Aminoguanidine alleviates gout in goslings experimentally infected with goose astrovirus-2 by reducing kidney lesions.

Goose astrovirus (GAstV)-2, a novel pathogen identified in 2018, mainly causes visceral gout in goslings, leading to approximately 50% mortality. At present, no commercial veterinary products are available to prevent and treat the disease. Our previous studies showed that nitric oxide (NO) and inducible NO synthase (iNOS) were markedly higher in the kidney and spleen of goslings infected with GAstV-2, but their effects during GAstV-2 infection remain unclear. In the present study, goslings were intraperitoneally injected with aminoguanidine (AG)-an iNOS inhibitor-to examine the role of NO during GAstV-2 infection. AG significantly decreased the serum NO concentration and iNOS mRNA expression in the kidney. Moreover, AG reduced the mortality, serum uric acid and creatinine content, and urate deposition in visceral organs and joints. Histopathological analysis demonstrated that AG reduced renal tubular cell necrosis, inflammatory cell infiltration, glycogen deposition in glomerular mesangium, and interstitial fibrosis, suggesting alleviation of kidney lesions. Furthermore, AG decreased the expression of renal injury markers such as KIM-1 and desmin; inflammatory cytokine-related genes such as IL-1ß, IL-8, and MMP-9; and autophagy-related genes and proteins such as LC3II, ATG5, and Beclin1. However, quantitative real-time PCR and immunohistochemistry showed that treatment with AG did not affect the kidney and liver viral load. These findings suggest that AG decreases the mortality rate and kidney lesions in goslings infected with GAstV-2 through mechanisms associated with autophagy and inhibition of inflammatory cytokine production in the kidney but not with GAstV-2 replication.

Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease.

BACKGROUND: Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method. RESULTS: Using our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines. CONCLUSIONS: Our study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry.

Prevalence of Astroviruses in Different Animal Species in Poland.

Astroviruses (AstVs) are small RNA viruses characterized by a high mutation rate, the ability to recombine, and interspecies transmission, which allows them to infect a multitude of hosts including humans, companion animals, and farmed animals as well as wildlife. AstVs are stable in the environment, and their transmission is usually through the fecal-oral route or via contaminated water and food. Although direct zoonotic transmission was not confirmed, interspecies transmission events have occurred or have been indicated to occur in the past between wild and domestic animals and humans. They cause large economic losses, mainly in the poultry sector, due to gastroenteritis and mortality. In young children, they are the second most common cause of diarrhea. This study involved 166 intestine samples and pools of spleen, lymph node, and kidney samples collected from 352 wild animals, 52 pigs, and 31 companion animals. Astroviruses were detected in the intestine samples and were separately detected in pools of tissue samples prepared for individual animals using a heminested RT-PCR protocol. Amplicons were subjected to Sanger sequencing, and a phylogenetic analysis of 320 nt RNA-dependent RNA polymerase (RdRp) fragments referring to known nt sequences of astroviruses was performed. Astroviral RNA was detected in the intestine samples and/or tissue pools of red foxes (nine positive intestines and six positive tissue pools), rats (two positive intestines and three positive tissue pools), a cat (one AstV detected in an intestine sample), pigs (eight positive tissue pools), and wild boars (two positive pools of spleens, kidneys, and lymph nodes). No astroviral RNA was detected in wild mustelids, dogs, or other small wild animals including rodents. A phylogenetic analysis revealed that the astroviruses detected during this study were mostly host-specific, such as porcine, canine, and rat astroviruses that were highly homologous to the sequences of reference strains. In one of two wild boars, an AstV distinct to porcine species was found with the highest nt identity to Avastroviruses, i.e., turkey astroviruses, which suggests potential cross-species transmission of the virus, as previously described. Here, we present the first detection of astroviruses in the population of wild animals, companion animals, and pigs in Poland, confirming that astroviruses are frequent pathogens circulating in animals in the field. Our study also suggests potential cross-species transmission of Avaastrovirus to wild boars; however, further molecular characterization is needed.

Characterization of natural co-infection with goose astrovirus genotypes I and II in gout affected goslings.

RESEARCH HIGHLIGHTS: Urate tophi were found in the kidneys, liver, spleen and lungs.IFA confirmed the co-expression of GoAstV-I and II antigens in the same kidney.

