Abstract
BackgroundLethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. However, little is known about the function of viral genes including the conserved UL55 gene among alpha herpes virus due to the obstacles in maintenance and manipulation of DEV genome in host cells.MethodsIn this paper, we constructed an infectious bacteria artificial chromosome (BAC) clone of the lethal clinical isolate duck enteritis virus Chinese virulent strain (DEV CHv) by inserting a transfer vector containing BAC mini-F sequence and selection marker EGFP into UL23 gene using homologous recombination. UL55 deletion and its revertant mutant were generated by two-step RED recombination in E. coli on basis of rescued recombinant virus. The function of UL55 gene in DEV replication and its effect on distribution of UL26.5 protein were carried out by growth characteristics and co-localization analysis.ResultsThe complete genome of DEV CHv can be stably maintained in E. coli as a BAC clone and reconstituted again in DEF cells. The generated UL55 deletion mutant based on DEV CHv-BAC-G displayed similar growth curves, plaque morphology and virus titer of its parental virus in infected Duck Embryo Fibroblast (DEF) cells. Immunofluorescence assay indicated that the loss of UL55 gene do not affect the distribution of UL26.5 protein in intracellular. These data also suggest infectious BAC clone of DEV CHv will facilitate the gene function studies of DEV genome.ConclusionsWe have successfully developed an infectious BAC clone of lethal clinical isolate DEV CHv for the first time. The generated UL55 gene mutant based on that demonstrated this platform would be a very useful tool for functional study of DEV genes. We found the least known DEV UL55 is dispensable for virus replication and UL26.5 distribution, and it could be a very promise candidate locus for developing bivalent vaccine. Experiment are now in progress for testifying the possibility of UL55 gene locus as an exogenous gene insertion site for developing DEV vectored vaccine.
Highlights
Lethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes
Growth characteristics of DEV Chinese virulent strain (CHv)-bacteria artificial chromosome (BAC)-GΔUL55 and duck enteritis virus Chinese virulent strain (DEV CHv)-BAC-GΔUL55R were compared to their parental virus DEV CHv-BAC-G as we previously described to determine the function of UL55 in DEV replication
PCR was performed subsequently to confirm the purification of BAC-recombinant virus. repA, sopB gene in BAC plasmid and selection marker enhanced green fluorescent protein (EGFP) gene can be amplified while TK gene is not presented in the genome of BAC-recombinant virus as expected
Summary
Lethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. Duck Viral Enteritis (DVE), known as Duck Plague (DP), is an acute, febrile, septic and contagious disease of ducks, geese, swans and many other species of birds within the order Anseriformes caused by Duck Enteritis Virus (DEV) [1]. After primary infection, the viruses persist in their host for life. They hide from the immune system in a latent state in which their genome is almost dormant. Even live attenuated DEV vaccines have been used in prevention and control of this lethal disease since 1960s, the DEV infection is not completely prevented [6,7,8,9,10]. Investigation of DEV gene functions and pathogenesis will be an intelligent policy to novel vaccine development and disease controlling
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