Isolation And Identification Of Goose Astrovirus And Its Pathogenicity To Goslings

Avian gout is a disease caused by astrovirus(Ast V)with clinical symptoms of urate deposition in the kidneys,liver and other parenchymal organs and joint swelling.Ast V is a non-encapsulated,single-stranded positive-stranded RNA virus.Its clinial signs and symptoms vary among different species due to the diversity of its infected hosts.So far,many Ast V strains have been isolated from different animals in the world,and Ast V can cause great harm to the global human and animal breeding industry.Among them,goose astrovirus(GAst V)is a pathogen that has endangered the development of domestic goose industry in recent years.However,little is known about this pathogen in China,and there are no effective drugs and vaccines to prevent and control it.The isolation and identification of the GAst V wild strain and its whole genome sequence analysis can provide theoretical basis for its epidemiological and genetic evolution analysis and the development of preventive vaccines.In this study,we obtained the pathological materials from dead goslings suspected of being infected with GAst V from a goose farm in Harbin,Heilongjiang Province.The virus could passage on LMH cells without cytopathic effects.For the goose embryo inoculation of the virus,the initial passage was unstable,and the goose embryo died after the 5th generation,mainly manifested as thickened and whitened allantoic membrane,small embryo became redness,and liver with light color of pale,yellow,and mottled,hemorrhaged in the thigh muscles.The identification by RT-PCR indicated that a GAst V strain was successfully isolated and named as GAst V HLJ strain.According to the whole genome information of GAst V strains published on Gen Bank,6 pairs of overlapping specific primers were designed to amplify the internal genome of GAst V HLJ isolates.At the same time,according to the instructions of 5’RACE and 3’RACE kits manual,5’UTR and3’UTR end sequences were amplified.The obtained product was purified and cloned into p MD18-T vector.After successful identification,the cloned genes were sequenced and spliced into the whole genome of the virus.The full-length genome of GAst V HLJ strain is 7252 bp,and the structure of the genome has typical characteristics of GAst V,including 5’UTR-ORF1a-ORF1b-ORF2-3’UTRpoly(A)tail.The two ends of the genome are non-coding regions,and the internal genes include three open reading frames,ORF1 a,ORF1b,and ORF2.The 5’-end non-coding region at positions1-245 and the 3’-end non-coding region at positions 7028-7252.The genome encodes 3 different genes,ORF1 a at positions 246-3350 consists of 3105 nucleotides,mainly encoding non-structural proteins;ORF1b at positions 3341-4891 consists of 1551 nucleotides encoding Rd Rp;ORF2 at positions 4910-7024 consists of 2119 nucleotides responsible for encoding nucleocapsid protein.GAst V HLJ strain ORF1 a and ORF1 b have a segment of nucleotide repeat sequence,namely AAAAAACTAG,and there is a 18 bp discontinuous sequence between ORF1 b and ORF2.In order to further understand the molecular biological activity of the GAst V HLJ strain,the molecular antigenicity of its main antigenic protein ORF2 was analyzed.It was found that the ORF2 protein appeared mutation and concentrated at the 3’ end,of which 229(Q-P),325(A-T)and 376(T-A)were its unique mutations.These may relate with the virus adapting to new regions,environments and climates.DNAStar software was used to analyze its hydrophilicity,hydrophobicity and antigenicity.Furthermore,28 antigenic epitopes were predicted with online software,and most of the epitopes are densely distributed at the 3’ end of the genome.To better understand the pathogenicity of GAst V HLJ strain,1-day-old goslings were subcutaneously injected into the neck,and then the clinical symptoms and pathological changes were observed.The clinical symptoms of the infected goslings during the whole experiment were lethargic and thin,but there was no death.At necropsy,different organs showed their unique pathological changes at different ages of infection.The pancreas of infected goslings was red,swollen and suspected of white urate deposition at 3 dpi;there was white substance attached to the surface of the liver at 7 dpi;the spleen of infected goslings showed mottled patterns at 13 dpi;the bursa of the infection goslings showed unsmoothness with white substances attached at 13 dpi.Histopathological examination showed that the renal tubular epithelial cells in most areas were degenerated and necrotic during 10-13 dpi.In the liver,a large number of inflammatory cells infiltrated near the central vein and portal area,large-scale necrosis foci formed by hepatocyte necrosis with the presence of eosinophilic red granules.Absolute quantitative detection of the virus in tissues and organs by real-time fluorescence quantitative PCR method showed that the virus could be detected in all organs within 21 days of infection,and there appeared significantly higher at the later stage of infection of 10-13 dpi.The highest viral load in the kidney indicates that it is the main target organ of astrovirus infection.However,there just detected moderate quantity of virus in the liver,maybe it is due to the serious degenerative changes of this organ.The animal model of GAst V infection in 1-day-old goslings was successful established and could provide theoretical and material basis for the prevention and control of the disease and the research on vaccines in the future.