Molecular characteristics and pathogenicity of a novel chicken astrovirus variant.

It is well-established that the genetic diversity, regional prevalence, and broad host range of astroviruses significantly impact the poultry industry. In July 2022, a small-scale commercial broiler farm in China reported cases of growth retardation and a 3% mortality rate. From chickens displaying proventriculitis and pancreatitis, three chicken astroviruses (CAstV) isolates were obtained and named SDAU2022-1-3. Complete genomic sequencing and analysis revealed the unique characteristics of these isolates from known CAstV strains in ORF1a, ORF1b, and ORF2 genes, characterized by an unusually high variability. Analysis of amino acid mutations in ORF1a, ORF1b, and ORF2 indicated that the accumulation of these mutations played a pivotal role in the emergence of the variant strain. Inoculation experiments demonstrated that affected chickens exhibited liver and kidney enlargement, localized proventricular hemorrhage, and a dark reddish-brown appearance in about two-thirds of the pancreas. Histopathological examination unveiled hepatic lymphocytic infiltration, renal tubular epithelial cell swelling, along with lymphocytic proventriculitis and pancreatitis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated viremia and viral shedding at 3 days post-infection (dpi). The proventriculus displayed the highest viral loads, followed by the liver, kidney, duodenum, and pancreas. Liver parameters (AST and ALT) and kidney parameters (UA and UN) demonstrated mild damage consistent with earlier findings. While the possibility of new mutations in the ORF2 gene of CAstV causing proventriculitis and pancreatitis warrants further investigation, these findings deepen our comprehension of CAstV's pathogenicity in chickens. Additionally, they serve as valuable references for subsequent research endeavors.

miRNA Expression Signatures Induced by Chicken Astrovirus Infection in Chickens.

miRNAs represent ubiquitous regulators of gene expression and play an important and pivotal regulatory role in viral disease pathogenesis and virus-host interactions. Although previous studies have provided basic data for understanding the role of miRNAs in the molecular mechanisms of viral infection in birds, the role of miRNAs in the regulation of host responses to chicken astrovirus (CAstV) infection in chickens is not yet understood. In our study, we applied next-generation sequencing to profile miRNA expression in CAstV-infected chickens and to decipher miRNA-targeted specific signaling pathways engaged in potentially vital virus-infection biological processes. Among the 1354 detected miRNAs, we identified 58 mature miRNAs that were significantly differentially expressed in infected birds. Target prediction resulted in 4741 target genes. GO and KEGG pathway enrichment analyses showed that the target genes were mainly involved in the regulation of cellular processes and immune responses.

Pathogenicity of a goose astrovirus 2 strain causing fatal gout in goslings.

Gosling gout has posed a serious threat to the development of the China's goose industry since the outbreak in mainland China in 2016; goose astrovirus (GAstV) was identified as the culprit pathogen. Two genotypes of this virus have been identified: GAstV-1 and GAstV-2, of which GAstV-2 is the main epidemic strain. Our current understanding of the pathogenic mechanisms of GAstV-2 remains limited. To assess pathogenicity, 1-day-old goslings were inoculated with the GAstV-2 YC20 strain via the subcutaneous, intranasal, and oral infection routes. All the goslings showed typical gout symptoms, with those in the oral infection group exhibiting earlier and more severe clinical symptoms, the highest mortality rate, and greatest weight loss. The blood biochemical indicators, viral loads in cloacal swabs and all representative tissues, and serum antibody titers of all infection groups increased significantly, and no significant differences in these parameters were observed among the three infection groups. Histopathological studies showed that the livers, kidneys, and spleens were the main damaged organs, and the pathological changes in the oral group were more severe than those in the other groups. Further analysis revealed that hepatic sinuses narrowed or became occluded as early as 1 day post-inoculation; urate deposition occurred in the renal tubules at 2 days post-inoculation (dpi), followed by necrosis of renal tubular epithelial cells; and lymphocytic infiltration appeared in the splenic tissue at 5 dpi. These results further our understanding of the pathogenic mechanisms of GAstV-2 and provide a reference for future studies.

Base composition, adaptation, and evolution of goose astroviruses: codon-based investigation.

