FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate

Abstract

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

Keywords: CRISPR/Cas9; FAdV-4; deletion of fiber-2; pathogenesis; protection; virus rescuing.

FIG 1

Generation and identification of the recombinant virus FAdV4-EGFP-rF2. (A) Strategy of the homology-dependent recombinant for generating FAdV4-EGFP-rF2 using a CRISPR/Cas 9 system. LMH cells were first transfected with sgRNA1 and sgRNA2. At 24 hpt (hours posttransfection), the LMH cells were infected with FAdV-4 and transfected with donor plasmid. The rescued FAdV4-EGFP-rF2 was then purified by limiting dilution assay. (B) PCR identification of FAdV4-EGFP-rF2. The DNA of unpurified FAdV4-EGFP-rF2 (lane 2), purified FAdV4-EGFP-rF2 (lane 3), and wild-type (WT) FAdV-4 (lane 4) was extracted and detected by PCR using specific primers. Lane 1 shows the DNA marker and lane 5 shows the negative control. (C) LMH cells infected with purified FAdV4-EGFP-rF2 and wild-type FAdV-4, or transfected with pcDNA3.1-Fiber-2 and pcDNA3.1-EGFP, respectively, were harvested at the indicated time points and lysed; the lysates were then detected by monoclonal antibodies against Fiber-2, Hexon, GFP, and GAPDH.

FIG 2

FAdV4-EGFP-rF2 replicated inefficiently in LMH, but efficiently in LMH-F2. (A) LMH cells were infected with FAdV4-EGFP-rF2 and FAdV-4 at an MOI of 0.1, respectively; viral supernatants collected from infected LMH cells at the indicated time points were then titrated by TCID₅₀. (B) LMH and LMH-F2 cells were infected with FAdV4-EGFP-rF2 at an MOI of 0.1, respectively, and the viral supernatants collected from infected cells at the indicated time points were then titrated by TCID₅₀. All tests were done in triplicate and repeated twice. Statistical analysis in this experiment was performed with a Student’s t test using GraphPad 5 software.

FIG 3

FAdV4-EGFP-rF2 without fiber-2 was highly attenuated in vivo. A total of 160 2-week-old SPF chickens were randomly divided into 4 groups (n = 40). Chickens in group I were inoculated with WT FAdV-4, while those in groups II and III were inoculated with FA4-EGFP and FAdV4-EGFP-rF2, respectively. All chickens were intramuscularly inoculated with 2.5 × 10⁴ TCID₅₀ of indicated virus in 200 μL of culture medium containing 1% FBS, and those in group IV, inoculated with the same volume of culture medium containing 1% FBS, were set as a negative control. Morbidity and mortality were recorded daily. Cloacal swabs and organ tissues from each group were collected at indicated time points for viral titration. (A) Percent survival for infected chickens. (B) Representative histological changes in liver tissues from chickens inoculated with FAdV4-EGFP-rF2 (yellow), FA4-EGFP (green), wild-type FAdV-4 (red), and negative control (black). (C) Viral shedding in the infected chickens. Viral loads in liver (D), spleen (E), and kidney (F) from the infected chickens. Statistical analysis in this experiment was performed with one-way ANOVA test using GraphPad 5 software.

FIG 4

Fiber-2 did not contribute to the induction of neutralizing antibody. Chicken sera were collected from chickens inoculated with FA4-EGFP (group II) and FAdV4-EGFP-rF2 (group III), respectively, at the indicated time points, and tested for titers of neutralizing antibodies. Panels A, B, and C show NT titers at 7, 14, and 21 dpi, respectively.

FIG 5

FAdV4-EGFP-rF2 provided efficient protection against FAdV-4 challenge. The survival chickens inoculated with FAdV4-EGFP-rF2, FA4-EGFP, and culture medium containing 1% FBS were challenged with a lethal dose of FAdV-4 at 21 dpi. The morbidity and mortality were recorded daily. Cloacal swabs and organ tissues from each group were collected at the indicated time points for viral titration. (A) Percent survival of the challenged chickens. (B) Representative gross lesions in heart and liver from negative-control chickens (top) and chickens previously inoculated with FAdV4-EGFP-rF2 (bottom) after challenge. (C) Viral shedding in cloacal swabs from challenged chickens. Viral loads in liver (D), spleen (E), and kidney (F) tissues from challenged chickens. Statistical analysis in this experiment was performed with a one-way ANOVA test using GraphPad 5 software.