A targeted mutation in ICP8 attenuates duck plague virus by disrupting replication and proposes a vaccine strategy

Figures

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  Fig. 1. In vitro characterization of ICP8 mutants. A. Sequence conservation analysis of residues 816-837 within the predicted DNA-binding domain of ICP8 across representative herpesviruses. Red asterisks denote targeted residues (H825 and F829/P830) selected for mutagenesis. B. Replication kinetics of wild-type (CHv), mutant (H825A, F829A/P830A), and revertant viruses in duck embryo fibroblast (DEF) cells infected at MOI = 0.01. Titers were determined at indicated time points post-infection (hpi). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test). C. Viral genome synthesis after 0.01 MOI DPV CHv, mutant and revertant viruses’ infection were quantified by UL30-probed qPCR following the formula: y = −3.3715x + 43.233, R² = 0.9991. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test). D. Viral protein expression profiles at 48 hpi (MOI = 0.1) analyzed by Western blot. β-tubulin served as loading control.

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  Fig. 2. Functional defects in replication compartment maturation and DNA synthesis induced by ICP8 mutations. A. Representative immunofluorescence images of ICP8 nuclear structures in duck embryo fibroblast (DEF) cells infected with wild-type DPV (CHv) at 24 hpi (MOI=1). Three distinct patterns are shown: pre-replication foci, replication compartments, and diffuse structures. B. Impaired replication compartment formation in cells infected with ICP8 mutants (H825A, F829A/P830A) compared to wild-type and revertant controls. Cells were fixed at 24 hpi and immunostained with mouse anti-ICP8 primary antibody followed by Alexa Fluor 488-conjugated secondary antibody. C. Quantitative analysis of ICP8 structure distribution from panel B. Data represent the percentage of each ICP8 morphology from three independent experiments (>100 cells/group); Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test). D. Spatial correlation of nascent DNA synthesis and ICP8 localization assessed by EdU co-labeling. Cells were pulsed with 10 μM EdU for 2 h prior to fixation at 24 hpi. E. Quantification of EdU-ICP8 co-localization…

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  Fig. 3. ICP8 mutations impair replication precursor assembly with helicase-primase complex components. A. Subcellular localization of EGFP-tagged ICP8 (WT, H825A, F829A/P830A) and mCherry-tagged UL5, UL8, or UL52 in BHK-21 cells. Scale bars: 10 µm. B. Nuclear foci formation following co-expression of ICP8-WT with helicase-primase subunits (UL5, UL8, UL52). Arrowheads indicate replication precursor foci. C. Quantification of replication foci formation efficiency from panel B. Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test). D. Co-immunoprecipitation (Co-IP) of HA-tagged ICP8-WT with FLAG/mCherry-tagged UL5, UL8, or UL52. Vector control lanes verify specificity. E-F. Co-IP analysis of interactions between mutant ICP8 (H825A, F829A/P830A) and (E) UL8 or (F) UL5. Input and IP fractions stained with anti-HA and anti-mCherry antibodies. G. Predicted hydrogen-bonding networks disrupted by H825A and F829A/P830A mutations. H. Proposed model of ICP8 mutants attenuates DPV. ICP8 mutations preserve binary interactions with UL5/UL8 but disrupt higher-order replication precursor…

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  Fig. 4. Graded virulence attenuation of ICP8 mutants in ducklings. A. Experimental design for in vivo evaluation. Fourteen-day-old ducklings were intramuscularly inoculated with indicated viruses at 10⁵ or 10⁷ TCID₅₀. B. Rectal temperature monitoring during the observation period (n = 10/group). C. Body weight changes relative to baseline following inoculation (n = 10/group). D. Survival curves of ducklings’ post-inoculation (n = 10/group).

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  Fig. 5. Pathological assessment of tissue protection. (A-B) Representative H&E-stained sections of major organs at 105 TCID₅₀ (A) or 10⁷ TCID₅₀ (B) Wild-type DPV, ICP8 mutants or revertant strains inoculation for 3 days. Black arrow indicates the representative lesions in selected organs. C-F. Organ indices (organ weight/body weight ratio) of the liver(C), spleen(D), thymus(E), and duodenum(F) at 3 dpi (n = 4/group), data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test).

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  Fig. 6. Systemic viral replication and shedding profiles. A. Quantification of viral loads in the liver, spleen, thymus, and duodenum at 3 days post-inoculation using probe qPCR targeted UL30 gene (n = 4/group). B. Viremia assessment: viral loads in peripheral blood collected at 3- and 5-days post-inoculation. (n = 4/timepoint; mean ± SD). C. Cloacal shedding analysis of viral loads in cloacal swabs collected at 3- and 5-days post-inoculation (n = 4/timepoint; mean ± SD). The symbols are defined as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (as determined by Student's t-test).