User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome

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.

Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

“…(E) Procedure for modification of FAdV‐4 fiber1 knob. The genome of FAdV‐4 in pKFAV4GFP originated from FAdV‐4 isolate NIVD2 (GenBank MG547384) 32 …”

“…(E) Procedure for modification of FAdV-4 fiber1 knob. The genome of FAdV-4 in pKFAV4GFP originated from FAdV-4 isolate NIVD2 (GenBank MG547384) 32 as two factors, and Dunnett's multiple comparisons test was performed to compare the difference of mean values between virus types at each MOI. p < 0.05 was considered statistically significant.…”

“…ORF19A coding sequence (CDS) was deleted and GFP CDS was replaced with that of mCherry in pKFAV4GFP to generate pKFAV4‐CX19A. 32 pMD‐FAV4Fs, a shuttle plasmid for FAdV‐4 fiber modification, carried MauBI‐SbfI fragment and short sequences flanking this region in the FAdV‐4 genome. 31 pFiber5‐35 carried the sequence encoding the fiber knob of HAdV‐35 and was used as the template for polymerase chain reaction (PCR) cloning of HAdV‐35 fiber knob.…”

“…The intermediate plasmid‐based system was employed for replacing the CMV promoter with human EF1a promoter to generate adenoviral plasmid pKFAV4‐EG. 32 pKFAV4‐EG was a progeny plasmid of pKFAV4‐CX19A and different from pKFAV4‐GFP in ORF0 and ORF19A deletion and the replacement of GFP promoter (the detailed procedure will be provided upon request). F1IJR‐F2 fragment was amplified by PCR with pKFAV4F1IJR‐GFP as the template and primers 1811MD‐FAV4FSf/r and inserted at the Mau BI/ Sbf I sites in pKFAV4‐EG to generate pKFAV4F1IJR‐EG by DNA assembly.…”

“…The seed virus was amplified in LMH cells, purified with the traditional ultracentrifugation method except that 10 m m citrate (pH 6.2) instead of 10 m m Tris‐Cl (pH 7.6) was used as the buffer solution. 32 Particle titer of purified virus was determined by measuring the content of genomic DNA, where 100 ng of genomic DNA is equivalent to 2.3 × 10 9 viral particles (vp) because a 43‐kb genome has a molecular mass of 2.6 × 10 7 . Infectivity titer was determined using LMH cells with the limiting dilution assay by counting GFP+ cells 30 hours post infection.…”