| China is the largest goose raising country in the world,and Guangdong Province is also the largest goose raising province.However,in recent years,there have been some new infectious diseases of goose in China,which caused huge economic losses to goose raising industry in China.Since 2002 when Breitbart first studied the virus community in the environment,the viral metagenomics has developed continuously,which greatly expanded people’s understanding of unknown viruses.Therefore,viral metagenomics can be used to investigate the virus community of geese in China,understand the virus species of geese,and master the variation and evolution of the virus,which will be helpful for the early warning of geese diseases.In this study,we collected 146 intestinal samples of goslings in Guangdong Province from 2018 to 2019.The composition of the enterovirus communities of gosling were analyzed based on viral metagenomics,and the differences of enterovirus communities between the clinical healthy goslings and the goslings with enteritis were compared.Study on the goose astrovirus has been carried out systematically.We understood the pathogenicity of goose astrovirus through the virus isolation and animal regression test,and the genomic features and genetic evolution features of goose astrovirus through the amplification of the whole genome of the virus;and established the detection method of specificity.The main results were as follows:(1)Viral metagenomics sequence and analysis on 10 intestinal samples of goslings with enteritis and 10 intestinal samples of clinical healthy goslings were analyzed,and a total of 55,237 contigs were obtained,of which 2682 were annotated as viral sequences.The annotated virus sequences were further divided into 39 virus divisions.Among them,there were 23 virus divisions annotated in vertebrate viruses,6 virus divisions annotated in bacteriophage,6 virus divisions annotated in plant viruses,3 virus divisions annotated in insect viruses and 1 virus division annotated in protist viruses.Compared with the intestinal virus communities of clinical healthy goslings and goslings with enteritis,astrovirus,parvovirus and adenovirus may be the cause of enteritis in goslings.(2)Two goose astroviruses of different genotypes were detected in the samples of goslings with enteritis,and a goose astrovirus and a new goose astrovirus were isolated through inoculating gosling embryos,which were named GoAstV＿QY/103 and GoAstV＿QY/126,respectively.After conducting experiment on pathogenicity of two goose astroviruses of different genotypes,we found that the goslings infected by GoAstV＿QY/103 only had slight diarrhea,while the goslings infected by GoAstV＿QY/126had symptoms of gout,and there was uric acid deposition in gallbladder after autopsy.Both of these two goose astroviruses could not cause death of goslings,and could continuously expel virus to the outside environment through feces,which was widely invasive to gosling groups.(3)The determination of total genome of these two goose astroviruses suggested that the genome length of GoAstV＿QY/103 was 7230nt and the genome length of GoAstV＿QY/126 was 7115nt.The structure was consistent with that of classic astroviruses,which was consisted of 5’UTR,3’UTR and three open reading frames ORF1a,ORF1b and ORF2.The results of homology analysis showed that the nucleotide homology of these two goose astroviruses was 58%and ORF2 protein genetic distance was 0.594,indicating that these two goose astroviruses are two different astroviruses.Compared with the amino acid sequence of capsid protein of the new goose astrovirus,GoAstV＿QY/126 and SD1 strain(MF772821),which could lead to fatal gout of goslings,we found that there was partially amino acid replacement.(4)SYBR Green real-time fluorescent quantitative RT-PCR test method for new goose astrovirus were established.This study amplified a conservative area of 1325bp from ORF2gene and cloned it into pMD18-T vector.Then diluted the plasmid by 10-times gradient as the standard template for real-time fluorescent quantitative PCR amplification to establish the standard curve of real-time fluorescent quantitative PCR.The standard template concentration was in the range of 6.88×10~1～6.88×10~9copies/μL,with good linear relationship,and the amplification correlation coefficient was 0.9994.This real-time fluorescent quantitative RT-PCR test method for new goose astrovirus had high sensitivity with a minimum detection concentration of 6.88×10~1copies/μL,and strong specificity which all showed negative in the detection of common goose viruses(H9N2 subtype avian influenza virus,Tembusu virus,goose parvovirus and duck reovirus).The repetitive result showed that the variation coefficient of repetition within the group was 0.25%-1.61%,and the variation coefficient of repetition between the groups was 0.55%-2.21%,which indicated good repeatability.To sum up,in this study,viral metagenomics were used to investigate the composition of gosling enterovirus community in Guangdong Province of China.It not only enriched the goose virus database,but also found a large number of viruses from other hosts,which provided a theoretical basis for the early warning of cross host viral infectious diseases of goose.Two geese astroviruses of different genotypes from the samples of goslings with enteritis were isolated,and through pathogenicity experiment and genetic evolution analysis,we provided the research basis for the study of epidemiology and molecular biology of goose astrovirus.A real-time fluorescent quantitative PCR method for good astrovirus were established,with high sensitivity,strong specificity and good repeatability,which could be used for clinical detection of new goose astrovirus. |
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