Molecular Epidemiological Investigation And Establishment Of Reverse Transcriptional Loop Mediated Isothermal Amplification Detection Method For Novel Astrovirus Caused Gout In Goslings

Since 2017,outbreaks of fatal gosling gout caused by the novel gosling astroviruses(GoAstVs)have occured in several provinces in China,causing considerable economic losses to poultry industry.To assess the infection status of GoAstVs causing gout,165 clinical samples were collected from 7 goose farms in Henan,Anhui and Hubei provinces during 2018-2019 in China.GoAstVs,goose parvovirus(GPV),reovirus(REOV),goose hemorrhagic polyomavirus(GHPV)and Tambusu virus(TMUV)were screened by PCR(RT-PCR)and their positive rates were statistically analyzed.The results showed that the positive infection rates of GoAstVs,GPV,REOV,GHPV and TMUV were 100 %,9.69 %,3.64 %,0 %,0 %,respectively,which proved that GoAstV was the pathogenic agent of epidemic gout again.Subsequently,in this study,seven GoAstVs strains were isolated from the samples and their whole genome was sequenced.The genomes of all seven GoAstV strains were 7170 nt long and encoded three open reading frames(ORFs),namely,ORF1 a,ORF1b,and ORF2.Sequence and phylogenetic analyses of the seven GoAstV strains showed that they clustered a branch with GoAstVs broken out recent and these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2.Moreover,the mutation rates of ORF1 a and ORF2 were high,and ORF1 a was highly mutated at loci 545-580.The tertiary structure of the mutated ORF2 protein occurred significant changes,and its antigenic epitope was highly mutated,which may be related to the pathogenicity of the virus and caused by antibody pressure from the host.These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.In order to better diagnose and monitor the epidemiology of gosling gout,a rapid,specific and sensitive reverse transcriptional loop mediated isothermal amplification reaction(RT-LAMP)method was established for detection of GoAstVs.Two sets of primers were designed and screened according to the conserved region of ORF1 b,and one primer set was finally selected to complete RT-LAMP detection in a water bath.The optimal reaction temperature and time were 61? and 30 min,respectively.The results of RT-LAMP were evaluated by agarose gel electrophoresis and dye indicator color rendering.The sensitivity of RT-LAMP method for detection of GoAstVs were based on the results of agarose gel electrophoresis and the naked eye detection of color change,respectively,which was the same as that of q RT-PCR method.The optimized RT-LAMP method was applied to the detection of clinical samples.The primers used in this study could specifically detect GoAstVs without cross-reaction with other common avian pathogens.These results confirm that GoAstVs RT-LAMP method is simple,rapid,specific and sensitive,which is very suitable for promotion and application in the field and laboratory of grassroots units.