The biological characteristics of DAstV molecular epidemiology and pathogenicity of duck astrovirus causing hepatitis in ducks and chickens in Southeast China.

A suspected outbreak of duck astrovirus (DAstV) disease occurred in a large Muscovy duck farm in Guangdong Province, China, in June 2022, which severely affected the production performance and health of Muscovy ducks. This study aimed to investigate the prevalence of DAstV disease in Southeast China. Herein, we employed semi-nested PCR ethodto screen 5203 swab and liver samples from 11 Muscovy duck farms in 5 provinces of China for the presence of DAstV. Among them, 1356 samples (26.06%, 1356/5203) tested positive for DAstV, out of which 11 DAstV strains were isolated after 10 generations of blind transmission through Leghorn male hepatoma (LMH) cells and performed their whole-genome sequencing. The alignment results showed that the 11 DAstV isolates exhibited relatively low homology (15.4%-75%) with the astrovirus isolates from other species published in GenBank, whereas their homology (nucleotide: 90.4%-99.99%; amino acid: 94%-99.8%) with the DAstV type 1 (DAstV-1) reference strain was higher, indicating considerable homology. The results indicated that DAstV-1 was the main pathogenic factor. Herein, we successfully recreated the clinical symptoms of natural infection in 28-day-old specific-pathogen-free (SPF) ducks using the DAstV-1-GDB-2022 strain. The primary clinical manifestations included liver enlargement, hemorrhaging, and disruptions in liver function. Additionally, we confirmed the cross-species transmission potential of DAstV-1, marking the first occurrence of clinical symptoms of DAstV in 28-day-old SPF chickens. Our findings provide new perspectives on the epidemiology and pathogenicity of DAstV-1 and may help in advancing the development of DAstV vaccines.

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.

Development of a multienzyme isothermal and lateral flow dipstick combination assay for the rapid detection of goose astrovirus II.

Introduction: Goose astrovirus (GAstV) is a newly emerging pathogen that is currently widespread among geese, causing visceral gout and leading to substantial gosling mortalities, posing a severe threat to the waterfowl industry. GAstV II is the predominant epidemic strain, characterized by its high morbidity and mortality rate. Consequently, there is an urgent necessity to develop an effective diagnostic approach to control the dissemination of GAstV II, particularly in clinical farms with limited laboratory resources. Methods: In this study, a novel multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combined assay was developed. Different primers designed specific targeting a highly conserved region within the viral RdRp gene for the detection of GAstV II. Primers optimized and MIRA-LFD assay analyzed its performance regarding limits of detection, specificity, and efficiency of detection. Results: The developed MIRA amplification is conducted at a constant temperature and accomplished within 10 minutes. Subsequent naked-eye observation of the LFD strips merely takes 5 minutes. The established MIRA-LFD method exhibits high specificity, with no cross-reaction with other pathogens and attains a detection sensitivity of 1 copy/µl, which is consistent with the reverse transcription quantitative PCR (RT-qPCR) assay. Further evaluation with clinical samples indicates that the accuracy of this MIRA-LFD method correlates well with RT-qPCR for the detection of GAstV II. Conclusion: In summary, the convenience, sensitivity, and rapidity of this newly developed detection method offer a significant advantage for on-site diagnosis of GAstV II.

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

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

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.

Occurrence of astrovirus in young racing pigeons and genome characterization of 2 new astrovirus genomes representing 2 new species.

Enteropathies are a serious concern in racing pigeons as they significantly impair performance in races and their training, and viruses are suspected to be one of the main factors. Astroviruses are well-known to be responsible for causing enteric disease in humans and various other animals including birds, although their prevalence and pathogenicity in pigeons is poorly understood. In this study, we investigated 2 groups of young racing pigeons (sick-study group and healthy-control group) to assess the correlation between the number of astrovirus genome copies in cloacal swabs and the occurrence of enteropathy. To determine this, we developed a novel TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for astrovirus detection and absolute quantitative analysis. We also performed high-throughput sequencing to obtain the complete genome sequences and establish the genetic similarity of the obtained strains to known astroviruses of poultry and other avian species. Two new complete genome sequences of pigeon astroviruses in the Avastrovirus genus were identified, representing 2 new species. These were found most closely related to astroviruses identified in Columbidae species and chickens. They share an average of 75.8% genome-wide pairwise identity and 57.6% and 64.6% capsid protein sequence identity with other unclassified columbid avastrovirus sequences in GenBank. Although the difference in prevalence of astrovirus in the study and control group was found statistically insignificant, there was a significant difference between the number of genome copies in positive samples from both groups. These unambiguous results leave the role of astroviruses as enteropathogenic factors in pigeons still undetermined.

