Avastrovirus Research Articles

Avian astrovirus caused mortality in pheasant (Phasianus colchicus, Linnaeus, 1758) farm inHungary.

We present the clinical symptoms, pathologic lesions and diagnostic possibilities of the avian astrovirus-related mortality in a pheasant colony. In addition to enteritis in chicks, we also confirmed acute nephrosis. The genome section of the astrovirus was detected and verified by polymerase chain reaction (PCR) testing. After sequencing the isolated genome section based on BLAST driver analysis (601-base pair-long) avian astrovirus has the same ORF-1b gene as turkey astrovirus 1 (TAstV1).

Establishment and application of quadruple fluorescence quantitative RT-PCR method for the identification of waterfowl astrovirus.

Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

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.

Rapid Rescue of Goose Astrovirus Genome via Red/ET Assembly.

The host-specific infection of Avian Astrovirus (AAstVs) has posed significant challenges to the poultry industry, resulting in substantial economic losses. However, few reports exist on the functional consequences of genome diversity, cross-species infectivity and mechanisms governing virus replication of AAstVs, making it difficult to develop measures to control astrovirus transmission. Reverse genetics technique can be used to study the function of viruses at the molecular level, as well as investigating pathogenic mechanisms and guide vaccine development and disease treatment. Herein, the reverse genetics technique of goose astrovirus GAstV/JS2019 strain was developed based on use of a reconstructed vector including CMV promotor, hammerhead ribozyme (HamRz), hepatitis delta virus ribozyme (HdvRz), and SV40 tail, then the cloned viral genome fragments were connected using Red/ET recombineering. The recombinant rGAstV-JS2019 was readily rescued by transfected the infectious clone plasmid into LMH cells. Importantly, the rescued rGAstV/JS2019 exhibited similar growth kinetics comparable to those of the parental GAstV/JS2019 isolate in cultured cells. Our research results provide an alternative and more effective reverse genetic tool for a detailed understanding of viral replication, pathogenic mechanisms, and molecular mechanisms of evolution.

Characterization of swine hepatitis E virus genotype 3 open reading frames

The goal of this study was a determination of structural organization features of swine hepatitis E virus genotype 3 open reading frames. Gene selective pressure for genes was estimated using the Tajima’s neutrality test as implemented in MEGA 6. Shannon entropy was used for diversity detection at each position of open reading frames. Shannon entropy plots representing quantitative diversity at each amino acid (nucleotide) position of 11 swine HEV-3 isolates aligned sequences were calculated by software BioEdit. For phylogenetic analysis, nucleotide distances were analyzed using the neighbour-joining algorithm. Analysis of nucleotide, non-synonymous and synonymous distances was performed using MEGA6. Parameters of mutational analysis for swine hepatitis E virus genotype 3 open reading frames ORF1 – ORF3 were determined using Tajima’s neutrality test. Shannon entropy technique revealed that ORF3 is characterized by the most number of diverse amino acid residues (21,7 %) comparing with ORF1, ORF2 coding regions. Analysis of non-synonymous distances in ORF3 allowed to determine an order of evolutionary events in the history of swine HEV-3 isolates. Avian astroviruses strains were used as control because phylogenetic relationships among them correspond to the evolution history of their host species. Tree topology for swine HEV-3 ORF3 based on non-synonymous distances differs from tree structures based on nucleotide and synonymous distances. Positive values of D Tajima’s factor for ORF1, ORF2 of swine HEV-3 show on operating positive selection onto these coding regions. Negative value of D Tajima’s factor for ORF3 coding region indicates onto purifying selection

Infection and innate immune mechanism of goose astrovirus.

