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This study focused on the construction of an infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus (DEV) strain 2085 and efficient generation of a vectored DEV vaccine expressing hemagglutinin (H5) of high pathogenicity H5N1 avian influenza virus (AIV) based on this infectious clone. Subsequently, the whole genome sequences of DEV strain 2085 were determined and compared with the whole genome of DEV VAC strain and sequences of DEV Clone-03 and CHv.
For construction of the DEV infectious clone, the mini-F vector sequences were inserted into the genome of DEV strain 2085 by homologous recombination in lieu of the UL44 (gC) gene. DNA of the resulting in recombinant virus v2085-GFPΔgC was electroporated into Escherichia coli strain Megax and subsequently strain GS1783, and a full-length DEV BAC clone (p2085) was recovered. Transfection of p2085 into chicken embryo cells resulted in DEV-specific plaques exhibiting green autofluorescence under UV excitation, indicating the successful generation of an infectious clone of DEV strain 2085. A gC-negative mutant, v2085ΔgC, was generated by deleting mini-F vector sequences by using Cre-Lox recombination, and a revertant virus v2085ΔgC-R was generated by co-transfection of p2085 with UL44 sequences produced through PCR. Finally, AIV H5 sequences were inserted into p2085 to generate a vectored DEV recombinant virus for expressing of H5, and high-level H5 expression of the v2085_H5 virus was detected by indirect immunofluorescence and western blotting. Plaque size determination showed that the sizes of v2085ΔgC plaques were significantly bigger (12%) over those of parental 2085 virus or the v2085ΔgC-R revertant virus (ANOVA, P<0.05), while plaque areas of v2085_H5 or v2085-GFPΔgC were significantly decreased. No significant difference was observed between parental or revertant DEV and mutant or recombinant DEV with respect to virus titers determined after trypsinization titration of infected cells, while virus titers of infectedcell supernatants revealed significant reductions in case of the gC-negative viruses of more than 700-fold when compared to parental 2085 or v2085ΔgC-R. Cell-associated virus titers of gCnegative DEV also showed significant reduction of 50-500-fold than parental 2085 or revertant DEV (ANOVA, P<0.05).
The nucleotide sequence was derived from the 2085 genome cloned as an infectious bacterial artificial chromosome (BAC) clone. The DEV 2085 genome is 160,649 bp in length and encodes 78 predicted open reading frames (ORFs)(GenBank ID: JF999965), a number identical to that of the attenuated DEV VAC strain (GenBank ID: EU082088.2). Comparison of the genome sequences DEV 2085 and VAC with partial sequences of the virulent CHv strain and the attenuated strain Clone-03 was carried out to identify nucleotide or amino acid polymorphisms that potentially contribute to DEV virulence. No amino acid changes were identified in 24 of the 78 ORFs, a result indicating high conservation in DEV independently of strain origin or virulence.
In addition, 39 ORFs showed only non-synonymous nucleotide substitutions. The remaining 15 ORFs had fragment insertion or deletions, frame-shift mutations or non-synonymous nucleotide substitutions with an effect on ORF initiation or termination. In 7 of the 15 ORFs with high and 27 of the 39 ORFs with low variability, polymorphisms were exclusively found in DEV 2085, a finding that likely is a result of a different origin of this strain (Europe) and the three other strains (Eastern Asia). Five ORFs (UL2, UL12, US10, UL47 and UL41) with polymorphisms were identical between the virulent DEV 2085 and CHv but different from VAC or Clone-03, and may, individually or in combination, represent DEV virulence factors.
We conclude that (1) absence of DEV gC results in increased plaque sizes in vitro, (2) gC plays a role in DEV egress, and (3) generation of an infectious DEV clone allows rapid generation of vectored vaccines, (4) DEV 2085 may represent an origin of Europe other than DEV VAC, Clone-03 and CHv of Eastern Asian, (5) UL2, UL12, US10, UL47 and UL41 may be related to virulence of DEV.