Influenza A virus (IAV) is widespread in wild bird reservoirs. Sixteen hemagglutinin subtypes are associated with wild waterfowl hosts; some subtypes are isolated infrequently, one of which is H12 IAV. In this study, we detected three H12 IAVs from Anascrecca and Anas formosa in Poyang Lake, China, in 2018, one of which was isolated. Phylogenetic analysis revealed that the genome sequences of the three H12 viruses belonged to the Eurasian lineage, except for PA genes and one NP gene, which belonged to the North American lineage. The growth kinetics showed that the H12 isolate grew better in A549 than MDCK cells. Moreover, although the H12 isolate cannot efficiently replicate in BALB/c mice, it can bind to both α-2,6 sialic acid (SA) and α-2,SA-linked receptors. In addition, we examined the phylodynamics of H12 viruses by Bayesian phylogeographic analysis. The results show that two major transmission routes of H12 IAVs were from Asia to Oceania and from Europe to South America, and Anas and Arenaria genera were the major hosts of the viral transmission. Our findings help us better understand the evolution of H12 IAV and highlight the need for the continued surveillance of IAVs circulating in wild birds.

Background: Duck adenovirus 3 (DAdV-3) is an emerging pathogen that has caused severe economic losses to the duck industry in China. Recently, the infection of ducks with serotype 4 fowl adenovirus (FAdV-4) has also been reported in China. Therefore, an efficient bivalent vaccine to control the diseases caused by DAdV-3 and FAdV-4 is extremely urgent. In our previous study, a recombinant FAdV-4 expressing Fiber-2 of DAdV-3 was generated and designated as rFAdV-4-Fiber-2/DAdV-3. Methods: Here, the recombinant virus rFAdV-4-Fiber-2/DAdV-3 was inactivated to serve as a bivalent vaccine, and its immunogenicity and protective efficacy against DAdV-3 were evaluated in Muscovy ducks. Results: The subcutaneous injection of rFAdV-4-Fiber-2/DAdV-3 could efficiently induce antibodies against Fiber-2 of DAdV-3 and neutralize antibodies against FAdV-4. After challenges with DAdV-3, in comparison with the non-immunized ducks, the immunized ducks did not show any bodyweight loss, gross lesions, or histopathologic change. Moreover, viral loads in livers and kidneys from immunized ducks were undetectable, whereas those in non-immunized ducks with challenge were significantly high. Conclusions: All these data demonstrate that the inactivated recombinant virus rFAdV-4-Fiber-2/DAdV-3 has the potential to be an efficient vaccine candidate against both FAdV-4 and DAdV-3, although efficacy for FAdV-4 needs to be confirmed experimentally.