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

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

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.

A Review of the Emerging White Chick Hatchery Disease.

White chick hatchery disease is an emerging disease of broiler chicks with which the virus, chicken astrovirus, has been associated. Adult birds typically show no obvious clinical signs of infection, although some broiler breeder flocks have experienced slight egg drops. Substantial decreases in hatching are experienced over a two-week period, with an increase in mid-to-late embryo deaths, chicks too weak to hatch and pale, runted chicks with high mortality. Chicken astrovirus is an enteric virus, and strains are typically transmitted horizontally within flocks via the faecal-oral route; however, dead-in-shell embryos and weak, pale hatchlings indicate vertical transmission of the strains associated with white chick hatchery disease. Hatch levels are typically restored after two weeks when seroconversion of the hens to chicken astrovirus has occurred. Currently, there are no commercial vaccines available for the virus; therefore, the only means of protection is by good levels of biosecurity. This review aims to outline the current understanding regarding white chick hatchery disease in broiler chick flocks suffering from severe early mortality and increased embryo death in countries worldwide.

Establishment of Duplex SYBR Green I-based Real-time PCR Assay for Simultaneous Detection of Duck Hepatitis A Virus-1 and Duck Astrovirus-3.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 101 copies/µl and 3.78 × 101 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

Artículo regular­Establecimiento de un ensayo dúplex de PCR en tiempo real basado en SYBR Green I para la detección simultánea del virus de la hepatitis A del pato-1 y del astrovirus del pato tipo 3. La hepatitis viral del pato (DVH) afecta principalmente a los patitos menores de 1 mes de edad, causa necrosis hepática, agrandamiento y hemorragia, y es altamente letal, lo que pone en grave peligro la industria del pato. La prevalencia del virus de la hepatitis A del pato (DHAV-1) y del astrovirus del pato tipo 3 (DAstV-3) está aumentando y la coinfección es común. Además, las características clínicas similares de las infecciones por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3 así como la alta frecuencia de coinfección dificultan el diagnóstico. En este estudio, para establecer un método para la detección rápida y simultánea por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3, se diseñaron dos pares de iniciadores específicos según sus regiones génicas conservadas. Se estableció con éxito un ensayo cuantitativo de PCR basado en SYBR® Green I que pudo detectar rápida y diferencialmente los dos virus. Además, el ensayo es muy específico y no muestró reacción cruzada con otros virus comunes. El límite de detección del método fue de 7.34 × 101 copias/µl y de 3.78 × 101 copias/µl para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3, respectivamente, lo que indica una alta sensibilidad. Se analizaron un total de 34 muestras clínicas utilizando el método establecido; las tasas positivas para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3 fueron del 14.71% y 8.82%, respectivamente y la de coinfección fue del 2.94% (1/34), que fue mejor que la obtenida con el método de PCR convencional. En resumen, el ensayo cuantitativo de PCR basado en SYBR Green I establecido en este estudio tiene alta especificidad, buena sensibilidad y precisión, alta viabilidad y es rápido. Por lo tanto, puede ser una herramienta poderosa para la detección de coinfecciones con el virus de la hepatitis A del pato y astrovirus del pato tipo 3 y para futuros estudios epidemiológicos.

New insights on Avian orthoreovirus and Chicken astrovirus co-infection in an Italian broiler flock: preliminary biomolecular and pathological results.

Common pathogens of intensive poultry farms, either parasitic or bacterial, such as Coccidiaor Salmonella, are well known and strictly controlled by veterinary management. This case study reports an unusual case of runting stunting syndrome (RSS) observed on a Sicilian poultry farm of broiler chickens during 2019. The investigation was carried out on five chickens which present delayed in body weight and growth performance. Animals showed also difficulty in deambulation and diarrhea. At necropsy, intestinal lesions were detected in three of the five clinical cases. Gut samples were collected and analyzed to identify potential pathogens responsible for the RSS. Presence of viruses was detected by using quantitative reverse transcription PCR (RT­qPCR), while selected tissues were fixed and embedded in paraffin wax according to routine procedures. All histological sections were stained with hematoxylin­eosin. RT­qPCR successfully detected both Chicken astrovirus (CAstV) and Avian orthoreovirus (ARV). Histology evidenced severe specific lesions on the intestinal mucosa in liver and kidneys. Chicken astrovirus and Avian orthoreovirus RNA was also detected in cecal tonsils, kidney and liver, thus implying their possible primary role in inducing the disease. Further studies are needed to evaluate the role of other possible factors (low biosecurity measures, e.g.) and, most of all, the consequences in terms of economic losses and animal health impairment.

