The Natural Large Genomic Deletion Is Unrelated to the Increased Virulence of the Novel Genotype Fowl Adenovirus 4 Recently Emerged in China

Abstract

Since 2015, severe hydropericardium-hepatitis syndrome (HHS), caused by a highly pathogenic fowl adenovirus 4 (FAdV-4), emerged in China. In our previous study, the FAdV-4 has been identified as a novel genotype with a unique 1966-bp nucleotide deletion (1966Del) between open reading frame 42 and 43. In this study, the natural 1966Del was frequently identified among 17 clinical isolates and other reported Chinese clinical strains. To investigate the relationship between 1966Del and the increased virulence of the novel FAdV-4, a CRISPR/Cas9 operating platform for FAdV-4 was developed for the first time in this study. Based on this platform, a Re1966 strain was rescued, inserted the relative 1966Del sequence of a nonpathogenic strain KR5. In the pathogenicity study, the Re1966 strain retained high virulence for specific-pathogen-free chickens, similar to the parental wild-type HLJFAd15, although the survival time of chickens infected with Re1966 was much longer. Therefore, the natural 1966Del was identified as a non-essential site for the increased virulence of the emerged novel FAdV-4. Although further research on the virulence-determining region or point within the genome of the novel FAdV-4 is needed, the CRISPR/Cas9 operating platform for the novel FAdV-4 was developed and successfully applied to edit the genomic DNA for the first time, and it provides a novel powerful tool for both basic virology studies and vaccine vector development of FAdVs.

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

“…To make things more intriguing, the highly pathogenic FAdV-4 loses ORF19 due to a 1966 bp deletion. However, it has been reported that the deletion of ORF19 does not contribute to the viral virulence [29,30]. We are interested in whether the remaining unique ORF19A influences the replication of the novel FAdV-4.…”

“…This platform was further modified and improved by Eva Nagy and her colleagues through introducing phage recombinase [10]. Recently, CRISPR/Cas9 was employed as a tool to introduce double-stranded breaks at targeting site in FAdV genome to increase the percentage of positive recombinants in eukaryotic cells [29]. The homologous recombination in bacteria with phage recombinase and counter-selection technique were combined and used for modifying the FAdV-4 genome [30].…”

A Novel Role for PX, a Structural Protein of Fowl Adenovirus Serotype 4 (FAdV4), as an Apoptosis-Inducer in Leghorn Male Hepatocellular Cell

“…Thus PX-induced apoptosis contributed at least in part to FAdV4-induced apoptosis, which facilitates viral replication. Up to now, some researchers have established a reverse genetic system to generate recombinant FAdV4 [34] or used the CRISPR/cas9 system to delete or insert some nucleotide fragments directly [4]. Therefore, more efforts will be required to investigate the role of PX in the pathogenesis of FAdV4 infection.…”

“…Interestingly, the FAdV4 strains that recently emerged in China had the same deletion of 1966 bp in the genome compared with the non-virulent FAdV4 viruses (ON1 strain) [2]. However, the experimental evidence shows that the natural deficiency of 1966 bp in the viral genome was not essential for the increased virulence of the recently isolated FAdV4 in China [4]. Currently, the virulence-associated factors of FAdV4 responsible for clinical HHS are still unknown.…”

Genetic characterization of fowl aviadenovirus 4 isolates from Guangxi, China, during 2017–2019

“…However, similar to our findings, all of the viruses previously isolated from China had a similar deletion of nucleotides ( Liu et al., 2016 ). It was speculated that this deletion might have a role in the pathogenicity of these isolates, but recent studies using the clustered regularly interspaced short palindromic repeat ( CRISPR ) and CRISPR associated protein 9 ( CRISPR – Cas9 ) system found that this deletion was dispensable for the high pathogenicity of such isolates ( Pan et al., 2018 ). The 6-bp deletion in all 9 isolates at position 28,784, when compared with the ON1 genome, was also present in HNJZ, indicating this deletion was specific to Chinese isolates.…”