Goose astroviruses (GoAstVs) are causative agents that account for fatal infection of goslings characterized by visceral urate deposition, resulting in severe economic losses in major goose-producing regions in China since 2017. In this study, we sought to unravel the intrinsic properties associated with adaptation and evolution in the host environment of GoAstVs. Consistent results from phylogenetic analysis and correspondence analysis performed on the codon usage patterns (CUPs) reveal 2 clusters of GoAstVs, namely, GoAstV-1 and GoAstV-2. However, multiple similar compositional characteristics were found, despite the high divergence between GoAstV-1 and GoAstV-2. Studies on the base composition of GoAstVs reveal an A/U bias, indicating a compositional constraint, while natural selection prevailed in determining the CUPs in the virus genome based on our neutrality plot analysis, reflecting high adaptive pressure to fit the host environment. Codon adaptation index (CAI) analysis revealed a higher degree of fitness to the CUPs of the corresponding host for GoAstVs than avian influenza virus and betacoronaviruses, which may be a favorable factor contributing to the high pathogenicity and wide distribution of GoAstVs in goslings. In addition, GoAstVs were less adapted to ducks and chickens, with significantly lower CAI values than to geese, which may be a reason for the different prevalence of GoAstVs among these species. Extensive investigations on dinucleotide distribution revealed a significant suppression of the CpG and UpA motifs in the virus genome, which may facilitate adaptation to the host's innate immune system by evading surveillance. In addition, our study reported the trends of increasing fitness to the host's microenvironment for GoAstVs through increasing adaptation to host CUPs and ongoing reduction of CpG motifs in the virus genome. The present analysis deepens our understanding of the basic biology, pathogenesis, adaptation and evolutionary pattern of GoAstVs, and contributes to the development of novel antiviral strategies.

Localization and distribution of goose astrovirus 2 antigens in different tissues at different times.

Goose astrovirus 2 (GAstV-2) causes visceral gout in goslings and has resulted in significant economic losses in the goose industry of China since its outbreak in 2017. To further investigate the distribution and localization of GAstV-2 in different tissues at different times, a monoclonal antibody (mAb)-based immunohistochemical (IHC) assay was developed to detect GAstV-2. A total of 80 1-day-old healthy goslings were inoculated with GAstV-2 via the oral (n = 40) and intramuscular routes (n = 40). GAstV-2 in the tissues of interest was detected using the established IHC assay. The results showed that positive signals were detected in most tissues at 1 day post-infection (dpi). Viral antigens were mainly distributed in the cytoplasm, and the staining intensity was higher in the renal tubular epithelial cells than in other cells. Taken together, our data demonstrated that GAstV-2 has a broad tissue tropism and primarily targets the kidneys. These results are likely to provide a scientific basis for further elucidation of the pathogenesis of GAstV-2.

Research Note: Isolation and complete genome analysis of a genotype II goose astrovirus in Sichuan Province, China.

The emergence of Goose astrovirus (GoAstV) has led to the gout in geese. This study aimed to isolate and identify the GoAstV from diseased goslings in Sichuan Province, China, followed by performing whole genome phylogenetic analysis of the isolate. The GoAstV was successfully isolated by inoculating the diseased gosling liver and kidney homogenate into the 11-day-old goose embryo allantoic cavity for 3 passages, and the isolate was named as GoAstV-C2 strain. The virus particles were spherical, without capsule, and the size was about 28 nm under transmission electronic microscope. The complete genome length of GoAstV-C2 was 7.035 nt, and the whole genome sequence phylogenetic analysis revealed that it belongs to the GoAstV genotype II (GoAstV-II) subgenotype IIc. The isolated GoAstV-C2 strain was able to be stably passaged in the goose embryo and uric acid sedimentation was observed. The complete genome bioinformation of GoAstV-C2 determined the evolutionary characteristics of the GoAstV isolated from Sichuan, China. This finding lays a foundation for the development of preventive measures, effective vaccines, and therapeutic drugs.

Genome Sequences and Characterization of Chicken Astrovirus and Avian Nephritis Virus from Tanzanian Live Bird Markets.

