Phylogennetic Analysis Of Goose Circovirus And Establishment Of A Duplex Real-Time PCR Detection Methods Of Goose Circovirus And Goose Astrovirus

Goose circovirus disease is an infectious disease caused by(Goose circovirus,GoCV)with diarrhea,slow growth and feathers disorders and other clinical symptoms.The virus mainly affects lymphoid tissues and organs in geese after infection,which leads to decreased immunity and then increases secondary infection.The virus is one of the potential pathogens endangering the goose industry.Until now,the disease has been reported in Zhejiang,Shandong,Taiwan,Guangxi,Guangdong and other provinces.The disease has not been reported in Anhui Province.So,this study conducted clinical detection of GoCV in disease materials from some areas of Anhui Province,and carried out genetic evolution analysis.First of all,the clinical samples of liver,bursa of Fabrica,spleen,small intestine and kidney of 25 disinfected geese were collected from different areas of Anhui province.These geese were characterized by stunted growth,wasting,and urate deposition in internal organs.A pair of primers based on Rep gene were designed by Primer Premier 5.0 software for GoCV detection.The results showed that a total of 6GoCV positive samples were detected.Then,three pairs of primers were designed.And the whole genome sequence of two of the GoCV strains was amplified.The results showed that the whole genome sequence was amplified successfully.And the nucleotide were submitted to Gen Bank named AAU109(Gen Bank accession number MW560282)and AAU111(Gen Bank accession number MW879363).Then,the whole-genome structure of the Open Reading Frame Finder(ORF)of GoCV was analyzed using the online website NCBI-Open Reading Frame Finder.The results showed that the two strains of GoCV encode four reading frames,namely V1,C1,V2 and C2.The V1 gene encodes replication-related REP protein,which is composed of293 amino acids.The C1 gene encodes capsid Cap protein,which is composed of 250 amino acids.Finally,the nucleotide and amino acid similarity analysis and phylogenetic analysis were carried out based on the whole genome,REP and Cap genes using Meg Align and Mega 6.0 software.Based on the whole-genome sequence analysis,the results showed that the nucleotide similarity and amino acid similarity of the two GoCV strains were 91% and 92%,respectively.Based on Rep gene sequence analysis,the nucleotide similarity and amino acid similarity of the two GoCV strains were 92.4% and 94.9%,respectively.Based on the Cap gene sequence analysis,the nucleotide and amino acid similarities between the Cap genes of the two isolates were97.3% and 96.3%,respectively.Phylogenetic showed that the AAU109 strain formed a small clade with the Shandong reference strain(Gen Bank accession number:KT387277.1),and the AAU111 strain formed a small clade with the Taiwan reference strain(Gen Bank accession number: AF536941.1).The phylogenetic analysis of Rep gene is similar to that of the whole genome.The results of phylogenetic analysis of Cap gene showed that the two strains of GoCV formed a separate cluster with the Northern Ireland GoCV(Gen Bank accession number:AJ304456.1).In addition,the recombination of Anhui strain AAU111 occurred in the Cap region.This study provides data support for further understanding the genetic evolution of GoCV in Anhui province.Due to the co-infection of GoCV and GAstV in clinical detection,a dual SYBR Green I fluorescence quantitative PCR method that can simultaneously and rapidly detect GoCV and GAstV was established for the first time.It provides technical support for the early diagnosis of the two viruses.At first,two pairs of specific primers were designed for GoCV Rep gene and GAstV ORF2 gene by software.Then,a simple,sensitive and highly specific dual SYBR Green I fluorescence quantitative PCR detection method was established.The method can distinguish GoCV(84.5?)and GAstV(81.0?)by the melting point of the melting curve.The results showed that,this method is not affected by Fowl adenovirus serotype 4(FAd V-4),Goose parvovirus(GPV),and Avian leukosis virus(ALV),which showed good specificity.The lowest values of GoCV and GAstV were 51.6 copies /?L and 37.5 copies /?L,respectively.The intra-group and inter-group coefficients of variation were all less than 1%,which indicates good repeatability.The results of clinical samples showed that the positive detection rate of the established method was 12%(3/25),which was higher than that of conventional PCR(4%,1/25).In summary,the genetic evolution of GoCV in some areas of Anhui province was analyzed in this study.And a duplex SYBR Green I fluorescence quantitative PCR method was established for the simultaneous detection of GoCV and GAstV.The results is useful for better understanding of the genetic evolution of GoCV in Anhui province,and also lay a foundation for the rapid diagnosis of GoCV and GAstV.