No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells

Liu, Xinglong; Zou, Xiaohui; Zhang, Wenfeng; Guo, Xiaojuan; Wang, Min; Lv, Yingtao; Hung, Tao; Zz, Lu

doi:10.1128/spectrum.00470-22

Cited by 3 publications

(6 citation statements)

References 51 publications

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“…The strong activity of the 37,061-nt promoter had been revealed by expressing reporter genes in our previous work [ 48 ]. This promoter mainly produced transcripts for GAM1, ORF19A-e865aa and ORF19A-t336aa ( Figure 7 and Figure S11 ).…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data

Wang

Zou

et al. 2023

Viruses

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The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.

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Section: Resultsmentioning

confidence: 99%

“…Wet-lab experiments provided evidence to support the PCR-cDNA results. In a previous study, we found two regions in the FAdV-4 genome that possessed promoter activity [ 48 ]. By determining the expression of reporter genes, we quantified their strength.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data

Wang

Zou

et al. 2023

Viruses

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“…The genome of FAdV is about 8–10 kb longer than that of HAdV. The regions encoding genus-specific genes are approximately 7 kb on the left and 10 kb on the right in FAdV-4 genome, and no genus-specific gene is essential for the virus replication [ 20 ]. In F2CF1K-CG or FAdV4-CG, about 4.7 kb of virus genome has already been deleted.…”

Section: Discussionmentioning

confidence: 99%

“…HAdV41-CG originated from human adenovirus 41, and the E1 and E3 regions in the genome were deleted and replaced with CMV promoter (CMVp) controlled GFP expression cassette and HAdV-5 ADP gene, respectively [ 26 ]. pKFAV4S-GFP is an adenovirus plasmid, and it carries deletions of ORF0, ORF1, ORF1B, ORF2 and ORF19A in FAdV-4 genome; and at the left deletion site in the genome, a CMVp controlled GFP expression cassette was inserted, which was flanked with two SwaI sites to facilitate transgene replacement [ 20 ]. pMD-FAV4Fs is an intermediate plasmid for FAdV-4 fiber modification, which contains fiber1 and fiber2 genes of FAdV-4 [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

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CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors

Sun

Zou

Guo

et al. 2022

Genes

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Fowl adenovirus 4 (FAdV-4) has the potential to be constructed as a gene transfer vector for human gene therapy or vaccine development to avoid the pre-existing immunity to human adenoviruses. To enhance the transduction of FAdV-4 to human cells, CELO fiber1 knob (CF1K) was chosen to replace the fiber2 knob in FAdV-4 to generate recombinant virus F2CF1K-CG. The original FAdV4-CG virus transduced 4% human 293 or 1% HEp-2 cells at the multiplicity of infection of 1000 viral particles per cell. In contrast, F2CF1K-CG could transduce 98% 293 or 60% HEp-2 cells under the same conditions. Prokaryotically expressed CF1K protein blocked 50% transduction of F2CF1K-CG to 293 cells at a concentration of 1.3 µg/mL while it only slightly inhibited the infection of human adenovirus 5 (HAdV-5), suggesting CF1K could bind to human cells in a manner different from HAdV-5 fiber. The incorporation of CF1K had no negative effect on the growth of FAdV-4 in the packaging cells. In addition, CF1K-pseudotyped HAdV-41 could transduce HEp-2 and A549 cells more efficiently. These data indicated that CF1K had the priority to be considered when there is a need to modify adenovirus tropism.

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Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody

Wang,

Zou,

Guo

et al. 2024

Genes

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The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

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No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells

Cited by 3 publications

References 51 publications

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data

CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody

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