Isolation and evolutionary analyses of gout-associated goose astrovirus causing disease in experimentally infected chickens.
ABSTRACT: 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.
Project description:<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>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.
Project description: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.
Project description: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.
Project description:Since 2017, a serious infectious disease characterized by visceral gout has emerged in China's main goose-producing regions. The disease has caused huge economic losses to China's goose industry. In our previous study, we determined that the pathogen causing gout in goslings is a novel goose-origin astrovirus, designated as AStV/SDPY/Goose/1116/17 (AStV-SDPY) strain. To investigate the effect of host age on the outcome of novel goose-origin astrovirus infection, 200 1-day-old healthy goslings were selected to be experimentally infected at 1, 5, 15, 25, and 35 D of age. It was shown in experimental infection that the AStV-SDPY strain was highly pathogenic in goslings aged 1 to 15 D, causing growth repression, severe visceral urate deposition, and even death, whereas goslings infected at 25 and 35 D of age showed mild symptoms. Histopathologic examination indicated that lesions occurred mainly in the kidney and liver of infected goslings, which is correlated to the severity of clinical signs and gross lesions. Viral RNA was detected in all representative tissues, and virus shedding was detected continuously within 15 D after inoculation. Higher viral copy number, especially in vital organs such as the liver and kidney, was developed in the goslings infected at 1 to 15 D of age than older geese. In addition, clinical chemistry and inflammatory cytokines showed that younger geese are more sensitive to AStV infection. Overall, our study demonstrates that the pathogenicity of AStV-SDPY in goslings is partly associated with the age of infection, laying a foundation for further study of the pathogenic mechanism of this virus.
Project description:Since the first isolation from human, astroviruses have been detected in many species. Wide host range and occasional cross-transmission of astrovirus pose a risk for zoonotic infection. Here, novel astroviruses were identified from goslings with recent epidemic gout disease in China. A virus, designated as GD, was efficiently isolated from a diseased gosling using LMH cells. Genome of GD amplified using 5' and 3' RACE was 7183nt in full length. Sequence analysis revealed the genome of GD was <60.8% homology with others deposited in Genbank. Moreover, GD could be neutralized by goose convalescent sera, and the gout associated symptom in goslings could be reproduced by GD infection. Our data demonstrated the goose astrovirus could be one of the causative agents of the ongoing gosling gout disease in China. The identification of the goose astrovirus not only diversified the astrovirus species, but also broadened the disease patterns caused by astroviruses.
Project description:In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/?L at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.
Project description: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.
Project description: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.
Project description:Chicken astrovirus (CAstV) infection is strongly associated with kidney disease, gout, "white chicks" hatchery disease, and runting and stunting syndrome (RSS). In the present study, 82.5% of 154 clinical samples from different provinces in China were positive for CAstV by RT-PCR. One CAstV isolate, designated as AAstV/Chicken/CHN/2017/NJ01, was successfully isolated from the small intestine of "Yellow" chickens using LMH cells. The genome sequence and structure analyses revealed that NJ1701 had the typical characteristics of avian astroviruses which was genetically distinct from other Avastrovirus. This isolate was classified as Group B subgroup i based on phylogenetic analysis of complete ORF2 (capsid) amino acid sequences. Meanwhile, growth depression and hatchability reduction were observed in the chicken embryo infection experiment. The results in the current study will contribute to our understanding of chicken astrovirus in China.
Project description:Avian astroviruses comprise a diverse group of viruses affecting many avian species and causing enteritis, hepatitis, and nephritis. To date, six different astroviruses have been identified in avian species based on the species of origin and viral genome characteristics: two turkey-origin astroviruses [Turkey Astrovirus type 1 (TAstV-1) and type 2 (TAstV-2)]; two chicken-origin astroviruses [Avian Nephritis Virus (ANV) and Chicken Astrovirus (CAstV)]; and two duck-origin astrovirus [Duck Astrovirus type 1 (DAstV-1) and type 2 (DAstV-2)]. ANV has also been detected in turkeys, ducklings, pigeons, and guinea fowl; and TAstrovirus-2-like viruses have also been found in guinea fowl. Astroviruses are commonly associated with enteric disease syndromes in poultry including runting-stunting syndrome of broilers (RSS), poult enteritis complex or syndrome (PEC or PES), poult enteritis mortality syndrome (PEMS), and enteritis in guinea fowl. The molecular characterization of the different avian astroviruses shows great genetic variability among each type, and this variability influences the ability to detect these viruses by molecular and serological techniques. In this chapter, we review the different aspects related to avian astroviruses, including molecular biology, pathogenesis, diagnosis, and control.