Abstract
Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.
Highlights
Duck Tembusu virus (DTMUV), family Flaviviridae, genus Flavivirus, is a newly emerging pathogen related to significant egg drop syndrome in laying ducks since 2010 [1]
The virus was passaged in chicken embryoblasts (CEFs) for 20 times to evaluate the genetic stability of the purified vBAC-C-KCE (Figure S1)
The E gene of DTMUV was inserted into the C-KCE genome based on the mating-assisted genetically integrated cloning (MAGIC) strategy
Summary
Duck Tembusu virus (DTMUV), family Flaviviridae, genus Flavivirus, is a newly emerging pathogen related to significant egg drop syndrome in laying ducks since 2010 [1]. The ORF encodes seven non-structural (NS) proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). Among these proteins, the E protein is the principal antigenic determinant that serves important functions in receptor binding and membrane fusion [3]. Not I site (bold), I-isce I sequence (underline). I-isce I sequence (bold), Homology arm H2 (underline). Primers used to the clone the E gene of DTMUV; b. Fragments from human elongation factor 1a (hEF1a) promoter to BGH ploy A were amplified from vector pEF6-v5/his with the primers, pCA-I-SceI-H1-F/pCA-I-SceI-H2-R, flanked by 50-bp homology arms and I-sceI restriction sites. An RT-PCR fragment encoding the E gene was amplified using primers E1-F/E1-R (Table 1). The PCR product was digested with BamH I and EcoR I and ligated to pRThGA, resulting in pRThGA-E
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