Isolation,Identification And Pathogenicity Of Goose Astrovirus From A Goose Farm In Chongqing

Goose astrovirus（GAst V）is an infectious disease of geese found China in recent years,mainly infecting gosling up to 4 weeks.Infection of goslings with goose astrovirus virus is characterised by urate deposits on the surface of internal organs and has been reported to be both horizontally and vertically transmissible.Once infected,the virus can spread rapidly and the mortality rate can be 50%.No specific drugs have been reported for goose astrovirus virus disease and no vaccine has been developed,making prevention of goose astrovirus virus disease more difficult.In order to understand the pathogenesis of this virus,this research was carried out to isolate and identify the virus from a goose farm in Chongqing,and to characterize the molecular biology of the virus.The results of the study are as follows:1.Isolation and identification of goose astrovirusIn this experiment,18 symptomatic goslings were collected from a goose farm in Chongqing,and extract total DNA and total RNA of liver,kidney and spleen were.tested by RT-PCR.In order to exclude infection by other viruses,PCR tests for GPV,MDPV,DPV,APMV,NDRV,DTMUV,DHV,NDV,AIV and ARV were performed,and the results showed that GAst V was positive and the rest were negative;The clinical samples collected were ground and inoculated in solid medium and no pathogenic bacteria were isolated;the tissue fluid was aseptically processed and inoculated with 11-day-old goose embryos for isolation and culture;by the fifth generation of transmission,the embryos appeared significantly reddened and embryo development was blocked.The results showed that the virus had an ID₅₀=1x10^-1.60/0.2ml.The results were positive for GAst V RT-PCR after each passaging.Primers were designed based on the whole gene sequence of GAst V XT1 strain,amplified by RT-PCR segmentation and ligated to p MD19-T and sent to tsingke for sequencing,and the sequencing results were spliced using DNAMAN to obtain the whole genome sequence of the strain.The gene length was 7114nt,containing three open reading frames ORF1a,PRF1b,and OFR2,as well as the 5’-UTR and 3’-UTR,and a conserved sequence（5’-AAAAAAC-3’）of the astrovirus.The structure is similar to that of the astrovirus.The whole genome sequence of the isolate was compared with the reported goose-derived astrovirus and the homology was 54.3%-98.7%.The Phylogenetic tree analysis showed that the isolate was on the same branch as strain G576,strain HNZZ-4 and strain ZJLD,with homology of 98.3%-98.7%,and belonged to the type I goose astrovirus.It is closer to the turkey astrovirus and duck astrovirus branches,with a homology of 58.6%-60.0%%.It is not on the same branch as type II goose astrovirus strains SCCD and TZ03,with only 54.3%-54.5%homology,and has the lowest homology with mammalian astrovirus virus,with only 36.9%-40.2%.The results of the whole genome evolutionary tree branch were similar to those of the ORF2 amino acid sequence evolutionary tree branch.The isolated strain had typical structural features of astrovirus and also caused gout in goslings in animal regression tests.The strain was identified as a type I goose astrovirus and was named TL01.2.Establishment of real-time quantitative PCR detection method for GAst V-ⅠTo facilitate the laboratory detection of goose astrovirus,a fluorescent quantitative RT-PCR assay was developed based on the conserved sequence of the ORF2 gene of type GAst V-Ⅰ:a specific primer was designed based on the conserved sequence of the ORF2 gene of GAst V,with a target fragment length of 132 bp.The fragment was amplified by common RT-PCR and ligated to p MD19-T vector to construct a The recombinant plasmid was used to create a standard quality plasmid.The real-time quantitative PCR was established,and the optimal reaction system and reaction conditions were obtained by optimising the reaction procedure.The sensitivity,specificity and reproducibility of the method were tested to check the reliability of the method.The linearity of the method was good when the plasmid concentration was between 1.23x10⁹ copies/μL and 1.23x10² copies/μL,with a linear regression equation of y=-3.3008x+35.977 and an amplification correlation coefficient of R²=0.9978.The results showed that the method has good specificity,and the intra-batch coefficient of variation was less than 1%,which is reproducible.The results showed that the established SYBR Green I dye method for real-time quantitative PCR is reliable and can be applied to tissue viral and clinical testing.3 GAst V-Ⅰpathogenicity experimentalThe GAst V-Ⅰwas subcutaneously injected into 2-day gosling at a dose of 0.2 ml/gosling.inoculation concentration 10^5.12copies/ml,The gosling were observed daily for clinical signs and growth differences after inoculation.The gosling were dissected at 1d,2d,4d,6d,8d,10d,12d and 14d and swabs were collected from the liver,spleen,pancreas,thymus,glandular stomach,bursa,kidney,lung,heart,brain and cloaca.Pathological sections were taken from the liver,spleen,pancreas,thymus,glandular stomach,bursa,kidney,lung,heart and brain of the affected gosling with obvious clinical signs for histological observation;the results showed that the gosling showed clinical signs and died on the fifth day after artificial infection,and urate deposits appeared on the surface of the internal organs of the gosling on the sixth day.The results showed that the virus was detectable in all organs of the body on the first day after inoculation and reached its highest level in all tissues on the sixth day,which was also the peak of detoxification in the goslings.The highest levels of virus were found in the liver and spleen during the first six days after inoculation,and in the kidneys after six days until the end of the test.In pathological sections,only the kidneys,liver and spleen showed significant lesions,while the rest of the organs showed no significant lesions.Goose embryos were inoculated with GAst V-Ⅰallantoic fluid for the embryo virulence test.The aseptic allantoic fluid was inoculated into 11-day-old goose embryos at a dose of 0.2 ml/embryo inoculation concentration 10^5.12copies/ml.Tissues were collected at 0.5d,1d,2d,3d,4d,5d,6d,7d,8d,9d,10d and 11d after inoculation,and the viral load of each tissue was monitored using q RT-PCR.The results showed that there was little variation in the levels of toxicity in the tissues,with the highest levels of toxicity in the embryos on the seventh day after inoculation,similar to the time point of the highest levels in the gosling.