Isolation And Identification Of Novel Goose Astrovirus ZM Strain And Establishment Of A Fluorescence Quantitative PCR Method

Novel goose astrovirus(GAst V)is a single-stranded positive-chain RNA virus,which causes gout disease characterized by a large amount of urate deposition in the joints and viscus of goslings and causes death of goslings.In this paper,genome sequencing analysis and animal regression test of GAst V ZM strain were carried out,and a quantitative PCR detection method for GAst V was established.It provides an important reference for the epidemiological investigation,prevention and control of goose astrovirus disease.In 2021,gout of goslings suspected to be caused by astrovirus occurred in a goose farm in Anhui Province.After RT-PCR detection and goose embryo isolation,a goose astrovirus strain named ZM strain was isolated.Then,whole genome sequencing analysis of GAst V ZM strain was performed.The results showed that the genome of ZM strain was 7175 nt with three open reading frames(ORF1a,ORF1b and ORF2).The nucleotide and amino acid homology of ORF2 gene of ZM strain were 96.7%～98.9%and 96.9%～99.0%,respectively,compared with GAst V strains isolated from 2014 to 2020.ZM strain was in the same evolutionary branch as the novel goose astrovirus circulating in China since 2016.The GAst V ZM strain was inoculated into LMH cells and continued to the fifth generation.The results showed that ZM strain proliferated well in LMH cells and did not cause cytopathy.Subsequently,the animal regression test of the goslings was carried out,2-days-old goslings were subcutaneously challenged with ZM strain.The results showed that GAst V infection could cause weight loss,depression,diarrhea and neurological symptoms in goslings.The death rate was 10%(2/20)at 7 days post-infection.The deposition of urate in the heart,liver,gallbladder,kidney and ureter of dead geese was obvious.Histopathological observation showed renal tubular epithelial cell collapse,degeneration necrosis and inflammatory cell infiltration.The results showed that the GAst V ZM strain had certain pathogenicity to goslings.In addition,a SYBR GreenⅠfluorescent quantitative PCR method was established for the detection of the novel goose astrovirus.Based on the ORF1b gene of GAst V,two pairs of primers were designed to detect and construct the recombinant plasmid,respectively,and the reaction conditions were optimized and the standard curve was established.The results show that the linear relationship of the standard curve R~2 is greater than 0.99,the GAst V amplification curve and the melting curve are single.The coefficient of variation between groups was small;The sensitivity test was 100 copies/μL.The method was used to detect the viral load of goslings infected with ZM strain and determine the viral load in kidney,liver and spleen.The results showed that the method had good specificity,sensitivity and repeatability.In summary,the GAst V ZM strain have high homology with the GAst V epidemic strains in recent years,which can cause obvious gout lesions in goslings.To provide technical support for the epidemiological investigation and prevention and control of the novel goose astrovirus.