Isolation, identification, and epidemiological characteristics of goose astrovirus causing acute gout in Guangdong province, China.

Goose astrovirus (GAstV) has been widespread in China since 2016, causing significant growth inhibition and gout symptoms in goslings and leading to substantial economic losses in the goose industry. To better understand the epidemiological characteristics of GAstV in Guangdong Province, 682 samples were collected from geese with suspected GAstV infection across different regions of Guangdong Province from January 2022 to January 2024. Virus isolation, identification, and genetic evolution analysis were performed. The results showed that all samples were GAstV positive, with 52.64% co-infected with GAstV-1 and GAstV-2, and 42.38% positive for GAstV-2 alone, indicating that GAstV-2 remains the most prevalent subtype. Additionally, three GAstV isolates were identified using molecular detection, immunofluorescence, and transmission electron microscopy on LMH cells or goose embryos. Compared with GDYJ2304 and other reported GAstV-2 strains, the ORF2 region of the GDYJ2210 isolates lacked 3 bases, and the replication ability of GDYJ2210 was significantly higher than that of GDYJ2304. Whole genome sequence alignment and genetic evolution analysis revealed that the GDFS2209 isolate was located in the GAstV-1 branch, with a sequence similarity of 89.70 to 99.00% to GAstV-1 reference strains. The GDYJ2210 and GDYJ2304 isolates were located in the GAstV-2 branch, showing a sequence similarity of 96.80 to 98.90% to GAstV-2 reference strains. These results demonstrated that the GAstV isolates were highly similar to each other despite being prevalent in 5 different regions of Guangdong Province. These findings enhance the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Isolation, identification, and pathogenicity of two novel goose astrovirus from goslings with severe gout in China.

Goose astroviruses (GAstVs) are important pathogens which can cause gout in goslings leading to huge economic losses for the goose farming industry in China. In 2023, an infectious disease characterized by visceral gout broke out in commercial goose farms in Guangxi and Guangdong provinces of China. In this study, two GAstV strains of GXNN and GDCS were successfully isolated from these two disease-ridden goose farms. The complete genomic lengths of these two strains were 7166 bp, and phylogenetic analysis showed that they were both GAstV-2 subtypes. The 3-dimensional structures of the capsid protein were predicted and six characteristic mutation sites at amino acid positions 60, 61, 228, 229, 456 and 523 were found within the strong antigenic regions. A recombination event occurred at 6833-7070 nt between the GAstV TZ03 and Turkey astrovirus CA/00 and this was detected in both the GXNN and GDCS strains. Another recombinant event occurred at 63-2747 nt between the GAstV XT1 and GAstV SDPY and this was detected in the GDCS strain. When 1-day-old goslings were infected with the novel GXNN and GDCS strains, they showed severe visceral gout. This was accompanied by enlarged spleens, liver hemorrhages and urate deposits in the kidneys and ureters and their blood urea nitrogen levels were significantly elevated. The mortality rates of the GXNN- and GDCS-infected groups were pathogenically high at 80 % and 60 %, respectively. These results will promote our understanding of the evolution and epidemic potential of GAstVs in China.

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.

Isolation and evolutionary analyses of gout-associated goose astrovirus causing disease in experimentally infected chickens.

