Evolution Analysis And Construction Of Infectious Clone Of Duck Circovirus Form Sichuan

Nowadays, DuCV is highly prevalent in the domestic duck population, and associated with considerable economic losses, with the main clinical symptoms including immunosuppression and stunting and feather abnormalities.Although controversial, lymphoid depletion predisposes the host to immunosuppression, and disease progression is further complicated by co-infections with other bacterial and viral pathogens. Therefore, it is of great scientific significance to investigate the genetic evolution of the DuCV and associated molecular mechanisms with its pathogenicity.1 Identification, genotyping analysis of duck circovirus In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8-99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparisons analyses (PASC) indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b,1c,2a,2b and 2c) based on the p-value of complete genome sequences. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution.2 molecular evolution analysis of duck circovirus Phylogenetic analyses indicated three isolates (GenBank No. EU344802, EU344805 and EU499309) were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-la and DuCV-2b isolates within the rep genes from 71nt to 581nt, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated purifying selection had been a major driving force in maintaining diversity among the DuCV.3. Construction and Virus Rescue of an Infectious Clone of Duck Circovirus To generate a genetic marker strain of DuCV for further understanding the pathogenesis and gene function of duck circovirus. In this study, the full-length genome of the GH01 isolate was amplified using PCR, and two copies of the genome, IC-1 and IC-2, were ligated in tandem into the pUC19 vector to construct an infectious molecular clone pIC-2DuCV, and a XhoI restriction enzyme site was inserted into the clone as a genetic marker. The linearized infectious DNA clone pIC-2DuCV was injected into 10 day old DuCV-negative ducklings though intramuscular and intravenous injection with lipofectamine. As a result, an infectious molecular clone pIC-2DuCV containing an engineered XhoI site that served as a genetic marker was successful obtained, and sera samples of ducklings with intramuscular and intravenous injection were detected positive at 10DPI, and distinguished the viral genome from the wild-type parent with the XhoI restriction enzyme site by sequencing. The results show that infectious DNA clone was infectious in ducklings, and was able to generate infectious DuCV particles with genetic marker.4. Characterization of the rescued virus in vivo In order to determine the rescue virus (designated PMDC) is usefull, we analyzed the pathogenicity of the PMDC together with the parental virus GH01 in DuCV-negtive ducklings. The ADWG of PBS group is significant higher than the PMDC and WT-DuCV groups, and the temperature is stable between 41.7℃ and 42.2℃; the clinical value of PMDC, WT-DuCV, PBS showed 12.5, 15.6 and 0; moreover, the viremia occur at 10 and 15 DPC of WT-DuCV and PMDC groups, and the antibodies could be detected at 15 and 21 DPC. The results showed that stunting and feather abnormalities occurred both in the rescued viruses and parental virus in the inoculated ducklings, it means that rescued viruses and parental virus showed no differences in pathogeniticity. The proliferation ability analysis showed that the titers of rescued viruses was lower than those of their parental viruses. This study showed that the rescued viruses PMDC have no significantly differences but lowerd in pathogeniticity and proliferation ability with parental virus.