Isolation and Characterization of a Duck Hepatitis A Virus Type 3 SH52 Strain in Ducklings

Abstract

Duck hepatitis A virus type 3 (DHAV-3), an avian-specific pathogen primarily impacting ducklings, poses a significant threat to the duck farming industry by causing high mortality rates. A DHAV-3 strain SH52 was isolated from the diseased ducks and phylogenetic analysis of the whole genome revealed that the DHAV-3 SH52 belongs to the prevalent strains in China. The DHAV-3 SH52 strain replicates at high levels in various organs of 8-day-old specific pathogen-free (SPF) shelducks, causing pathological damage and leading to high lethality in 8-day-olds following intramuscular infection. The findings of this study provide an ideal animal challenge model for evaluating the efficacy of DHAV-3 vaccines in the future.

Keywords: duck hepatitis A virus type 3; pathogenicity; phylogenetic analysis.

Figure 1

Identification and isolation of the DHAV-3 SH52 strain. (A) Tissue samples were tested by RT-PCR and PCR for common duck viruses. The RT-PCR and PCR analysis revealed 286 bp of the DHAV-3 target sequence in lane 11. Lanes 2 to 10 and lanes 14 to 25, which included DPV, DuCV, DAdV-3, NDRV, DTMUV, DERSV, DHAV-1, and their respective positive controls (P) and negative controls (N), extracted from the healthy ducklings, showed no interference with the target sequence. Lane 1 was served as the DL2000 marker (M). (B) DHAV-3-positive samples were inoculated into SPF duck embryos. The inoculated duck embryos died within 72 h post-inoculation, exhibiting systemic edema and hemorrhages, while the control duck embryos exhibited no gross pathological lesions.

Figure 2

Phylogenetic analysis based on the whole gene of DHAV-3. Sequences were aligned by Clustal W method and phylogenetic trees were generated using the NJ method in MEGA 12.0 software. For each strain, the GenBank accession number, strain type, and name are displayed.

Figure 3

Survival of ducklings inoculated with different titers of DHAV-3 SH52. Ducklings i.m. inoculated with different titers of DHAV-3 SH52 exhibited varying survival rates. Survival rates in experimental groups inoculated with 10^6.5 ELD₅₀ to 10^1.5 ELD₅₀ were 0%, 0%, 40%, 50%, 100%, and 100%, respectively. No deaths occurred in the control group.

Figure 4

Histological changes in ducks infected with DHAV-3 SH52 strain. (A) Livers from challenged ducklings showed enlargement with multifocal subcapsular petechiae. (B) Kidneys displayed swelling and cortical hemorrhage with gray-white necrotic foci. (C) Spleens exhibited congestion presenting diffuse dark-red discoloration. (D) The heart was enlarged with pericardial dilation and patchy hemorrhages visible on the epicardium. (E) The lungs were congested and edematous. (F–J) Control specimens demonstrated absence of gross lesions.

Figure 5

The virus titers of different tissues. SH52 replication was detectable in livers, kidneys, spleens, hearts, and lungs of ducks that died within 3 days post-challenge in the 10^6.5 ELD₅₀ group. Experimental data were presented as mean ± standard deviation. Error bars represent the standard deviation of the mean (n = 3 represents the virus titers of three tissue homogenates prepared from three different ducklings); tissue viral loads were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 9.5 software.

Figure 6

Pathological alterations in experimental tissues. (A) Liver degeneration and necrosis with cellular atrophy and cytolysis. Scale bar = 100 μm. (B) Kidney tubular epithelial degeneration and necrosis featuring nuclear pyknosis and minimal inflammatory infiltrate. (C) Disrupted periarteriolar lymphoid sheaths with lymphoid hyperplasia in spleen tissues. (D) No obvious pathological findings in the heart. (E) Interstitial hemorrhage in the lungs, proliferation of fibrous connective tissue, and inflammatory cell infiltration; (F–J) Absence of significant pathology in control specimens.