Etiological Identification And Establishment Of Fluorescence Quantitative PCR Method Of Novel Astrovirus

Several large-scale epidemics have significantly harmed human life,health,and social and economic development in recent years.Most infectious diseases are caused by zoonotic viruses,such as the Ebola virus,Zika virus,and the novel coronavirus circulating for the past three years.Poultry is usually in close contact with humans and is an essential food source for humans.While the development of the intensive breeding industry has brought huge benefits,a variety of new avian infectious diseases such as highly pathogenic avian influenza have also been emerging,increasing the risk of zoonotic pandemics.Zoonoses caused by viruses are of a wide variety,usually capable of causing a pandemic in a short period of time and difficult to control,posing great risks to human health and safety.Poultry host a wide range of viruses,including not only those known to cause zoonoses,such as avian influenza virus,but also a large number of new and unknown viruses.Traditional virus detection methods usually have their limitations,such as the PCR method can not detect unknown or large variations of the virus,electron microscope observation method is low sensitivity.In recent years,the emergence of viral metagenomics based on high-throughput sequencing technology provides a new technical means to solve these problems.Viral metagenomics technology takes all viral nucleic acids in environmental samples as detection objects.It can rapidly detect a large number of known viruses,but also identify relatively dispersed unknown viruses with low abundance.It has been widely used in various fields of virus research,such as new virus discovery,epidemic prevention and control,and virus community analysis.Studies have shown that astrovirus is one of the major pathogens causing gastroenteritis in children.Besides infecting humans,astrovirus can also infect sheep,cattle,dogs,cats,ducks,geese,and other mammals and poultry.In poultry,astrovirus mainly infects young animals and causes gout and other intestinal diseases,with a potential threat to cause zoonotic diseases In March 2020,a goose gout disease with similar symptoms and a fatality rate of more than 50percent broke out at a farm in Shanghai,China,where astrovirus infection had not been detected.The results of bacterial culture and PCR test of common viruses showed that the bacteria and common avian pathogenic viruses such as avian adenovirus,goose hemorrhagic polyomavirus,goose reovirus,goose coronavirus,goose circovirus,duck tambusu virus,newcastle disease virus,and avian influenza virus were harmful.To identify the pathogen of this disease,the library of the liver,kidney,and fecal samples of dead goslings was constructed using viral metagenomics technology,and the viral community composition in dead goslings was further analyzed.Potential new pathogenic viruses were discovered and identified.A new type of astrovirus was isolated by goose embryo culture,and its pathogenicity was verified.An SYBR GreenⅠfluorescence quantitative PCR method for detecting new astrovirus was established,which provided technical support and an etiological basis for the prevention and control of zoonotic diseases possibly caused by novel astrovirus and was of great significance for the prevention and control of potential emerging infectious diseases.The main contents and results of this study are as follows:1.Viral community analysis of liver,kidney,and fecal samples of dead goslings and discovery and identification of potential new pathogenic viruses:Except for plant,insect,invertebrate virus,and phage,the virus community of liver,kidney,and fecal samples collected from 7 dead goslings mainly included:Parvoviridae,Picornaviridae,Retroviridae,Hepadnaviridae,Poxviridae,Astroviridae,Marseilleviridae,Herpesviridae and Iridoviridae.The results show that:The virus community in the tissue and fecal samples of dead goslings showed high diversity,and Parvoviridae was the dominant DNA virus.Among RNA viruses,Picornaviridae and Astroviridae are prevalent,both of which can cause avian diseases.In this study,two strains of astrovirus were identified by viral metagenomics and named CHSH01（Gen Bank No.OM569656）and CHSH02（Gen Bank No.OM569657）.Their genome length was 7154 and 7330 nucleotides（excluding poly A tail）,and their genomic structural characteristics were significantly different.At the nucleotide level,CHSH01 had a low genome-wide sequence identity with other strains（68.27%）,and CHSH02 had a high sequence identity with other strains（99.20%）.Phylogenetic and recombination analyses showed that CHSH02belonged to goose source astrovirus type 2,the dominant species causing gout in geese in China.CHSH01 belonged to a new type and was a potential recombinant strain.One of the parent strains was an astrovirus from bar-headed geese,a migratory bird.This suggests that the CHSH01 may spread across species between poultry and wild birds.Preliminary epidemiological investigations indicated that the two strains were circulating in the same goose farm,and co-infection had occurred.Specific primers were designed according to the sequences of different strains to detect the dead goslings by RT-PCR.The results showed positive samples in the samples,which was consistent with the results of viral metagenomic sequencing.2.Isolation and pathogenicity verification of novel astrovirusesAfter homogenizing,the samples tested positive for the CHSH01 and eliminated bacterial contamination through bacterial culture.The centrifuged supernatant was inoculated into the allantoic cavity of the goose embryo at ten days of age for isolation and culture.After injection,some embryos died（50%mortality）and showed severe developmental retardation.RT-PCR was used to detect CHSH01 strain in goose embryo allantoic fluid and to detect several common viruses that cause gosling disease in clinical practice.The amplified products were sequenced and compared,and the results showed that the CHSH01 strain was positive while other viruses were negative.In conclusion,this study successfully isolated a new strain of astrovirus CHSH01and proved its pathogenicity.3.Establishment of SYBR GreenⅠreal-time fluorescence quantitative PCR method for detection of novel astrovirusThe whole genome sequence of the CHSH01 strain was compared with that of other astroviruses.Two pairs of primers were designed according to the conserved sequences of structural proteins to construct recombinant plasmid standard and real-time PCR detection of SYBR GreenⅠ.The optimal reaction conditions and system were determined by optimizing annealing temperature and primer concentration,the standard curve was established,and its specificity,sensitivity,repeatability,and stability were verified.The results showed that the concentration of recombinant plasmid standard was about 6.6×10¹¹copies/μL,the optimal annealing temperature was 60℃,and the optimal primer concentration was 0.05μM.When the standard attention was 1×10²-1×10⁹copies/μL,the Ct value had an excellent linear relationship with the logarithm of the copy number of the recombinant plasmid with a slope of-3.053 and a correlation coefficient（R²）of 0.9982.The specific test results showed that the method could detect the CHSH01 strain but could not amplify the five viruses,such as the goose influenza virus and goose parvovirus.Sensitivity test results showed that the detection limit of this method was 10²copies/μL,and the detection sensitivity was 100 times higher than that of conventional PCR.The results of repeatability and stability experiments showed that the coefficient of variation between groups and within groups was less than 2.5%,and the method had good repeatability and stability.A total of 105 feces,tissues,and organs of dead goslings were collected from a farm in Shanghai.The detection results showed that the positive rate of standard PCR was significantly lower than that of quantitative fluorescence PCR.In conclusion,the real-time fluorescence quantitative PCR detection method of the CHSH01 strain established in this study has a good detection effect,which provides reliable technical support for the research and identification of new astrovirus and the prevention and treatment of infectious diseases.Conclusions:In this study,the viral community composition in the liver,kidney,and fecal samples of dead goslings was analyzed by viral metagenomic technique,which enriched the viral diversity.Two strains of astrovirus were found,one of which was a new recombinant strain.The new astrovirus CHSH01 was isolated,and its pathogenicity was verified by inoculation and culture in the allantoic cavity of the goose embryo.Finally,an SYBR GreenⅠreal-time PCR method was established to detect the CHSH01 strain.