-four cases of white chick syndrome (WCS) in broiler breeders producing affected progeny were reported from seven hatcheries in Ontario, Canada, between 2009 and 2016, with 43 of those originating from two hatcheries owned by a single company. WCS cases were identified by the presence of typical chicks in the hatchery that were generally weak with pale to white down, enlarged abdomens, and occasionally brown wiry fluff on the dorsum of the neck. Affected embryos and chicks had characteristic gross and histologic liver lesions, and livers were positive for chicken astrovirus (CAstV) RNA by real-time reverse transcriptase PCR. Affected broiler breeder flocks experienced egg production drops of 0% to 21% and hatchability drops of 0% to 68.4%. The amino acid sequence of the region encoding the capsid gene of WCS viruses demonstrated all Ontario CAstV to be in Group B, Subgroup Bii.

Isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings.

Astroviruses are recognized as a leading cause of gastroenteritis in humans and animals. They are also associated with extra-intestinal diseases, such as hepatitis in ducklings, nephritis in chickens, and encephalitis in cattle. In February 2017, a fatal infection of goslings characterized by visceral urate deposition was reported in the Shandong province, China. Our systematic investigation led to the isolation of an astrovirus, designated AAstV/Goose/CHN/2017/SD01, and similar disease was reproduced by experimental infection of healthy goslings, fulfilling Koch's postulates. The isolated astrovirus replicated well and resulted in 100% mortality of goose embryos. Complete genome sequence analysis revealed that the isolate was genetically distinct from known astroviruses and closely related to members of the avastrovirus genogroup II. Experimental infection showed that the isolate was highly pathogenic in goslings, causing clinical signs, growth repression and in many cases mortality. Histopathological examination indicated that lesions occurred mainly in the kidneys of infected birds. However, virus-specific genomic RNA was detected in all representative tissues, and virus shedding was detected up to 12 days after inoculation, suggesting that the isolate was able to spread systemically and replicate efficiently in vivo. Collectively, our study demonstrates, for the first time, the etiological role of a genetically distinct astrovirus in the fatal infection of goslings.

A novel group of avian Avastrovirus in domestic geese, China.

Using an ORF1b-based astrovirus-specfic reverse transcription (RT)-PCR assay, a novel astrovirus-like was detected from domestic geese in China. Pairwise comparisons and phylogenetic analyzes suggested that a novel group of goose astrovirus, different with previously known astroviruses in the genus Avastrovirus, was found circulating in geese. This study has expanded our understanding about the role of domestic waterfowls as reservoirs for diverse astroviruses.

Complete genome sequence of a novel avastrovirus in goose.

We report the complete genome sequence of a new avastrovirus of goose-origin (FLX). The 7299-nt-long genome consisted of three overlapping open reading frames (ORFs) that were in different reading frames. Pairwise comparisons showed that the FLX genome was 59% identical to its closest relatives and that the levels of amino acid identity shared by FLX with other astroviruses did not exceed 54% in ORF1a, 66% in ORF1b, and 50% in ORF2, respectively. Phylogenetic analysis based on the amino acid sequence of the full-length ORF2 demonstrated that FLX was highly divergent from all other avastroviruses. At the amino acid level the complete capsid region of FLX shared genetic distances of 0.574-0.719 with three official avastrovirus species, suggesting that it can be classified as a member of a novel species in the genus Avastrovirus.

A Review of the Strain Diversity and Pathogenesis of Chicken Astrovirus.

Although a relatively recently emerged virus, identified only in 2004 as a separate species of avian astrovirus, chicken astrovirus (CAstV) has been associated with poor growth of broiler flocks, enteritis and diarrhea and is a candidate pathogen in cases of runting stunting syndrome. More recently CAstV has been implicated in cases of two other diseases of broilers as the sole etiological agent, namely severe kidney disease of young broilers with visceral gout and the "White Chicks" hatchery disease. Examination of the strains of CAstV associated with the two latter diseases reveals they are closely related genetically. This review will discuss the pathogenesis of CAstV in relation to strain diversity and the effects of vertical versus horizontal transmission, virus load, co-infections and age of bird at infection, all factors that may impact upon disease severity.

Complete genome sequence analysis of chicken astrovirus isolate from India.

