Isolation And Identification Of Goose Astrovirus And Establishment Of Real-time Quantitative PCR Method

Astrovirus(AstV)belongs to the family Astroviridae,including Mamastrovirus and Avastrovirus.AstV is one of the leading causes of diarrhea in young animals and can lead to severe organ damage and even death in birds.Since 2017,there have been many reports about fatal infection of goslings characterized by visceral and articular gout in Chinese commercial goose farms.At autopsy,large amounts of white urate deposits were found on the visceral surface and articular cavity.The mortality rate ranges from 20 to 30 per cent,up to 50 per cent,hampering the healthy development of the goose industry.?n this study,the pathogen that caused the goose gout epidemic was separated and identified,and the research on pathogen detection and prevention was carried out,which is of great significance for effectively controlling the occurrence and development of goose gout disease.1 ?solation and identification of goose astrovirusThe infection of fatal goslings was excluded by bacteriological examination after dissecting,and then the infected material was collected and inoculated into the 12-day-old goose embryo for virus isolation,while the identification of virus isolates was carried out by PCR and construction of evolutionary tree.A goose-derived astrovirus strain was isolated by inoculation of goose embryo and named AstV/Goose/CHN/2019/AHMG(AHMG for short).?n the genetic evolutionary tree based on the ORF2 amino acid sequence,AHMG and other goose astrovirus strains share a branch,and the amino acid homology is greater than 98%.1-day-old healthy goslings had a subcutaneous injection with the tissue homogenate harvest from goose embryo.The goslings showed diarrhea,anorexia,depressed spirit and other symptoms,which was similar to the symptoms of natural infection,causing a morbidity of 27%and mortality of 7%.At autopsy,typical white urate deposition was seen in the viscera and joints.Microscopic examination revealed hepatocyte steatosis and renal tubular epithelial cells shed.The above results indicated that goose astrovirus was the cause of gosling gout.2 Establishment of SYBR Green ? real-time fluorescence quantitative PCR methodTo construct standard plasmid,a fragment of 110 bp was amplification by PCR according to goose astro virus ORFlb area.The concentration of plasmid was 2.3×10¹⁰ copies/?L.Specificity,sensitivity and repeatability tests were carried out to optimize the primer concentration and other related conditions.The results showed that the reaction had a great amplification efficiency when adding 1.0 ?L primers with the concentration of 2.0 ?mol/L,and the Ct value of the standard curve showed a good linear relationship with the concentration of the standard plasmid,while the correlation coefficient was 0.990 and the amplification efficiency was 104.5%.The specificity test of other common pathogenic goose viruses showed that only when goose astrovirus as the substrate template appeared a high and smooth amplification curve.This method is 1000 times more sensitive than normal PCR.?n the repeated test,the coefficient of variation between and within groups was less than 1.00%.Clinical samples testing results show that positive detection rate of the built SYBR Green ? real-time fluorescence quantitative PCR was 100%,while the positive rate of ordinary RT-PCR method was 85%.These results indicate that the established SYBR Green ? real-time fluorescent quantitative PCR method is equipped with good repeatability,specificity and high sensitivity,which can be used for goose astrovirus pathogen detection and quantitative analysis.3 Expression of goose astrovirus capsid spike proteinA pair of specific primers was designed based on the C-terminal region of goose astrovirus ORF2,and the target fragment with a length of 723 bp was amplified by PCR using goose astrovirus AHMG strain as template.The recombinant capsid spike protein expression vector pET32a(+)-S was constructed,and transformed into E.coli BL21(DE3)competent cells for prokaryotic expression.There was a maximum expression of the recombinant protein when inducing for 6 hours with 1.5 mmol/L ?PTG.Then the induced supernatant and precipitation were respectively taken for SDS-PAGE,and the result showed that the recombinant protein was an inclusion body protein.Finally,Western Blot was conducted to identify the recombinant protein,with His-tag protein as an antibody.After color development,the target band appeared at the relative molecular weight of the protein of about 52 kDa,and the band was single and free of impurity,which was consistent with the expected results.The above results indicate that the successful expression of AHMG capsid spike protein,which will be helpful to the establishment of serological detection methods for goose astrovirus in the future.?n summary,a goose-derived astrovirus strain was successfully isolated by inoculation of goose embryo.The results of animal regression test showed that the astrovirus from goose was the cause of novel viral gout in gosling.A real-time fluorescent quantitative PCR method for goose astrovirus was established,and the capsid spike protein of goose astrovirus was successfully expressed by the prokaryotic expression system of E.coli.These results provide an effective method for the rapid diagnosis of gout in goslings caused by astrovirus,and also lay a foundation for further research on the biological characteristics and pathogenesis of gout.