Astroviruses are a common cause of gastroenteritis in humans and animals. They are also associated with extraintestinal infections, including hepatitis in ducklings, nephritis in chickens, as well as fatal meningitis and encephalitis in humans and other mammals. Since 2014, outbreaks of disease characterized by visceral gout and swelling of kidneys have been reported in goslings and ducklings in China, with the causative agent revealed to be a novel avian astrovirus designated goose astrovirus (GoAstV). In the present study, this novel gout-associated GoAstV was identified in diseased goslings from 2 farms in Hunan province, China. Three genomes were successfully sequenced and analyzed and were shown to have high identities of 99.7 to 99.8% between each other, with some specific amino acid alterations revealed in open reading frame 2 when compared with other gout-associated GoAstVs. Two strains were further efficiently isolated in the DF-1 chicken fibroblast cell line with high virus titers of 1011 viral genomic copies per mL of culture media. A pilot virus challenge study using GoAstV in chickens demonstrated that this virus can cause clinical visceral gout in chickens, indicating its ability to cross the species barrier. Based on the phylogenetic analyses of capsid sequences, the identified GoAstVs were proposed to be classified into 2 genotypes, GoAstV1 and GoAstV2, and the novel gout-associated GoAstVs were all clustered in GoAstV2. Further Bayesian inference analyses indicated a nucleotide substitution rate of 1.46 × 10-3 substitutions/site/year for avian astrovirus based on open reading frame 2 sequences, and the time to the most recent common ancestor of GoAstVs was estimated to be around 2011. This is the first report to confirm GoAstV can infect chickens while also providing an estimation of the evolutionary rates of Avastroviruses.

Pathogenicity of novel goose-origin astrovirus causing gout in goslings.

BACKGROUND: A novel goose-origin astrovirus (GoAstV) has broken out across China in recent years, causing gout in goslings with a mortality rate of around 50%. However, our understanding of the dynamic distribution, tissue tropism and pathogenesis of GoAstV is incomplete. In order to assess its pathogenicity, one-day-old goslings were inoculated separately with GoAstV via oral and subcutaneous injection routes. RESULTS: Clinical symptoms, gross and microscopic lesions, blood biochemical parameters and viral loads were detected and recorded for 20 days after infection. Typical gout was observed in experimental goslings. GoAstV can be replicated in tissues and cause pathological damage, especially in the kidney, liver, heart and spleen. Virus-specific genomic RNA was detected in blood, cloacal swabs and all representative tissues, and virus shedding was detected up to 20 days after inoculation, suggesting that GoAstV has a wide tissue tropism and spread systematically after inoculation. The viral copy numbers examined in kidney were the highest, followed by spleen and liver. CONCLUSION: This experiment determined the accurate value of viral loads and biochemical indicators of GoAstV-induced goslings. These findings increase our understanding of the pathogenicity of GoAstV in goslings and provide more reference for future research.

Successful Treatment of Covid-19 Associated Cytokine Release Syndrome with Colchicine. A Case Report and Review of Literature.

We describe the case of a 42 year old, healthy patient with Covid-19 who despite improvement in his respiratory symptoms developed a mild to moderate cytokine release syndrome (CRS) and an associated monoarticular gout flare. Since the patient refused admission to the hospital and had stable vital signs, we chose to treat him with a safe anti-inflammatory and non-immunosuppressive therapy. To hit two birds with one stone, we considered colchicine, as it has systemic anti-inflammatory effects and is also effective in gout flare. Unexpectedly, 48 hours after treatment, not only did his ongoing fever and toe pain disappear, he also had significant improvements in his general state of health and all his inflammatory markers including fibrinogen, ferritin, D-dimer, and IL-6 levels normalized. To our knowledge, the use of colchicine in Covid-19 and CRS has not been reported. This observation merits the consideration of colchicine as a safe, inexpensive and oral medication for the treatment of mild to moderate CRS in Covid-19 patients. More importantly, in Covid-19 patients with early lung involvement colchicine may be an appropriate candidate to prevent CRS in adjunction with routine antiviral agents. Indeed, multicenter, randomized controlled studies are required to evaluate the benefits of this therapy.

Rapid and visual detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription loop-mediated isothermal amplification.

To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription-quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.

Genomic and phylogenetic characteristics of a novel goose astrovirus in Anhui Province, Central-Eastern China.