OBJECTIVE: Chicken astroviruses have been known to cause severe disease in chickens leading to increased mortality and "white chicks" condition. Here we aim to characterize the causative agent of visceral gout suspected for astrovirus infection in broiler breeder chickens. METHODS: Total RNA isolated from allantoic fluid of SPF embryo passaged with infected chicken sample was sequenced by whole genome shotgun sequencing using ion-torrent PGM platform. The sequence was analysed for the presence of coding and non-coding features, its similarity with reported isolates and epitope analysis of capsid structural protein. RESULTS: The consensus length of 7513 bp genome sequence of Indian isolate of chicken astrovirus was obtained after assembly of 14,121 high quality reads. The genome was comprised of 13 bp 5'-UTR, three open reading frames (ORFs) including ORF1a encoding serine protease, ORF1b encoding RNA dependent RNA polymerase (RdRp) and ORF2 encoding capsid protein, and 298 bp of 3'-UTR which harboured two corona virus stem loop II like "s2m" motifs and a poly A stretch of 19 nucleotides. The genetic analysis of CAstV/INDIA/ANAND/2016 suggested highest sequence similarity of 86.94% with the chicken astrovirus isolate CAstV/GA2011 followed by 84.76% with CAstV/4175 and 74.48%% with CAstV/Poland/G059/2014 isolates. The capsid structural protein of CAstV/INDIA/ANAND/2016 showed 84.67% similarity with chicken astrovirus isolate CAstV/GA2011, 81.06% with CAstV/4175 and 41.18% with CAstV/Poland/G059/2014 isolates. However, the capsid protein sequence showed high degree of sequence identity at nucleotide level (98.64-99.32%) and at amino acids level (97.74-98.69%) with reported sequences of Indian isolates suggesting their common origin and limited sequence divergence. The epitope analysis by SVMTriP identified two unique epitopes in our isolate, seven shared epitopes among Indian isolates and two shared epitopes among all isolates except Poland isolate which carried all distinct epitopes.

Detection and molecular characterization of enteric viruses in enteritis-affected commercial broiler chickens in India.

A study was conducted to detect and characterize the enteric viruses (chicken astrovirus, avian nephritis virus and avian orthoreovirus) present in flocks of commercial broiler chickens suffering from enteritis in Haryana, India. The intestinal contents were collected from 65 enteritis-affected flocks (cases) and tested by reverse transcription PCR (RT-PCR). Of these 65 cases, 35 (53.80%) were positive for a single virus and 26 (40.00%) for two viruses. The remaining four samples were negative for all three viruses tested. Of the 65 cases, 57 were positive for chicken astrovirus (CAstV) while 30 cases had avian nephritis virus (ANV). None of the cases were positive for orthoreovirus. Comparison of 12 CAstVs of this study with previously published CAstV sequences revealed nucleotide identities ranging from 73.20 to 98.00%. The nucleotide identities ranged between 83.10-95.50% when nine ANVs of this study were compared with previously reported ANV sequences. The amino acid sequences of CAstVs in comparison to previously published sequences revealed certain unique changes. Phylogeny based on polymerase gene revealed that CAstVs and ANVs of this study were under the same monophyletic clade. In conclusion, a large number of broiler chicken flocks experiencing enteritis were positive for CAstV and ANV by RT-PCR. The presence of more than one enteric virus in enteritis-affected flocks and changes at the genetic level in these viruses may affect the severity of disease.

Avian nephritis virus (ANV) on Brazilian chickens farms: circulating genotypes and intra-genotypic diversity.

Avian nephritis virus (ANV), which belongs to the family Astroviridae, is associated with different clinical manifestations (including enteric disorders). Despite being frequently found in the avian industry worldwide, information regarding genetic features of these viruses in Brazil is scarce. Therefore, sixty fecal sample pools (5-6 birds of the same flock), representing 60 poultry farms from six Brazilian States, were screened using an astrovirus-specific hemi-nested-PCR assay targeting the conserved ORF1b gene, followed by nucleotide sequencing of amplified products. PCR and phylogenetic analysis confirmed the detection of 21 positive samples to ANV (35 %). In order to investigate the genetic diversity represented by these viruses, amplification, cloning and phylogenetic analysis of the deduced amino acid sequence of ORF2 gene were attempted. Eight samples were successfully cloned (generating 32 clones in total) and sequenced. Based on phylogenetic analysis of ORF2, sequences defined in this study were classified into three genotypes: genotype 5, which has already been described in birds, and two other novel genotypes, tentatively named genotype 8 and 9, all of which occurred in single or mixed infections. Moreover, high intra-genotypic diversity and co-circulation of distinct strains in a same host population were observed. This study revealed the presence of new strains of ANV in Brazilian poultry and their circulation in commercial chicken flocks.

A metagenomic comparison of endemic viruses from broiler chickens with runting-stunting syndrome and from normal birds.

Runting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterize to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual virus has been identified as the main agent of RSS and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. This paper attempts to characterize the viral pathogens associated with 2-3-week-old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Analysis of the viromes identified a total of 20 DNA and RNA viral families, along with 2 unidentified categories, comprised of 31 distinct viral genera and 7 unclassified genera. The most abundant viral families identified in this study were the Astroviridae, Caliciviridae, Picornaviridae, Parvoviridae, Coronaviridae, Siphoviridae, and Myoviridae. This study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus 1 and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study.