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Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Recently, the disease of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) has spread widely and resulted in huge economic loss to poultry industry. Although the genome of FAdV-4 has two fiber genes (Fiber-1 and Fiber-2), the exact role of the genes in the infection of FAdV-4 is barely known. In this study, through superinfection resistant analysis and interfering assay, we found that Fiber-1, but not Fiber-2, was the key factor for directly triggering the infection of FAdV-4. The truncation analysis further revealed that both of the shaft and knob domains of Fiber-1 were required for the infection. Moreover, the sera against the knob domain could block FAdV-4 infection, and the knob-containing fusion protein could provide efficient protection against the lethal challenge of FAdV-4 in chickens. All the data demonstrated the significant roles of Fiber-1 and its knob domain in directly mediating the infection of FAdV-4, which established a foundation for identifying the receptor of FAdV-4 and developing efficient vaccines against FAdV-4. Importance: Among 12 serotypes of FAdV, FAdV-1, FAdV-4 and FAdV-10 all carry two Fiber genes (i.e., Fiber-1 and Fiber-2) whereas other serotypes have only one. As important viral surface proteins, the Fibers play a vital role in the infection and pathogenesis of FAdV. However, the importance of the Fibers to the infection and pathogenesis of FAdV may be different from each other. Recent studies reveal that Fiber-2 is identified as a determinant of virulence, but which Fiber triggers the infection of FAdV-4 almost remains unknown In this study, Fiber-1 was identified as a key factor for directly mediating the infection of FAdV-4 through its shaft and knob domains, whereas Fiber-2 did not play roles in triggering FAdV-4 infection. The results suggest that Fiber-1 and its knob domain may serve a target for identifying the receptor of FAdV-4 and developing efficient drugs or vaccines against FAdV-4.

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) have caused a huge economic loss to the poultry industry globally since 2013. Although the Fiber-2 has been identified as a key virulent related factor for FAdV-4, little is known about its molecular basis. In this study, we identified the efficient interaction of the Fiber-2 with the karyopherin alpha 3/4 (KPNA3/4) protein via its N-terminus of 1–40aa. The analysis of the overexpression and knockout of KPNA3/4 showed that KPNA3/4 could efficiently assist the replication of FAdV-4. Moreover, a fiber-2 -edited virus FAV-4_Del with a deletion of 7–40aa in Fiber-2 was rescued through the CRISPR-Cas9 technique. In comparison with the wild type FAdV-4, FAV-4_Del was highly attenuated in vitro and in vivo . Notably, the inoculation of FAV-4_Del in chickens could provide full protection against the lethal challenge with the wild type FAdV-4. All these findings not only give novel insights into the molecular basis for the pathogenesis of Fiber-2 but also provide efficient targets for developing antiviral strategies and live-attenuated vaccine candidates against the highly pathogenic FAdV-4.

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