Isolation,Identification And Recombination Analysis Of Simian Adenoviruses

ObjectiveThe purpose of this study:(1)DNA Extraction and PCR amplification from feces have serious interference because feces contains a large amount of protein,inorganic,fat,undigested food fiber,digestive residue,and intestinal exfoliated cells and bacteria.In order to improve efficiency,we established a simple and rapid DNA extraction method of virus in monkey feces,named the Guanidine Isothiocyanate method;and compared with the QIAamp DNA Stool Mini Kit;(2)Developed a molecular epidemiological study of simian adenovirus,to assess the prevalence and possible cross-species transmission of monkey adenovirus in non-human primate colonies,and analyzed the evolution and recombination of the isolated monkey adenovirus strains by genome sequencing and bioinformatics,in order to illuminate the evolutionary rule of monkey adenovirus,It lays an important foundation for the further study of the biological characteristics,pathogenic mechanism and diagnostic reagents of monkey adenovirus.(3)The isolated monkey adenovirus is prepared for the construction of the adenovirus vaccine vectors subsequently.Method:1.The establishment DNA extraction method from monkey fecal sampleThe samples of fresh monkey faeces in a zoo in Yunnan province were collected,and were extracted with Guanidine Thiocyanate method and QIAamp DNA Stool Mini Kit respectively.The results were compared from DNA concentration,DNA purity(OD260/OD280 ratio),DNA extraction rate and the detection rate of monkey adenovirus.2.PCR and sequencing screening out SAdv positive stool samplesEstablishment the detected adenovirus methods using PCR from fecal DNA,comparing the two Primer HexF and HexR,Hex01F and Hex01R PCR for adenovirus DNA detection from simian fecal DNA extrated by QIAamp DNA Stool Mini Kit.Select one pair of primers to with high sensitivity and specificity of the adenovirus,pcr detection for adenovirus DNA detection from simian fecal DNA extrated by the Guanidine Isothiocyanate method.PCR Positive product combine sequencing,identification of whether it is monkey adenovirus,and comparison the difference in the detection rate of the adenovirus of two methods to extract stool samples DNA.,3.Epidemiological analysis of the adenovirus of monkeys in a zoo in Yunnan province.Based on the result of PCR and sequencing detection.Evaluated the prevalence of adenovirus in monkeys in a zoo in Yunnan province.4.Isolation and culture of SAdV strainWe used Vero cells and A549 cells to isolate and culture the viruses found in the PCR-positive samples.After plaque purification,we continued to passage many times until the infected cells appeared cytopathic effect(CPE),and extracted the whole genome of the virus.5.Genome sequencing and phylogenetic analysis of Cynomolgus adenovirus 1 UK/UK-1/2004 strainThe whole genome sequence of Cynomolgus adenovirus 1/UK/UK-1/2004 strain was sequenced by the Sanger’s dideoxy chain termination and the Ion Torrent platform.Then the sequence assembly and annotation,comparative genomics,evolutionary analysis and recombinant analysis of this strain were carried out.Result1.The results shows that DNA extraction from monkey stool samples extracted by Guanidine Thiocyanate method compared with QIAamp DNA Stool Mini Kit was low.But less sample portions were used in the former method,but the concentration of DNA is the same in the two methods.2.Out of the 23 positive sample detected by the QIAamp DNA Stool Mini Kit,only five samples were positive using the HexF and HexR Primer PCR detecting for fecal DNA resulting in a false positive result of 77.3%.Using the primer Hex01F and Hex01R PCR detection only four positive stool samples,identified by sequencing has 3 adenovirus DNA,account for a 25%false positive rate.Use Hex01F and Hex01R PCR for fecel DNA extraction from isothiocyanate DNA,only 23 samples were positive from the initial38 positive sample done by PCR sequencing.The rate of DNA extraction from monkey stool samples extracted by Guanidine Thiocyanate method compared with QIAamp DNA Stool Mini Kit kit was low,The statistical significance calculated shows that that Guanidine Thiocyanate method is more suitable for the extraction of adenovirus DNA in stool samples.3.The prevalence of simian adenovirus of 26 positive samples in zoo among NHPs scales up to 32.5%,and most are asymptomatic infections.Including 1 ring tailed lemurs、18 macaques、5 gibbon、1 orangutan、1 baboon.Among the 12 types of adenovirus confirmed by sequencing,sample No.27 was human adenovirus 43,No.31 was human adenovirus type 45 or 73,suggesting that human adenovirus can infect non-human primates.4.Samples were isolated and cultured in A549 cells,and amplified after plaque purification.Only sample No.43 CPE was observed.The plaque was identified as a single pure adenovirus by PCR.The Hexon fragment was amplified and sequenced to be a Simian adenovirus 6.It needs further purifying and enlarge cultivation to confirm the pure adenovirus isolate.5.The genomic evolutionary analysis of Cynomolgus adenovirus 1 UK/UK-1/2004 showed that this virus belongs to a new SAdV-B type.Simplot recombinant analysis showed that Cynomolgus adenovirus 1 UK/UK-1/2004 may be a new type of monkey adenovirus,which is formed by the recombination of SAdV-A1 327 and SAdV-A1163 in the DNA sites of Hexon protein.Conclusion:Guanidine Thiocyanate method is more suitable for extracting adenovirus genomic DNA from simian stool samples.In a zoo of Yunnan Province,the prevalence of simian adenovirus among NHPs scales up to 32.5%,and most are asymptomatic infections.Human adenovirus type D was also detected from the samples,which indicated a possibility of human adenovirus to cross barriers between humans and NHPs.Moreover,complete genome phylogenetic analysis indicates that Cynomolgus adenovirus 1/UK/UK-1/2004 belongs to SAdV-B,whilst recombination analysis shows that Cynomolgus adenovirus 1 UK/UK-1/2004 probably originated from intraspecific recombination of SAdV-B.Thus,it’s more advisable to keep a close watch on adenovirus infections among NHPs to find its evolution regularities and possibilities of interspecies transmission.And this research has a great value in studying the vector of monkey adenovirus vaccine.