Identification And Characterization Of Goose Astrovirus In Parts Of Heilongjiang Province

Since 2016,there have been reports of gout in goslings caused by astrovirus in various parts of China.Due to the lack of effective prevention and control measures,a large number of goslings have died,and the morbidity and mortality remain high,causing heavy losses to the goose raising industry.Since 2018,the laboratory has received samples of goslings from all parts of Heilongjiang province for inspection.For this purpose,the laboratory has carried out a series of studies on goose astrovirus.The specific research contents and results are as follows.Total RNA was extracted from the liver or kidney of goslings and reversed into c DNA,and specific RT-PCR primers were designed to identify the goose astrovirus by molecular biological methods.Viruses from astrovirus positive samples were isolated with 11-day old goose embryos and transmitted to goose embryos for stable death.PCR was used to identify whether goose astrovirus was propagating in goose embryos and the viruses of positive goose embryos were collected.Fifteen pairs of primers were designed to sequence the whole genome of the virus strains,and the homology and evolutionary tree analysis were performed.The most lethal strain to goose embryo was named as Astv/Goose/Heilongjiang/ZYL02/2019,and the LD₅₀was measured by goose embryo.The healthy goslings were infected by oral administration and subcutaneous injection,and the pathogenicity to the goslings was determined to judge whether there are the same pathological changes as the sent diseased materials.The ORF2 capsid protein of this strain was expressed by prokaryotic expression system,which was used as the coated antigen coated enzyme plate to establish an indirect ELISA method for detection of goose serum astrovirus antibody,which was used to monitor the antibody titer of immunized goslings.SDA 15A VG adjuvant and 10%aluminum-containing adjuvant were used to prepare the immunization stock of the strain respectively.Goslings were immunized with 0.25ml and 0.5ml respectively.After 7 days of immunization,20ELD₅₀was used to challenge the virus to determine the protective effect of different immune adjuvants and immune doses on goslings.Three strains of nephropathogenic goose astrovirus were isolated from the positive samples of goose embryos.The full genome length of the three strains were 7181nt,7182nt and 7190nt respectively,and the full gene sequence had been uploaded to NCBI（accession numbers:OM811458,OM811459 and OM811460）.Genetic and evolutionary analysis showed that the three viruses were in the same branch as the known goose astrovirus type 2,and their homology was higher than 95%,and homologous recombination had not occurred.The lethal dose(LD₅₀)of the selected virus strain was 10^-1.7.The infected goslings showed typical clinical symptoms and pathological changes caused by goose astrovirus,and the incidence rate was up to 95%.The development of resistant goslings was also significantly affected.The strain showed a broad tropism for tissues of young geese,with high viral loads in liver and kidney tissues of 4.57×10¹⁰copies/g and 3.16×10¹⁰copies/g.Pathological examination showed urate deposits in joints,subcutaneous and visceral tissues.Histological examination showed vacuolar degeneration of liver cells,degeneration and necrosis of renal tubular epithelial cells,exfoliation of intestinal mucosa epithelial cells,and diffuse infiltration of glial cells in brain tissue.The cut-off value of indirect ELISA method was 0.298 based on the reading value of 72negative serum samples.The established indirect ELISA method had excellent specificity,sensitivity and repeatability.The 3-day-old goslings were inoculated with the inactivated strain treated with 0.5ml SDA 15A VG adjuvant,and the serum indirect ELISA OD450nm was 0.404after 4 days of immunization,which was positive.After 7 days of immunization,the protection rate could reach 75%by 0.2ml 20ELD₅₀.In conclusion,three nephropathogenic goose astrovirus strains were isolated and characterized in this study successfully,and proved that this virus is the main pathogen causing gout in goslings in some areas of Heilongjiang Province.The molecular biological and immunological methods for the detection of the virus and its antibodies were established,and the obtained strains were proved to have good immunogenicity.It provides a reference for the study of egg yolk antibody and pathogenesis of goose astrovirus.