Goose astrovirus (GAstV, genus Avian Astrovirus, family Astrovirus) was first discovered in 2005, but was not considered as a pathogen of gosling gout until 2016. Since then, goose astrovirus has erupted in Chinese goslings, causing at most 50% of gosling deaths. By December 2022, the disease had become epidemic and prevailed in goose farms in Jiangsu, Shandong, Anhui, Henan, Guangdong, Liaoning, Sichuan and other places in China. The disease mainly affects goslings within 3 weeks old. The typical symptoms of goose astrovirus are large deposits of urate in the viscera, joint cavity and ureter surface of infected goslings. Goose astrovirus infection can trigger high levels of iNOS, limiting goose astrovirus replication. The ORF2 domain P2 of the goose astrovirus activates the OASL protein, limiting its replication. Goose astrovirus can also activate pattern recognition receptors (RIG-I, MDA-5, TLR-3), causing an increase in MHC-Ia, MHC-Ib and CD81 mRNA, activating humoral and cellular immunity, thereby hindering virus invasion. Goose astrovirus also regulates the activation of IFNs and other antiviral proteins (Mx1, IFITM3, and PKR) in the spleens and kidneys to inhibit viral replication. The innate immune response process in goslings also activates TGF-β, which may be closely related to the immune escape of goose astrovirus. Gaining insight into the infection and innate immune mechanism of goose astrovirus can help researchers study and prevent the severe disease in goslings better.

Molecular detection of chicken astrovirus in broiler chicken, Malaysia

The emergence of avian diseases can cause major economic problems due to production losses and mortality in domestic poultry. Astrovirus is frequently associated with enteric diseases in poultry, being isolated from cases of runting-stunting syndrome (RSS) of broiler chickens, poult enteritis complex (PEC), and poult enteritis mortality syndrome (PEMS) of turkeys. Avian astrovirus can be detected in chickens from both healthy and poorly performing flocks. In Malaysia, information and reports regarding chicken astrovirus (CAstV) in poultry are limited. The objective of this study is to perform a phylogenetic study on the avian astrovirus isolated from a suspected case in 2019 and to determine the subgroups of avian astrovirus strains that existed in Malaysia. Reverse Transcription Polymerase chain reaction (RT-PCR) was performed based on the partial ORF1b gene and the nucleotide sequence was analyzed. Phylogenetic analysis showed that this isolate was clustered together with CAstV strains from several strain from USA, Malaysia and others. Furthermore, the isolate from broiler chicken showed 97.2% to 99.4% of its nucleotide identity with isolates from the American strains, compared to the previously CAstv Malaysia strain, which shared 94.8% to 95%.Therefore, the current study provides important information on the epidemiology of CAstV and highlights the importance of control strategies against CAstV-infected poultry in Malaysia.

Complete genome sequence and phylogenetic analysis of a goose astrovirus isolate in China.

Astroviruses are considered the cause of gastroenteritis in humans and animals. Studies in recent years show avian astroviruses are also associated with duckling hepatitis, gosling gout, and chicken nephritis. In this study, a GAstV strain, designated as JS2019/China, was detected in dead goslings from a commercial goose farm in Jiangsu province of China. Viral strain was proliferated in goose embryos and sequence analysis showed the isolated strain had a classical structure arrangement and a series of conserved regions compared with other GAstVs. Sequence comparison and phylogenetic analysis of whole genome and ORF2 revealed that JS2019/China belongs to the GAstV-1 group, which consists of most of the GAstV strains. Amino acid analysis indicated that some mutants might have an impact on viral protease capacity, such as V505I and K736E of ORF1a and T107I, F342S, and S606P of ORF2. Taken together, a novel GAstV strain was isolated and genomic analysis and protein polymorphism analysis indicated that some amino acid mutants might affect the viral virulence.

Prevalence and genetic diversity of coronaviruses, astroviruses and paramyxoviruses in wild birds in southeastern Kazakhstan