Goose astrovirus (GAstV) causes a novel disease characterized by urate deposition in the viscera and joints in goslings in many provinces of China, leading to huge economic losses in the goose industry. To better understand the genetic diversity of GAstV in the Anhui Province, Central-Eastern China, 48 kidney samples from goslings with gout were subjected to reverse-transcription polymerase chain reaction (RT-PCR) analysis for detecting GAstV, and phylogenetic analysis of whole genomes and ORFs was performed. Thirty-five samples were GAstV-positive, indicating that the virus is a frequent cause of gout. The whole genomes of 5 GAstV strains were successfully sequenced and named AHAU1-5. The sequenced genomes and those of reference GAstV strains in GenBank displayed 97.4-99.8% similarity. The isolates had high nucleotide sequence similarity with the GAstV reference strain SDPY. A phylogenetic analysis showed that AHAU1 and AHAU4 were closely related to the reference strain SDPY; AHAU2, AHAU3, and AHAU5 formed separate branches. Furthermore, recombination analysis revealed putative recombination sites in the Jiangsu strains that originated from strains in the Anhui and Shandong Provinces, accompanied by the recombination of different strains in the Anhui Province. This study is the first to carry out systematic phylogenetic analysis of GAstV isolated in the Anhui Province, Central-Eastern China. By improving our understanding of the diversity of GAstV in the Anhui Province, these results provide a basis for the prevention and control of its spread.

Coinfection of parvovirus and astrovirus in gout-affected goslings.

Outbreaks of gosling gout have occurred in China since 2017 and caused a considerable economic impact on the poultry industry. While gosling astrovirus (GoAstV) is believed to be the main causal pathogen of gout, the full-blown disease of gout cannot be well reproduced by infecting the goslings with GoAstV, suggesting the possibility of other infectious agents being involved with the development of gosling gout. To assess other possible infectious agents, we collected tissues from gout-affected goslings in 12 goose farms in China, followed by PCR detection of GoAstV, goose reovirus (GRV), goose parvovirus (GPV), fowl adenovirus (FAdV), goose circovirus (GcoV), Tembusu virus (TMUV) and goose haemorrhagic polyomavirus (GHPV). Our data showed that all gout-affected goslings carried both of GoAstV and GPV determined by PCRs, and this was further confirmed by fluorescence multiplex immunohistochemical staining, and phylogenetic analysis of ORF2 gene of GoAstV and VP3 gene of GPV. In addition to the haemorrhage in the kidney, liver, spleen and lung of the gout-affected goslings, histological examinations showed also extensive infiltration of heterophil myelocytes in the kidney, liver, spleen, bursa of Fabricius, thymus, lungs and pancreas. Our findings strongly suggest that coinfection of GoAstV and GPV increases the severity of gout. While this is the first study to report GPV in gout-affected goslings, further studies including infection model are warranted to investigate the role of GPV and its coinfection with GoAstV in the development of gosling gout.

Goose astrovirus infection affects uric acid production and excretion in goslings.

In 2018, a new goose astrovirus (GAstrV) was reported in China, which causes 2 to 20% deaths in 4- to 16-day-old goslings causing great damages to the livestock industry. Gout is the typical feature of GAstrV infection in goslings. However, the mechanism of gout formation remains unclear. In the present study, 2-day-old goslings were infected intramuscularly with GAstrV for 14 D. One quarter of the infected goslings died, and typical gout pathological changes were found in the dead infected goslings. Pathological changes were observed in the morphology of the kidney and liver, such as degeneration, necrosis, and inflammatory cell infiltration. Accordingly, a high virus load was found in both organs. The serum level of uric acid in the inoculated goslings was higher, whereas no differences were found in levels of creatinine, calcium, and phosphorus. Moreover, the xanthine dehydrogenase (XOD) and adenosine deaminase (ADA) activities and the mRNA levels of xanthine dehydrogenase, adenosine deaminase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1 in livers increased, wheres the multidrug resistance-associated protein 4 mRNA level and Na-K-ATPase activity in the kidneys decreased. These results showed that GAstrV infection could cause lesions on the liver and kidney and then increase the expression or activity of enzymes related to uric acid production in the liver and decrease renal excretion function, which contribute to hyperuricemia and gout formation.

