Abstract
A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.
Highlights
Fowl adenoviruses (FAdV) belong to the family of Adenoviridae and the genus of Aviadenovirus [1].Currently 12 types of FAdV have been identified (FAdV-1 to -8a and -8b to 11), which are further classified into five species (FAdV-A to -E) [2]
FAdVs are believed to be the pathogens causing adenoviral gizzard erosion (AGE), inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) in chickens, the outcome of anFAdV infection depends on many factors including
Plasmid pKFAV4 was constructed in the laboratory previously [13], which contained the complete genome of the novel fowl adenovirus 4 (FAdV-4) (Genbank accession No MG547384). pAd5GFP was an E1/E3-deleted HAdV-5-based adenoviral plasmid constructed previously in the laboratory [14], which contained an Enhanced Green Fluorescent Protein (GFP) expression cassette controlled by the human cytomegalovirus (CMV) promoter and SV40
Summary
Fowl adenoviruses (FAdV) belong to the family of Adenoviridae and the genus of Aviadenovirus [1]. Genus-common genes are essential for virus replication in vivo or in vitro cell culture. The function of these genes should be conserved among the family and be analogous to that of Mastadenovirus. The reverse genetics system is a valuable approach to study viral genes of FAdV, and some systems have been established and used to construct gene transfer vectors and vectored vaccines [9,10]. Most of these systems work through homologous recombination, which needs the activity of intracellar recombinase. We characterized some genus-specific genes of FAdV-4 by using this system
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