The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 100 copy/μL, while DRC-LFA achieved limit of 101 copies/μL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.

To investigate the distribution and genetic variation in four vertically transmitted duck pathogens, including duck hepatitis B virus (DHBV), duck circovirus (DuCV), duck hepatitis A virus 3 (DHAV-3), and avian reoviruses (ARV), we conducted an epidemiology study using PCR and RT-PCR assays on a duck population. We found that DHBV was the most prevalent virus (69.74%), followed by DuCV (39.48%), and then ARV (19.92%) and DHAV-3 (8.49%). Among the 271 duck samples, two, three or four viruses were detected in the same samples, indicating that the coinfection of vertical transmission agents is common in ducks. The genetic analysis results showed that all four identified DuCV strains belonged to genotype 1, the DHAV-3 strain was closely clustered with previously identified strains from China, and the ARV stain was clustered under genotype 1. These indicate that different viral strains are circulating among the ducks. Our findings will improve the knowledge of the evolution of DuCV, DHAV-3, and ARV, and help choose suitable strains for vaccination.

Duck hepatitis A virus 3 (DHAV-3) is a wild endemic virus, which seriously endangers the duck industry in China. The present study aims to elucidate the mechanism of duck resistance to DHAV-3 infection. Both resistant and susceptible ducks were challenged with DHAV-3 in this experiment. The histopathological features and serum biochemical indices (ALT and AST) were analyzed to estimate liver injury status at 6, 12, 15, and 24 h post-infection (hpi). The dynamic transcriptomes of liver were analyzed to explain the molecular regulation mechanism in ducks against DHAV-3. The result showed that the liver injury in susceptible ducks was more serious than that in the resistant ducks throughout the four time points. A total of 2,127 differentially expressed genes (DEGs) were identified by comparing the transcriptome of the two populations. The expression levels of genes involved in innate immune response increased rapidly in susceptible ducks from 12 hpi. Similarly, the expression of genes involved in cytokine regulation also increased at the same time points, while the expression levels of these genes in resistant ducks remained similar between the various time points. KEGG enrichment analysis of the DEGs revealed that the genes involved in cytokine regulation and apoptosis were highly expressed in susceptible ducks than that in resistant ducks, suggesting that excessive cytokine storm and apoptosis may partially explain the mechanism of liver injury caused by DHAV-3 infection. Besides, we found that the FUT9 gene may contribute to resistance towards DHAV-3 in resistant ducklings. These findings will provide insight into duck resistance and susceptibility to DHAV-3 infection in the early phases, facilitate the development of a strategy for DHAV-3 prevention and treatment, and enhance genetic resistance via genetic selection in animal breeding.