Investigation On Uric Acid Elevation And Immune-related Genes Expression Caused By Goose Astrovirus Infection

Astroviruses are non-enveloped,single-stranded,positive-sense RNA viruses belonging to the Astroviridae family.They can infect mammals and birds which cause diarrhea and viral enteritis in infants and young animals.Since 2016,an acute infectious disease with goose gout as the main symptom has emerged in China’s goose industry.Untill February 2017,the disease has occurred and spread in goose farms in Jiangsu,Shandong,Anhui,Henan,Guangdong,Liaoning provinces and other places in China.The disease mainly affects gooses under 3 weeks of age which characterized by a large amount of urate deposits on the surface of visceral organs,joint cavities,and ureters of diseased goose,leading to significant economic loss.It is reported that the occurrence of the disease is possibily due to the excessive use of high protein feed,but studies have shown that the occurrence of the disease has nothing to do with the feed of goose,and reducing the protein content in the feed can’t control the disease.In our previous research,a goose astrovirus(GAstV)strain was successfully isolated from diseased goose tissues and named JSHA.It was proved the cause of this gout outbreak by virus inoculation of goose embryos and animal regression experiments.In the study,a pair of 489bp specific primers were designed and synthesized based on the conserved sequence of ORF1b of JSHA strain.The corresponding target fragments were amplified by PCR.Random primers were used to construct a digoxigenin-labeled cDNA probe.Dot blot hybridization on the membrane was used to detect the labeling efficiency and specificity.The results showed that the probe could detect 0.1pg/μL of total RNA and had good specificity,which can be used for in situ hybridization experiments.The above-mentioned probes were used to detect GAstV mRNA in paraffin sections of liver,kidney,spleen and intestinal tissues of goslings obtained in animal regression experiments.A large number of positive signals were found in the cytoplasm and nucleus of kidneys.There were also hybridization signals in the nucleus and cytoplasm of lymphocytes and macrophages in the spleen,and in the cytoplasm of liver cells.No hybridization signal was found in the intestine.The above results indicate that the virus is widely distributed in the renal tubular epithelial cells of the kidney,and it is likely to replicate in the epithelial cells.The virus distribution is consistent with our previous qPCR results.Gout is a typical feature of goose infection with GAstV.The above experiments show that the virus is mainly distributed in the liver and kidney,but how the virus damages the liver and kidney and causes the mechanism of uric acid in animals is not clear.In response to this problem,this study tested the activity and expression levels of key enzymes in the uric acid synthesis pathway.The results showed that the activity of xanthine dehydrogenase(XOD),adenosine deaminase(ADA),mRNA expression,Ribosyl pyrophosphate amidotransferase(PRPPAT)and phosphoribosyl pyrophosphate synthetase 1(PRPS1)mRNA levels were significantly higher than those in the control group.The mRNA level and Na-K-ATPase activity of renal multidrug resistance-related protein 4(MRP4)were lower than those in the control group.The above results indicate that GAstV infection will cause liver and kidney disease,increase the activity of enzymes that affect uric acid production in the liver,and increase uric acid production in the body;at the same time,reduce the excretion of uric acid by the kidneys,leading to hyperuricemia and gout occur.Some studies have shown that astrovirus infection can trigger the body’s immune response mechanism,but the current research on the immune response caused by goose astrovirus infection is still lacking.In this experiment,pattern recognition receptors(RIG-I,MAVS,MDA5,TLR3,MYD88,IRF7),cytokines(IL-1β,IL-8.IL-6,TGF-β,iNOS,IL-10,IFN-α),antiviral proteins(OASL,IFITM3,MIX1)a total of 16 innate immune-related genes,and MHCla,MHClla,CD4+,CD8+a total of 4 acquired immune-related genes mRNA expression.The experimental results showed that the mRNA expressions of RIG-I,MAVS,MDA5,TLR3,MYD88,and IRF7 were significantly up-regulated in the spleen of challenged goose,among which MDA5,TLR3,MYD88,and IRF7 were significantly up-regulated at 14dpi,indicating that astrovirus infection Toll-like receptors and RIG-I-like receptor-mediated signaling pathways are stimulated to initiate the innate immune response.MRNA expression of IL-1β,IL-8,TGF-β,iNOS was significantly up-regulated at 14dpi,and mRNA expression of IL-10 was significantly down-regulated;mRNA expression of IFN-α,OASL,IFITM3,and MIX1 was significantly up-regulated,and OASL was extremely up-regulated Significantly;mRNA expression of MHCla,MHClla,CD8+was significantly up-regulated.After challenge,the expression trend of innate immune-related genes in the liver of geese was basically the same as that of the spleen,except that the mRNA expression of IL-1β was significantly down-regulated,and the mRNA expression of IL-10 was not significantly different from that of the control group,indicating that various immune factors such as interferon,Interleukins and antiviral proteins are activated after GAstV invades the body,forming a strong immune response against the virus through synergy.This study revealed the dynamic changes in the expression of immune-related genes from the transcriptome level,suggesting that innate and adaptive immune response may play an important role in goose astrovirus infection.Investigating the immune mechanism of astrovirus will provide the theoretical basis for prevention and treatment of goose astroviruses.