Deletion of the N-terminal 400 amino acids of DPV pUL36 attenuated virulence and conferred effective protection against virulent challenge

Figures

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  Fig. 1. Construction of recombinant viruses and assessment of the replication characteristics of the in vitro mutant strain Δ400. (A) Amino acid sequence alignment of DPV pUL36 and its homologous proteins. (B) Schematic diagram of the construction of recombinant viruses Δ400 and Δ400-R. (C, E) TCID50 measurements of different strains. (D, F) Genomic copy number measurements of different strains. (* P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant).

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  Fig. 2. In vivo challenge experiment of the Δ400 mutant strain. Ducklings were inoculated with Δ400, Δ400-R, and the wild-type strain at doses of 105 TCID50, 106 TCID50, and 107 TCID50, respectively; their body temperature, body weight, and survival rate were monitored for 10 days post-inoculation.

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  Fig. 3. Detection of viral load of the Δ400 mutant strain. On the 3rd and 5th days post-infection, the copy numbers of the DPV UL30 gene in the brain, spleen, and duodenum of ducklings inoculated with a viral dose of 107 TCID50 were determined. (** P < 0.01, **** P < 0.0001).

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  Fig. 4. Histopathological characteristics of tissues infected with the Δ400 mutant. Duodenum, spleen, and brain of a duck inoculated with a viral dose of 107 TCID50 were collected on the 5th day post-infection for histopathological observation.

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  Fig. 5. Immunological evaluation of mutant strain Δ400. (A) Ducklings were immunized with the deletion strain at varying TCID50 doses, and neutralizing antibody titers were determined on the 7th and 14th days post-immunization. (B) Experimental flow chart for investigating the immune protection efficacy of mutant strain Δ400: 14-day-old ducklings were immunized with Δ400 at a dose of 105 TCID50, followed by a challenge infection with 100 LD50 of a virulent DPV strain 14 days post-immunization. Subsequently, the body temperature, body weight, and survival rate of the ducklings, as well as the pathological changes in tissues and organs, were monitored. (C) Body temperature monitoring of ducklings after challenge. All ducklings in the mock group died on day 6, precluding statistical analysis at subsequent time points. Therefore, statistical significance analysis was only performed between the Δ400 mutant and mock groups for the initial five days. (D) Body weight monitoring of ducklings after challenge. Similarly, only data from the initial five days were used for statistical comparison between the Δ400 mutant and mock groups. (E) Survival rate monitoring of ducklings after challenge. (F)…

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  Fig. 6. Determination of neutralizing antibody titers induced by the Δ400 mutant strain. (A) Experimental flow chart for the determination of neutralizing antibody levels induced by the Δ400 mutant strain. (B) 14-day-old ducklings were immunized with 10⁵ TCID50 of the Δ400 mutant strain. Blood samples were collected on days 3 and 5, and in weeks 1, 2, 3, and 4 post-immunization. Sera were separated for the neutralizing antibody assay. (**** P < 0.0001, ns: not significant).