The enteric chicken astrovirus (CAstV) and avian nephritis virus (ANV) are the type species of the genus Avastrovirus (AAstV; Astroviridae family), capable of causing considerable production losses in poultry. Using next-generation sequencing of a cloacal swab from a backyard chicken in Tanzania, we assembled genome sequences of ANV and CAstV (6918 nt and 7318 nt in length, respectively, excluding poly(A) tails, which have a typical AAstV genome architecture (5'-UTR-ORF1a-ORF1b-ORF2-'3-UTR). They are most similar to strains ck/ANV/BR/RS/6R/15 (82.72%) and ck/CAstV/PL/G059/14 (82.23%), respectively. Phylogenetic and sequence analyses of the genomes and the three open reading frames (ORFs) grouped the Tanzanian ANV and CAstV strains with Eurasian ANV-5 and CAstV-Aii viruses, respectively. Compared to other AAstVs, the Tanzanian strains have numerous amino acid variations (substitutions, insertions and deletions) in the spike region of the capsid protein. Furthermore, CAstV-A has a 4018 nt recombinant fragment in the ORF1a/1b genomic region, predicted to be from Eurasian CAstV-Bi and Bvi parental strains. These data should inform future epidemiological studies and options for AAstV diagnostics and vaccines.

Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China.

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Pathogenicity of goose astrovirus genotype 2 in chickens.

Goose astrovirus genotype 2 (GAstV-2) is the causative agent causing severe visceral gout and joint gout in goslings, with mortality rates of affected flocks up to 50%. To date, continuous GAstV-2 outbreaks still pose a great threat to goose industry in China. Although most researches on GAstV-2 have focused on its pathogenicity to geese and ducks, limited studies have been performed on chickens. Herein, we inoculated 1-day-old specific pathogen-free (SPF) White Leghorn line chickens with 0.6 mL of GAstV-2 culture supernatant (TCID50 10-5.14/0.1 mL) via orally, subcutaneously and intramuscularly, and then assessed the pathogenicity. The results revealed that the infected chickens presented depression, anorexia, diarrhea, and weight loss. The infected chickens also suffered from extensive organ damage and had histopathological changes in the heart, liver, spleen, kidney, and thymuses. The infected chickens also had high viral load in tissues and shed virus after the challenge. Overall, our research demonstrates that GAstV-2 can infect chickens and adversely affect the productivity of animals. And the viruses shed by infected chickens can pose a potential risk to the same or other domestic landfowls.

Rapid diagnosis of different goose astrovirus genotypes with Taqman-based duplex real-time quantitative PCR.

The epidemic of goose astrovirus (GoAstV) caused huge losses to the poultry industry. Epidemiological studies in China revealed 2 circulating genotypes of GoAstV, but there is a lack of differential diagnosis tools. By analyzing all published genomes of GoAstV, this study designed specific PCR primers and Taqman probes to recognize different genotypes of GoAstV. After optimization and verification, this study developed a Taqman-based real-time quantitative PCR method that is capable of differential diagnosis. The established qPCR exhibited detection limitations of 100 copies/µL or 10 copies/µL, respectively, for GoAstV genotype 1 and genotype 2, and showed no false positive for other common avian viruses. This method was then used to analyze 72 samples collected from different regions in Jiangxi, and the results were verified by genome sequencing and phylogenetic analysis. These results revealed a complex coinfection of GoAstV different genotypes in China, highlighting the importance of long-term focus on the prevalence and genome evolution of GoAstV.

Chicken Astro virus (CAstV): Isolation and characterization of new strains in broiler flocks with poor performance.

Chicken astroviruses (CAstV) were associated with retarded growth, enteritis, kidney diseases, and white chick syndrome. In the current study, we aimed to evaluate the effect of CAstV infection on growth, performance, and gross and histopathological picture of commercial chicken flocks suffering increased culling rate and decreased performance. Samples were collected for virus isolation, identification, and sequencing on day one, 15 days, and 30 days of age. Body weight, feed conversion rate, and mortality rates were determined. A gross examination was performed, and tissue samples from the liver, intestine, kidneys, heart, and lungs were kept in formalin for histopathological evaluation. Embryos inoculated with CAstV revealed dwarfism, and edema. The cytopathic effect on CAstV inoculated cells included aggregation,, and sloughing. The isolated Egyptian isolates shared the highest nucleotide homology (93%) with the Korean isolate Kr/ADL102655-1/2010 and showed the most distant relation to the Indian isolate Indovax/APF/1319 with 82-83% homology. Body weight exhibited significant reduction with a decrease in feed conversion rate in CAstV infected flocks. Gross examination of CAstV-infected chickens revealed white feathered chicks on day one, and poor body condition in older chickens as well as swollen kidneys. Histopathological examination of CAstV-infected birds showed mild proventriculitis, shortening of intestinal villi, enteritis, focal hepatocellular necrosis, pericarditis, myocarditis, and proliferative response in lung tissue. Kidneys showed interstitial nephritis, urate deposition, and glomerular hypercellularity. CAstV is a chicken pathogen that could be related to decreased performance, and screening of flocks for CAstV might be an essential step for breeders.

