Establishment Of Rapid Identification And Detection Methods For Four Important Pathogens In Ducks And Molecular Epidemiology Of Duck Tembusu Virus And Duck Circovirus In Guangxi

The duck Tembusu virus（DTMUV）,duck circovirus（DuCV）,Muscovy duck reovirus（MDRV）,and novel duck reovirus（NDRV）are common pathogens in Chinese poultry farming.In recent years,DTMUV,DuCV,MDRV,and NDRV have become more prevalent due to the dramatic increase in farming scale,poor feeding management,and pathogenic variation,especially the incidence of DTMUV and DuCV in China has reached more than 81.6%,making the development of poultry farming industry suffer a heavy blow.The similarity of some diseases and the fact that DuCV,MDRV,and NDRV belong to immunosuppressive viruses,they are easy to cause mixed infections with other viruses and bacteria clinically,which increases the difficulty of identification.To this end,a rapid identification assay for the four viruses was developed,and molecular epidemiological studies were conducted on DTMUV and DuCV.To provide basic data for epidemic prevention and control and molecular characterization of the pathogen.1.Establishment and Application of Multiplex q RT-PCR Assay for DuCV,DTMUV,MDRV,and NDRVFour pairs of specific primers and probes were designed for the Rep,C,S3,and S3 genes of DuCV,DTMUV,MDRV,and NDRV,respectively.By optimizing the annealing temperature,primer,and probe concentrations,a multiplex q RT-PCR assay was developed to simultaneously detect the four pathogens.It was specific for the DuCV,DTMUV,MDRV,and NDRV only,and the lower limit of detection was 1.51×10¹ copies/μL with intra-and inter-assay coefficients of variation less than 1.54%.For the 404 clinical samples tested,the positive rates of DuCV,DTMUV,MDRV,and NDRV were26.0%,9.9%,4.0%,and 4.7%,respectively,and the mixed infection rates of DuCV and DTMUV,DuCV and MDRV,DuCV and NDRV,MDRV and NDRV,DTMUV and MDRV,and DTMUV and NDRV were 2.7%,1.2%,1.2%,1.0%,0.5%,and 0.7%,respectively,providing a technical means for rapid identification and detection of DuCV,DTMUV,MDRV,and NDRV.2.Establishment and Application of Multiplex d PCR Assay for DTMUV,DuCV,and NDRVTo detect DTMUV,DuCV,and NDRV simultaneously,three pairs of specific primers and probes were designed for their C,Rep,and S3 genes,respectively,and the reaction conditions were optimized to develop a multiplex digital PCR（d PCR）.It can specifically detect DTMUV,DuCV,and NDRV with a minimum detection limit of 1.3 copies/μL,which is 10 times higher than multiplex q PCR,and the intra-and inter-assay coefficients of variation are between 0.06%～1.94%.173 clinical samples were detected,and the positive rates of DTMUV,DuCV,and NDRV were 18.5%,29.5%,and 14.5%,respectively,which were about 4%higher than the multiplex q PCR.The Kappa values of the clinical test results of multiplex d PCR and q PCR were0.85,0.89,and 0.86,respectively,indicating that the two methods have a good agreement and provide a new tool for pathogen detection.3.Genetic Diversity Analysis of Duck Tembusu Virus in Guangxi During 2019 to 2022To study the molecular genetic characteristics of DTMUV,duck tissue samples were collected in Guangxi from 2019 to 2022,and some positive strains were selected for amplification analysis.A total of 10 DTMUV strains with a complete genome length of 10992 bp were obtained.The similarity of ORF,E,and NS5 genes for the nucleotide and amino acid were86.4%-99.8%and 96.0%-99.8%,85.8%-99.5%and 94.8%-100%,87.0%-99.9%and 97.4%-99.9%between Guangxi and the reference strains.E protein analysis showed that there were 14 amino acid sites mutated,among which positions 43,108,113,150,153,326,403,464,487,and 501 were unique for Guangxi the strain.The results of the ORF phylogenetic tree showed that the Guangxi strains were distributed in subgroup 2.1,while the Guangxi-originated GX2011 and GX2015 strains belonged to subgroup 2.2,indicating that the evolutionary trends of DTMUV were not completely consistent and the recombination signals were detected in some Guangxi strains.The estimated rates of the genetic evolution of ORF,E,and NS5 genes were 8.77×10^-4,1.43×10^-3,and 1.30×10^-3substitutions per site per year,respectively.To provide basic data for understanding the prevalence of DTMUV and developing effective prevention and control measures.4.Molecular Epidemiology of Duck Circovirus in Guangxi During 2019 to 2022To understand the genetic variation of DuCV,54 positive strains were selected for PCR amplification and analysis among 761 clinical samples collected from 2019 to 2022 in Guangxi.The DuCV positivity rate was 22.5%by testing,and the genome size was from 1988 to 1996 bp.The nucleotide similarities of complete genome,Rep,Cap,and ORF3 genes between the Guangxi DuCV and the reference strain were 71.9%-99.6%,83.2%-99.9%,52.8%-99.9%,and 86.4%-100%,and the recombination existed between DuCV-GX13-2019,DuCV-GX14-2020,DuCV-GX47-2022 and other strains.Genetic evolutionary analysis showed that the Guangxi strains were DuCV-1and DuCV-2 genotypes,with DuCV-1 being the main dominant strain and DuCV-1b being the most widely prevalent.To provide basic data for mastering the epidemic and control of DuCV in Guangxi.In summary,this study successfully developed the identification and detection technology regarding DuCV,DTMUV,MDRV,and NDRV,and mastered the molecular epidemiological characteristics of DTMUV and DuCV Guangxi strains.