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.

Keywords: CRISPR/Cas9; genomic deletion; natural deletion; novel FAdV-4; virulence.

Figure 1

Investigation of the prevalence of the natural large genomic deletion in the novel genotype FAdV-4 that recently emerged in China. (A) Phylogenetic analysis of FAdV clinical isolates based on the sequence of hexon-L1. Seventeen clinical isolates were characterized as FAdV-4: HLJFAd15 (KU991797) and HLJDAd15 (KX538980) were isolated from chickens and ducks from Heilongjiang Province; JLFAd15, JLDH-1, JLDH-2 were isolated from chickens from Jilin Province; and HNNL15, HNQX15, HNSQ15, HNTK15, HNYZ15, HNDF15, HNZK15, HNZM15, HNLY15, HNDC15, HNLH15-1, and HNLH15-2 were isolated from chickens from Henan Province. (B) Partial genome organization from pVIII to GAM-1 of FAdV-4 isolates. The natural 1966 bp deletion mutant, including the lipase reading frame, presented between ORF42 and ORF43 in 17 clinical, highly pathogenic isolates and another two reported Chinese clinical strains, JSJ13 (KU991797) and HB1510 (KU587519), but did not appear in KR5 (HE608152) and ON1 (GU188428) isolated outside of China.

Figure 2

Strategy of the CRISPR/Cas9 platform for the novel genotype FAdV-4. Co-transfection with purified HLJFAd15 genome DNA, ReA-EGFP/1966Del-ReB sequences containing the recombinant arms, and pX330-sgRNA into LMH cells were used to rescue recombinant viruses. The ReEGFP strain was rescued first for screening of the sgRNA with the highest efficiency in targeting the site where the deletion occurs. Then, the Re1966 strain containing the 1966Del sequence of KR5 (HE608152) was rescued.

Figure 3

The cleavage ratio of variant sgRNAs targeting the site of the deletion. (A) Purified virus particles obtained by CsCl2 gradient centrifugation. Scale bar= 100 nm. (B) The viral genome DNA was of high quality and purity, with a molecular size of approximately 43 kb. (C) Cleavage ratios were detected by calculating the percentage of GFP-positive clones among the total virus clones. sgRNA1-4 showed efficient cleavage effects for editing FAdV-4 DNA (>80%). The sgRNA2 showed a cleavage ratio of 98.3 ± 1.6% and was used for the subsequent targeting.

Figure 4

Stability and growth properties of the rescued viruses. Recombinant viruses ReEGFP/Re1966 with insertion of a EGFP expression cassette or the natural deletion 1966 bp sequence were both rescued by the CRISPR/Cas9 platform. (A) The ReEGFP strain was able to continuously express GFP for at least 10 passages. Scale bar= 200 μm. (B) The 1966Del insertion was also detected in the rescued Re1966 virus by PCR. A 658 bp sequence was amplified from the wild-type strain (Lane 2), and a 2624 bp sequence with the 1966Del insertion was detected in the Re1966 strain (Lane 1). (C) There was no significant difference in any of the growth properties in vitro among the 3 rescued viruses: ReEGFP, Re1966, and wild-type.

Figure 5

Analyses of the pathogenicity of the rescued Re1966 for SPF chickens. All of the SPF chickens were observed for 10 dpi. The chickens in the wild type and Re1966 strain groups died within 2 to 5 dpi (A), and the mortalities of both the groups were 100% (B). The survival time of chickens infected with Re1966 was greatly prolonged relative to that of the birds infected with the wild-type strain. (C) In the necropsy examination, all of the dead chickens in both infected groups showed severe hydropericardium syndrome, hepatitis (swollen and friable liver), proventriculus erosion, and kidney enlargement kidney, and there was no significant difference in these features between the two infected groups. All of the SPF chickens in the negative control group remained alive and did not show any clinical signs.

Figure 6

Histopathology and virus loads in tissues of chickens inoculated with the rescued viruses. (A) Histopathology assays of the major targeted tissues by FAdV-4 infection. Generally, there was no significant histopathology presented in tissues of chickens in the control group. However, degeneration, vacuolar necrosis, and basophilic inclusion bodies presented in liver cells, large numbers of vacuolar necrosis showed in kidney cells, and partial lymphocyte necrosis appeared in the lamina propria of the proventriculus mucous membrane in the chickens inoculated with viruses. There was no significant difference in these features between chickens inoculated with the wild-type strain and those inoculated with the Re1966 strain. (B) Virus loads in different tissues of chickens inoculated with FAdV-4. The Re1966 strain showed the same distribution and replication kinetics as the wild-type strain in the different tissues of SPF chickens except for a higher number of virus copies in spleen (* p < 0.05).