Genetic And Phylogenetic Analysis Of Feline Astroviruses In Some Areas Of Anhui Province And Establishment Of Real Time RT-PCR Detection Method

Feline astrovirus(FeAstV)is RNA virus.The virus is associated with cat gastroenteritis and exists dangers of zoonosis,causing serious harm to social cats.The genome structure consists of three open reading frames(ORFs),namely ORF1a,ORF1b and ORF2,in which the genetic distance between complete ORF2 sequences is the classification criterion for astroviruses.The virus can cause invisible infection and the research on the virus is few,so reference documentation is very limited.At present,no genetic analysis of FeAstV in Anhui Province has been reported,and no inexpensive and efficient method has been established for large-scale detection of the virus.In order to provide theoretical basis for FeAstV prevention and control.The purpose of this study is to explore the genetic evolution analysis and homology analysis of FeAstV,and to establish a real-time quantitative RT-PCR detection method based on SYBR Green?.A total of 75 fecal samples of domestic cats with or without diarrhea were collected from the Anhui province,and two positive samples were detected.The complete genome and ORF2 of the two strains were sequenced and phylogenetically analyzed.Phylogenetic tree analysis revealed that all strains could be classified into three different clusters;therefore,the mean amino acid genetic distances(p-dist)of ORF2 among the three clusters were estimated.The results suggested that the two strains and other FeAstV strains were grouped into three genotypes,with AH-1-2020 belonging to a novel genotype.High similarity was observed in ORF2 between porcine astrovirus type 1 and AH-1-2020.Furthermore,inter-specifc recombination between porcine astrovirus type 1 and FeAstV was observed.We,therefore,inferred that inter-specific transmission may exist between pigs and cats;however,further studies are required to verify this.This is the first report on the genetic characterization and phylogenetic analysis of FeAstVs in the Anhui province and would further the current understanding of the genetic diversity and epidemiology of FeAstVs.Specific primers were designed based on the conserved region of the FeAstV ORF1b gene.Experiments for specificity,sensitivity,and repeatability of the assay were carried out.In addition,the assay was evaluated using clinical samples.Specificity analysis indicated that the assay indicated good specificity of the assay.Sensitivity analysis showed that the SYBR Green I-based real-time RT-PCR method could detect as low as 3.72x10~1copies/?L of template,which is 100-fold more sensitive compared to the reverse transcription PCR.Both intra-assay and inter-assay variability were lower than 1%,indicating good reproducibility.Furthermore,an analysis of 75 fecal samples showed that the positive detection rate of SYBR Green I-based real-time RT-PCR was higher than that of reverse transcription PCR,indicating the high reliability of the method.The assay is cheap and effective.Therefore,it could provide support for the detection of FeAstV in large-scale clinical testing and epidemiological investigation.