Detection and characterization of chicken astrovirus associated with hatchery disease in commercial day-old turkeys in southwestern Nigeria.

Infectious diseases are a major obstacle to profitable poultry production in Nigeria due to the mortality and severe economic losses they cause. In particular, they are a potent threat to attainment of the food security goals of government and national self-sufficiency in food production. Thus, there is a need for continuous monitoring of the nation's poultry population for these diseases. As part of an ongoing investigation of enteric viruses associated with poor performance or hatchery diseases in commercial poultry in southwestern Nigeria, intestinal contents from 97 condemned or runted day-old commercial turkey poults were examined for turkey astroviruses, infectious bronchitis virus, chicken astrovirus (CAstV), avian nephritis virus, avian rotavirus, avian reovirus, fowl adenovirus, and chicken parvovirus by virus isolation, electron microscopy (EM), polymerase chain reaction (PCR), and reverse transcription PCR. The samples were collected from five commercial hatcheries and five farms located in southwestern Nigeria. While all samples tested negative for other viruses, CAstV was detected in the majority (83.5%) of the birds, although some pleomorphic virus-like particles with surface projections that appeared fringed or fimbriated were observed in five of the cell culture samples by EM. Phylogenetic analysis revealed these CAstV strains belonged to the Bi clade. These findings not only implicate CAstV as the major cause of hatchery condemnations in commercial turkeys in southwestern Nigeria but also highlight the need for experimental studies to further establish its role in this disease condition.

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.

Serodetection of astroviruses in runted commercial broilers and turkeys in southwest Nigeria.

Infections with divergent strains of astroviruses appear to be endemic in commercial poultry. In order to investigate enteric viruses associated with hatchery condemnations in Nigerian poultry, an indirect immunofluorescence test with CAstV-612- (Group A), CAstV-11672- (Group B) and ANV-1-infected cells was used to screen sera obtained from commercial broilers (n = 164) and turkeys (n = 97) in farms and hatcheries in southwest Nigeria. Of the 261 sera tested, 16 (6.1%) were positive for CAstV antibodies after immunofluorescent staining with CAstV-11672-infected cells. Thirteen (81.3%) of the positive sera were from broilers with three (18.7%) being from turkeys. Conversely, all tested sera were negative for CAstV-612 and ANV-1 antibodies. Since CAstV-11672, a group B CAstV is known to be antigenically and genetically distinct from CAstV-612 that belongs to group A, these findings reveal that the circulating serotype of CAstV in commercial broilers and turkeys in southwest Nigeria belongs to group B of CAstV. Education of veterinary personnel and poultry farmers about this emerging virus and its impact on commercial poultry in Nigeria, as well as continuous monitoring of chicken and turkey flocks for infections caused by it are therefore imperative in order to facilitate the implementation of effective prevention and control measures.

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.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

First report of a novel goose astrovirus outbreak in Cherry Valley ducklings in China.

A highly acute disease broke out in ducklings in Shandong Province in March 2019. The disease was characterized as visceral gout, with a mortality rate of 30%. The causative agent, which has given rise to similar symptoms in goslings, has been confirmed to be a novel goose astrovirus. The novel goose astrovirus, which was designated as the SDXT strain, was identified from a diseased duck farm using duck embryo primary cells in an experimental infection test. Genomic sequence analysis, as well as phylogenetic analysis of the viral proteins, revealed that the SDXT strain was closely related to a novel goose astrovirus of the Avastrovirus three species. These results indicate that the novel goose astrovirus may cross-host infect ducklings. Further studies are needed to define its host range and transmission route.

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.

Establishment and application of a TaqMan-based one-step real-time RT-PCR for the detection of novel goose-origin astrovirus.

The outbreak of an infectious disease characterized by severe symptom of gout has set great threat to several major goose-producing regions in China since December 2016. The causative agent for the novel infection has been identified was a novel goose-origin astrovirus (GoAstV). Lack of effective detection methods indeed hinders further research, as well as prevention and control of GoAstV. Keep this in mind, a TaqMan-based one-step real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for rapid detection of GoAstV was developed. Primers and probe were targeting the capsid protein gene sequence (ORF2). The method is capable of detecting quite low number of targeting nucleic acid as low as 10 copies/µL. What's more, it is also of great specificity and repeatability for GoAstV detection. No cross-activity was found with other goose-origin viruses. The assay had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.48% to 0.99%. A total of 340 GoAstV specimens from different regions of China were used in this study to verify the feasibility and effectiveness of this method in clinical diagnosis. The results indicated that qRT-PCR is a highly sensitive, specific and repeatable method for quantitative detection of GoAstV, which can be used to detect this virus, thereby facilitating epidemiological investigations of gout in goslings.