Characterization and genomic analysis of emerging astroviruses causing fatal gout in goslings.

Since February 2017, severe outbreaks of fatal gout caused by novel gosling astroviruses (GoAstVs) have occurred in several Chinese provinces, causing a considerable economic impact on the poultry industry. To assess the infection status of GoAstVs causing gout, 165 clinical samples were collected from goslings from seven farms located in different Chinese provinces, and they were screened for viral infection. Seven GoAstV strains were completely sequenced. The positive infection rates of GoAstV, goose parvovirus, reovirus, goose haemorrhagic polyomavirus and Tembusu virus were 100%, 9.69%, 3.64%, 0% and 0%, respectively, indicating the role of GoAstV in gout. The genomes of all seven GoAstV strains were 7170-nt long and encoded three open reading frames (ORFs), namely, ORF1a, ORF1b and ORF2. Sequence and phylogenetic analyses of the seven GoAstV strains showed that these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2. Moreover, the mutation rates of ORF1a and ORF2 were high, and ORF1a was highly mutated at amino acid loci 545-580. The tertiary structure of the mutated ORF2 protein was smooth, and its antigenic epitope was highly mutated, which may be related to the pathogenicity of the virus and caused by antibody pressure from the host. These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.

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.

Novel Goose Astrovirus Associated Gout in Gosling, China.

In 2017, an emerging disease outbreak with high mortality in goslings occurred in China. A novel goose astrovirus was isolated and identified as the causative agent of this disease. Genomic analysis revealed that this virus strain was significantly distinct from published astrovirus strains.

Isolation and genomic characterization of gosling gout caused by a novel goose astrovirus.

A severe infectious disease characterized with gout, haemorrhage and swellings of kidneys has affected goslings around the major goose-producing regions in China since November 2016. A Novel goose-origin astrovirus (AStV), designated as AStV/SDPY/Goose/1116/17 (AStV-SDPY) strain, was isolated from diseased goslings, and experimental reproduction of gout was successful using the AStV-SDPY strain. Additionally, the AStV-SDPY was conducted for its full genome sequencing characterization using next-generation sequencing (NGS) technique on Illumina HiSeq platform. A complete genome of the AStV-SDPY was 7,252 nt in length and encoded three viral proteins. Phylogenetic analysis revealed that AStV-SDPY strain belongs to an independent branch of avian astroviruses, and the nucleotide homology among AStV-SDPY and other classic avian astrovirus strains was only 48.8%-68.2%. Results of above data indicated the causative agent of the gosling gout occurring in China is a novel divergent goose astrovirus.

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.

Role of chicken astrovirus as a causative agent of gout in commercial broilers in India.

Several outbreaks of gout were reported in commercial broilers in India during 2011 and 2012, causing up to 40% mortality in the birds. Gross and histopathological observations confirmed gout. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis from kidney samples of gout-affected birds indicated the presence of chicken astrovirus (CAstV) in 41.7% of cases and a mixed infection of CAstV and avian nephritis virus (ANV) in 36.4% of cases. CAstV isolated from gout-affected kidneys by inoculating embryonated specific pathogen free (SPF) eggs showed dwarfing in embryos and a cytopathic effect in chicken embryo kidney cells. Inoculation of 1-day-old SPF and broiler chicks with CAstVs caused gout and mortality between 4 and 10 days post inoculation. Virus isolation and qRT-PCR analysis showed the presence of only CAstV in inoculated chicks. Sequence analysis of capsid genes indicated a major group of Indian CAstVs that displayed 92.0 to 99.2% intergroup amino acid identity and 83.9 to 90.4% identity with subgroup Bi CAstVs of UK origin. We designated this group Indian Bi. Analysis of the partial polymerase amino acid sequences of our isolates indicated two groups of CAstVs (Indian 1 and 2) that displayed 90.2 to 95.5% amino acid identity between them. We thus report for the first time that, in addition to infectious bronchitis virus and ANV, CAstVs are a causative agent of gout.