Mutations in VP0 and 2C Proteins of Duck Hepatitis A Virus Type 3 Attenuate Viral Infection and Virulence

Figure 1. A schematic representation of the duck hepatitis A virus (DHAV)-3 genome. Comparative genomic analysis showed that there were two nucleotide substitutions (T1142A and C4334A) during the adaptation process, which led to two amino acid mutations in the VP0 (at residue 164) and 2C (at residue 71) proteins, respectively.

Figure 2. Viral titers of the virulent parental DHAV-3 strain and related embryo-adapted strain in the allantoic fluid of ducklings. The relative viral RNA load in the allantoic fluid was measured using qRT-PCR analyses. Each bar represents the mean ± SD (n = 3). The Student’s t-test was used to calculate the p values and 95% confidence levels. ** p < 0.01.

Figure 3. (A) Survival rate of ducklings inoculated with the virulent DHAV-3 strain, related embryo-adapted strain, or phosphate-buffered saline (PBS). Ducklings were inoculated through the intramuscular route. (B) Gross pathology of the liver tissues of ducklings after viral infection. At 36 hpi, the livers of the diseased ducklings were harvested for gross pathological assessment. The liver tissues of ducklings inoculated with the virulent strain exhibited obvious gross lesions, with punctate hemorrhaging and ecchymosis, whereas the liver tissues of the ducklings inoculated with the attenuated strain or PBS had no obvious lesions.

Figure 4. Histopathologic lesions detected in the liver tissues of ducklings inoculated with the virulent DHAV-3 strain (A–F), related embryo-adapted strain (G–L), and PBS (M–R). The livers had vacuolation and necrosis of hepatocytes (black arrows in B and C), a large number of infiltrating inflammatory cells, and multifocal confluent coagulative necrosis (black arrows in D and E).

Figure 5. In vitro growth kinetics of the virulent strain and its embryo-passaged strain of DHAV-3 in the liver tissues of ducklings. The relative viral RNA load in liver tissues was measured by qRT-PCR. Each bar represents the mean ± SD (n = 3). The Student’s t-test was used to calculate the p values and 95% confidence levels. ** p < 0.01, and *** p < 0.001 (***) vs. the control group.

Figure 6. Dynamic activation of pattern recognition receptors (PRRs) genes in the livers of inoculated ducklings. Expression levels of these genes were detected by qRT-PCR analyses and analyzed using the 2-ΔΔCt method. Each bar represents the mean ± SD (n = 3). The Student’s t-test was used to calculate the p values and 95% confidence levels. * p < 0.05, ** p < 0.01.

Figure 7. Dynamic activation of IFNs genes in the livers of inoculated ducklings. Expression levels of these genes were detected by qRT-PCR analyses and analyzed using the 2-ΔΔCt method. Each bar represents the mean ± SD (n = 3). The Student’s t-test was used to calculate the p values and 95% confidence levels. * p < 0.05, ** p < 0.01, and *** p < 0.001 (***) vs. the control group.

Figure 8. Dynamic activation of proinflammatory cytokines and anti-inflammatory cytokines genes in the livers of inoculated ducklings. Expression levels of these genes were detected by qRT-PCR analyses and analyzed using the 2-ΔΔCt method. Each bar represents the mean ± SD (n = 3). The Student’s t-test was used to calculate the p values and 95% confidence levels. * p < 0.05, ** p < 0.01, and *** p < 0.001 (***) vs. the control group.