Wild birds are natural reservoirs of many emerging viruses, including some zoonoses. Considering that the territory of Kazakhstan is crossed by several bird migration routes, it is important to know pathogenic viruses circulating in migratory birds in this region. Therefore, the aim of this study was to identify the host range, diversity and spatial distribution of avian paramyxoviruses, coronaviruses, and astroviruses in free-ranging wild birds in the southeastern region of Kazakhstan. For this purpose, we collected tracheal and cloacal swabs from 242 wild birds belonging to 51 species and screened them using conventional PCR assays. Overall, 4.1% (10/242) and 2.9% (7/242) of all examined birds tested positive for coronaviruses and astroviruses, respectively. Coronaviruses were found in the orders Pelecaniformes (30%; 3/10), Charadriiformes (30%; 3/10), Columbiformes (20%; 2/10), Anseriformes (10%; 1/10), and Passeriformes (10%; 1/10). All detected strains belonged to the genus Gammacoronavirus. Astroviruses were detected in birds representing the orders Passeriformes (57%; 4/7), Coraciiformes (14%; 1/7), Charadriiformes (14%; 1/7), and Columbiformes (14%; 1/7). Paramyxoviruses were observed in only two birds (0.8%; 2/242). Both strains were closely related to the species APMV-22, which had not been previously detected in Kazakhstan. Phylogenetic analysis of the partial RdRp gene sequences of the virus strains revealed three different clades of astroviruses, two clades of coronaviruses, and one clade of paramyxoviruses. The results of this study provide valuable information on the diversity and spatial distribution of paramyxoviruses, coronaviruses, and astroviruses in wild birds in southeastern Kazakhstan and highlight the importance of further thorough monitoring of wild birds in this region.

A Review of the Emerging Poultry Visceral Gout Disease Linked to Avian Astrovirus Infection.

Avian astroviruses, including chicken astrovirus (CAstV), avian nephritisvirus (ANV), and goose astrovirus (GoAstV), are ubiquitous enteric RNA viruses associated with enteric disorders in avian species. Recent research has found that infection of these astroviruses usually cause visceral gout in chicken, duckling and gosling. However, the underlying mechanism remains unknown. In the current article, we review recent discoveries of genetic diversity and variation of these astroviruses, as well as pathogenesis after astrovirus infection. In addition, we discuss the relation between avian astrovirus infection and visceral gout in poultry. Our aim is to review recent discoveries about the prevention and control of the consequential visceral gout diseases in poultry, along with the attempt to reveal the possible producing process of visceral gout diseases in poultry.

Surveillance and genetic diversity analysis of avian astrovirus in China.

Avian astroviruses (AAstVs) have caused major problem for poultry breeding industries in China in recent years, and the goose gout caused by goose astrovirus has produced particularly great economic losses. To better understand the prevalence and genetic diversity of AAstVs in China, 1210 poultry samples collected from eight provinces were tested with reverse transcription-polymerase chain reaction (RT-PCR) to detect AAstV infections in different poultry populations. Then, Open reading frames 2 (ORF2) was amplified by specific primers, and the genetic evolution was analyzed. Our surveillance data demonstrate the diversity of AAstVs in China insofar as we detected 17 AAstVs, including seven chicken astroviruses (CAstVs), five avian nephritis viruses (ANVs), two goose astroviruses (GoAstVs), two duck astrovirus (DAstVs), and one new AAstV belonging to Avastrovirus Group 3. The positive rate of AAstV infection was 1.40%. Host analysis showed that CAstVs and ANVs were isolated from chickens, DAstVs and GoAstVs were isolated from ducks. Host-species-specific AAstVs infections were also identified in numerous samples collected at each stage of production. This study provides further evidence to better understand the epidemiology of AAstVs in different species of poultry in China.

Isolation, Identification, and Genomic Characterization of Chicken Astrovirus Isolates From China.

Chicken astrovirus (CAstV) infection can cause diarrhea, nephritis, stunted growth, and “white chickens” condition, resulting in economic losses to the poultry industry. Currently, a few CAstVs were isolated and a few full-length genome sequences of CAstV have been deposited in the GenBank. In the present study, two CAstV isolates (AAstV/Chicken/CHN/2017/NJ01 and AAstV/Chicken/CHN/2018/CZ01) were successfully isolated by using LMH cells, and we molecularly characterized these two CAstV isolates and observed the effect of these two isolates on hatchability using chicken embryo infection experiment. The genetic analysis demonstrated that these two strains had the typical characteristics of avian astroviruses, which were composed of three open reading frames, 5′UTR, and 3′UTR. The full-length genome sequence showed a high-degree identity at nucleotide level of 97.5–98.7% among Chinese isolates suggesting their common ancestors and limited sequence divergence. Sequence analysis of ORF2, which encodes the capsid protein associated with classification of avian astrovirus, revealed our two isolates belonging to CAstV Bi subtype. At the amino acid level, the complete capsid region of Chinese strains shared genetic distances of 0.03–0.04 with FP3 strains isolated from the UK, suggesting their common origin. Meanwhile, hatchability reduction was observed. These results provided novel insights into the molecular epidemiology and hatchability effect of CAstV.