Global analysis of gene expression profiles and gout symptoms in goslings infected with goose astrovirus.

While blocking inflammation is an effective way to ease the symptoms of gout disease in humans, the treatment and prevention of gout in goslings infected with goose astrovirus (GAstV), a recently emergent condition, remain unclear. In this study, we investigated the reprogramming of the host genes as a result of GAstV infection by combining analysis of the global transcriptome and metabolic network pathways in the kidneys of goslings infected with GAstV. We showed that as GAstV replication increased in vivo, the regulation of key enzymes in the host metabolism progressively increased, flowing metabolites into the purine/pyrimidine biosynthesis pathways. Furthermore, we found that GAstV: 1) inhibits the host oxidation-reduction response by inhibiting the expression of the catalase gene; 2) activates the Toll-like receptor 2 pathway to enhance the immune inflammatory response; and 3) activates the key enzyme in lactic acid synthesis to produce lactate accumulation which inhibits the host's antiviral response, so as to facilitate the replication of the virus itself. This study provided the first insight into the overall metabolic requirements of GAstV for replication in vivo by combining transcriptome with metabolic network pathway information.

Identification and characterization of a novel avian nephritis virus variant in chickens with enteritis in Hunan province, China.

Avian nephritis virus (ANV) infection is associated with diarrhea, uricosis, stunting, tubulonephrosis, interstitial nephritis, and mortality of chicken flocks, leading to economic losses in the poultry industry. In this study, an ANV strain designated as HNU-ANV-ML-2020 was identified in tissue samples collected from chickens with severe enteritis on a poultry farm in Hunan province, China, and analyzed. The genome of HNU-ANV-ML-2020 is 6943 nucleotides in length. It showed the highest sequence identity (88.1%) to ANV strain CHN/GXJL815/2017 (MN732559) from Guangxi province, China, while it showed less than 86% identity to other astrovirus (AstV) genome sequences available in the GenBank database. The capsid protein of this virus showed the highest sequence identity to ANV strains HQ330482 and HQ330498 from the UK (81.2% and 81.06%, respectively), while it showed only 73.9% identity to MN732559 and less than 80% identity to the capsid proteins of other AstVs available in GenBank. Further phylogenetic analysis demonstrated that HNU-ANV-ML-2020 belongs to group 4, together with ANV strains identified in Australia, Brazil, the UK, and the Netherlands. Furthermore, ANV strains identified in chickens in China were found to be separated into four distinct groups/genotypes, indicating substantial genetic divergence and a complex circulation pattern in China. The virus characterized in the present study is a novel ANV variant identified for the first time in Hunan province, China.

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.

Development of an indirect competitive ELISA method based on ORF2 detecting the antibodies of novel goose astrovirus.

Goose astrovirus (GAstV) characterized by articular and visceral gout, is an emerging pathogen with a wide distribution on mainland China, leading to serious economic losses in the goose-raising industry. Because vaccines to prevent GAstV infections are not available currently, early diagnosis is critical when treating symptomatic geese and in preventing GAstV transmission. In this context, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) to detect GAstV-specific antibodies from geese was developed, and the detections were optimized. A series of experiments proved that the ic-ELISA shows excellent diagnostic performance and discriminatory power with high sensitivity and specificity. The ic-ELISA for GAstV detection was applied on 67 field serum samples, and comparing the detection results with the virus neutralization test verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the virus neutralization test was 80%, demonstrating the proposed ic-ELISA method could be a useful and effective tool for the diagnostic, serological epidemiological investigation and immune monitoring of the GAstV in goose-producing regions.