| Avian astrovirus(AAstV)belongs to the genus Astrovirus of Astroviridae,which contains many subtypes and is an important pathogen threatening poultry.It not only affects the normal growth and development of poultry,but also causes chick nephritis,duck viral hepatitis,goose gout,diarrhea,growth inhibition and other symptoms,and has the risk of infecting humans.In recent years,the astroviruses of waterfowl,such as Duck astrovirus(DAst V)and Goose astrovirus(GAst V),have become more and more popular in China,bringing huge economic losses to the waterfowl breeding industry.In addition,with the emergence of new epidemic strains such as DAst V-3,the prevention and control of AAst V has also brought severe challenges.In order to further understand the prevalence of waterfowl astrovirus in China,1012duck and goose samples were collected from 14 provinces in China,including Guangdong,Heilongjiang and Hubei,and detected by reverse transcription polymerase chain reaction(RT-PCR).The results showed that 115 AAst V nucleic acid positive samples were detected,and the overall positive rate was 11.36%.AAst V was also detected in various links such as breeding,slaughtering,retail and wholesale,indicating that AAst V has been prevalent in China.In addition,DAst V was detected from goose samples in this study,which confirmed that DAst V had the phenomenon of cross host transmission.This study enriched the epidemiological data of waterfowl astrovirus in China,suggesting that we should strengthen the surveillance of waterfowl astrovirus.In order to further understand the genomic characteristics of waterfowl astrovirus,this study used second generation sequencing to sequence the whole genome of 8 selected AAst V strains detected,and a total of 8 whole genome sequences were obtained,with the total genome length in the range of 7.2-7.5 kb.Genetic evolution analysis showed that among the 8 strains of waterfowl astrovirus,4 strains were DAst V-3,3 strains were DAst V-4,and 1 strain was GAst V.The whole genome sequence analysis showed that DAst V-3 strain had high homology with the reference strain KJ020899 sequence,DAst V-4 strain had high homology with the reference strain JX624774 sequence,and GAst V strain had high homology with the reference strains MZ819185,OL762471 and OL762472 sequence.It shows the complexity and genetic diversity of the epidemic of waterfowl astrovirus in China,and provides a basis for the follow-up study of waterfowl astrovirus.In order to further deepen the research on DAst V-3 and meet the demand for rapid on-site detection of waterfowl astrovirus,this study successfully isolated and identified a DAst V-3 strain using duck embryos by chorioallantoic membrane inoculation,and designed specific probes and primers according to the RNA dependent RNA polymerase gene(Rd Rp)of DAst V-3,and established a fluorescent RT-RAA detection method for DAst V-3.The results showed that the established RT-RAA method could rapidly detect pathogens within 20 minutes under 39℃constant temperature,and had no cross reaction with H9 subtype avian influenza virus,Newcastle disease virus,goose stellate virus and other common viruses.The minimum detection limit was 10~1 copies/μL;The detection rate of RT-RAA method was higher than that of conventional RT-PCR method,and the detection time was shorter than that of RT-PCR method.The results showed that the method established in this study was highly sensitive,specific and reproducible,and did not need expensive instruments.It provided a rapid detection method with strong specificity,good repeatability and high sensitivity for the early monitoring and epidemiological investigation of DAst V-3. |
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