Molecular Epidemiological Investigation And Establishment Of Diagnostic Technique On Pigeon Circovirus

The Pigeon circovirus(PiCV)belongs to the genus Circovirus and the family Circoviridae.PiCV contains two main open reading frames(ORFs),which encode capsid protein(Cap)and replication-associated protein(Rep).PiCV alone is not capable of inducing symptoms of disease in pigeons,but it can cause severe immunosuppression which results in insufficient immune responses to various pathogens such as viruses,bacteria,fungi,and parasites,thus causing subsequent infection,and showing corresponding clinical symptoms.The mortality rate caused by subsequent infection can reach 100%,which has brought a huge threat to the healthy breeding of pigeons in our country.In recent years,the competitions of racing pigeons are becoming increasingly popular in China.However,the research on the diseases of racing pigeons is insufficient,especially the prevalence and genetic evolution of PiCV in Chinese racing pigeons.In order to grasp this information,this thesis aims to conduct research on PiCV molecular epidemiology investigation,whole genome cloning,sequencing,and genetic variation.Moreover,a visual LAMP detection method has also been established.The specific research contents are as follows:1.Epidemiological investigation of PiCV infection in Chinese racing pigeons from several provincesThe aim of this part is to investigate the PiCV infection and prevalence in racing pigeons in some regions of China.Between 2016 and 2019,a total of 571 serum samples collected from healthy pigeons and 51 tissue samples collected from sick pigeons(including reduced racing performance,depression,respiratory problems,vomiting,diarrhea,untidy feathers,neurological symptoms,or even sudden death)were obtained from 39 racing pigeon clubs in11 provinces or municipalities of China,such as Beijing,Shaanxi,and Hebei.The PCR method established in this experiment was used for pathogen detection.The results showed that the positive rates of PiCV in healthy and sick pigeons were 18.6% and 27.4%,respectively,and the total positive rate was 19.3%.In addition,the positive rate of each province was also analyzed.Importantly,the inapparent infection in healthy racing pigeons might become a major hidden danger in the healthy breeding of racing pigeons,which should be paid enough attention.This part of research provides the novel epidemiologic data for PiCV strains among racing pigeons in China.2.Sequence analysis for the full genome of PiCVIn this study,67 full-genome sequences of PiCV were obtained.The results showed that the genomes of PiCV in racing pigeons were highly diverse through comparing with the reference sequences published by NCBI.The highly diverse mainly reflected in:(1)The sequence homology among the epidemic strains identified in this study was lower than that of domestic and foreign epidemic strains.(2)A positive selection site and many new amino acid mutations were found at the C-and N-terminus of the Cap protein.(3)The ATT and GTG were highly suspected to be the start codons of the Cap protein,which might cause different efficiencies in initiating translation of Cap protein.The work in this part is contribute to our understanding of the variations of the virus.It also provides reference data for the development of subunit vaccines base on the Cap protein in future.3.Recombinant and genetic evolution analysis of PiCV strains from Chinese racing pigeonsGen Bank database and the full genome sequences of PiCV identified in this study were used to analyze the recombination events using RDP4,Simplot,and MEGA5 software.The results showed that 10 recombination events related to the epidemic strains of the Chinese racing pigeons were identified,including 23 recombinant strains,which accounted for 34.3%of the epidemic strains;Their parent strains might come from Chinese meat pigeons,Polish or Japanese racing pigeons,or feral pigeons from the United States;Genetic evolution analysis showed that all PiCV strains could be divided into 8 groups.The epidemic strains from Chinese racing pigeons were distributed in 7 groups.Moreover,it appeared that the endemic strains of the same loft were distributed in different genetic branches.This part indicated that viral recombination had a significant role in the evolution of PiCV,and it also reflected that the complexity of the genetic evolutionary relationship of the epidemic strains among Chinese racing pigeons,which indicates that the prevention and control of this virus is difficult;Furthermore,it also suggests that the breeding of racing pigeons in our country should pay more attention to biosafety work at the time of pigeon racing and introduction to prevent the spread of the virus among different countries.4.Establishment of visual LAMP detection methodThe sequences obtained in this study were compared with all reference sequences.The conserved region was selected for LAMP primer design.Moreover,in order to reduce the pollution of aerosol during detection,Calcein and manganese chloride were added into the LAMP reaction system in this study to establish a visual LAMP detection method.The result can be identified according to liquid color after reaction: bright green is positive,and orange yellow is negative.The limitation of this method can reach 10 copies/μL.The method has high sensitivity and good reproducibility,and no cross reaction is found with other common diseases.This study laid a foundation for the development of a rapid detection kit for PiCV.This study demonstrates the prevalence of PiCV in racing pigeons in China,the sequence information,the recombination events,and the genetic evolution of the PiCV strains.This study provided reference data for the prevention of PiCV and the development of subunit vaccine,and also contributed to the further study of this virus in the future.In addition,the visual LAMP detection method established in this study can provide technical support for the prevention and early diagnosis of PiCV.