Whole Genome Sequence Analysis And Infection Investigation Of Waterfowl Circovirus In Guangdong Province

Duck circovirus(DuCV)and Goose circovirus(GoCV)belong to the circovirus of circoviridae,both of which can infect the immune organs of the corresponding host and cause the immunity decreased,increased the probability of infection of other pathogens or aggravate the clinical manifestations of avian influenza and other diseases,also has potential harm to the waterfowl feeding industry,and can cause some economic losses.In order to understand the infection status of circovirus in waterfowl in some parts of Guangdong,the genotype of DuCV and the sequence difference between GoCV and DuCV were established.Based on the establishment of DuCV real-time quantitative PCR method,DuCV infection and the whole genome of DuCV and GoCV were sequenced and analyzed,and the differences between DuCV and GoCV sequences and related genetic information were clarified.1.The establishment of real-time PCR detection method for duck circovirus and preliminary applicationIn order to achieve the rapid detection of duck circovirus(DuCV),a pair of specific primers were designed and synthesized in its conserved region according to the DuCV genome sequence registered in GenBank,and the corresponding 212 bp fragment was cloned into pMD-18T the recombinant plasmid pMD18DuCV was amplified by SYBR Green I fluorescence incorporation.The specific curve of pMD18DuCV concentration was calculated by pMD18DuCV concentration and Ct value.The linear regression equation was deduced.The correlation coefficient(R~2)was 0.9968,and the DuCV real-time PCR method was established.After repeated and sensitive test,the results showed that the repeatability was good and the detection limit was 67.2 copies/?L.The nucleic acid or cDNA of DHV,Escherichia coli,Newcastle disease virus(NDV),H9 subtype avian influenza,goose pestivirus(GPV)were tested for specificity.The results showed that the specificity was good.The results of the preliminary application showed that the detection rate of DuCV real-time PCR was 26.4%(62/235),which was 11.1%higher than that of conventional PCR method15.3%(36/235),which was better than conventional PCR.Real-time quantitative PCR and conventional PCR was performed on 235 samples from54 farms.62 positive samples ofDuCV were found,the positive rate of DuCV was 26.4%(62/235),DuCV infection rate was 38.3%(54/141)in duck flock,and the infection rate of DuCV in goose flock was 8.5%(8/94).The positive rate of GoCV was 13.6%(32/235),and the infection rate of GoCV was 20.2%(19/94)in goose flock,and the infection rate of GoCV was 9.2%(13/141)in duck flock.In the flock of 141 ducks,the highest positive rate of Muscovy duck was 66.7%(8/12)at the age of 4-8 weeks;The highest positive rate of teal was 50.0%(7/14))in 0-4 weeks;The highest positive rate of White duck was 40.0%(6/15)in 4 weeks or older;The highest positive rate of shelduck was 46.7%(7/15)in 8 weeks;The highest positive rate of mud duck was 12.5%(1/8)in 8 weeks.In the flock of 94 geese,the highest positive rate was 30.8%(8/26)in 0-4 weeks old of geese.The total positive farms accounted for 42.6%(23/54)of DuCV,the positive rate of different varieties of farms was11.8%~83.3%.2.Analysis of Genetic Variation of Waterfowl CircovirusAccording to the DuCV and GoCV genome sequences registered in GenBank,two pairs of reverse specific primers were designed,respectively.The results showed that,the target fragment size of DuCV was 1840bp and 635bp respectively.The target fragment size of GoCV was 1784bp and 284bp respectively.The target fragment identified by PCR was cloned into pMD-18T vector.The recombinant plasmids were determined by PCR,the sequences were ligated and analyzed by DNAMAN,DNAStar and MEGA6.Six strains of DuCV and eight strains of GoCV genome sequences were compared with the departmental sequences registered in GenBank,the results showed that the nucleotide sequence homology between the DuCV genome sequences was between 82.8%and 98.6%,the GoCV genome sequences was between 91.7%and 99.3%.The genetic distance of JM09D-2015 strain was the closest to that of Korean isolate D11JW002.The genetic distance between QY115G-2016 and Zhejiang isolate xs1 was the closest;DuCV,GoCV and CIAV,StCV,SwCV,RaCV,PiCV,GuCV,etc.are in different branches.