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.

The Isolation and Molecular Characterization of an Astrovirus From "Yellow" Chickens, China.

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.

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

ABSTRACT 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.

Investigation of suspected Newcastle disease (ND) outbreaks in Egypt uncovers a high virus velogenic ND virus burden in small-scale holdings and the presence of multiple pathogens

ABSTRACTHighly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchitis virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (n = 16) comprising small-scale holdings. Phylogenetic analysis of the fusion protein gene of 11 NDV-positive samples obtained from this group assigned all viruses to genotype 2.VIIb and point to four different virus populations that were circulating at the same time in one governorate, indicating independent epidemiological events. In group B, comprising large commercial broiler farms (n = 10), virulent NDV was not present, although in six farms NDV vaccine-type virus (genotype 2.II) was detected. Besides, in both groups, co-infections by IBV (n = 10), AIV H9 (n = 3) and/or avian reovirus (ARV) (n = 5) and avian astrovirus (AastVs) (n = 1) could be identified. Taken together, the study confirmed clinical ND suspicion in small scale holdings, pointing to inefficient vaccination practices in this group A. However, it also highlighted that, even in an endemic situation like ND in Egypt, in cases of suspected ND vaccine failure, clinical ND suspicion has to be verified by pathotype-specific diagnostic tests.RESEARCH HIGHLIGHTSVelogenic NDV circulates in small-scale poultry holdings in Egypt.Viral transmission occurred among neighbouring farms and over long distances.Co-infections with multiple pathogens were identified.Pathotype specific diagnostic tests are essential to verify ND suspicions.

A novel group of avian astroviruses from Neotropical passerine birds broaden the diversity and host range of Astroviridae

Metagenomics is helping to expand the known diversity of viruses, especially of those with poorly studied hosts in remote areas. The Neotropical region harbors a considerable diversity of avian species that may play a role as both host and short-distance vectors of unknown viruses. Viral metagenomics of cloacal swabs from 50 Neotropical birds collected in French Guiana revealed the presence of four complete astrovirus genomes. They constitute an early diverging novel monophyletic clade within the Avastrovirus phylogeny, representing a putative new astrovirus species (provisionally designated as Avastrovirus 5) according to the International Committee on Taxonomy of Viruses (ICTV) classification criteria. Their genomic organization shares some characteristics with Avastrovirus but also with Mamastrovirus. The pan-astrovirus RT-PCR analysis of the cloacal samples of 406 wild Neotropical birds showed a community-level prevalence of 4.9% (5.1% in passerines, the highest described so far in this order of birds). By screening birds of a remote region, we expanded the known host range of astroviruses to the avian families Cardinalidae, Conopophagidae, Furnariidae, Thamnophilidae, Turdidae and Tyrannidae. Our results provide important first insights into the unexplored viral communities, the ecology, epidemiology and features of host-pathogen interactions that shape the evolution of avastroviruses in a remote Neotropical rainforest.

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

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.

The Broad Host Range and Genetic Diversity of Mammalian and Avian Astroviruses.

Astroviruses are a diverse family of viruses that infect a wide range of mammalian and avian hosts. Here we describe the phylogenetic diversity and current classification methodology of astroviruses based on the ORF1b and ORF2 genes, highlighting the propensity of astroviruses to undergo interspecies transmission and genetic recombination which greatly increase diversity and complicate attempts at a unified and comprehensive classification strategy.