First establishment of a Duck Model for in vivo and in vitro studies of West nile virus (Kunjin subtype).

We present a method for constructing a Kunjin virus (KUNV) infectious clone capable of stable proliferation in bacterial systems. This KUNV infectious clone successfully produces infectious viral particles within host cells. In vitro, the recombinant virus demonstrates the ability to replicate in primary duck cells and induces 100 % mortality in duck embryos. In vivo, the recombinant virus exhibits virulence in our established duckling model, leading to clinical signs such as hepatic haemorrhage, splenomegaly, and pancreatic haemorrhage. Furthermore, the recombinant rKUNV induced a mortality rate of 20 % in ducklings, thereby providing a robust model for studying the virus's pathogenicity in horses or human. To expand upon this foundation, we have developed KUNV replicons tagged with luciferase and mCherry, which confirm successful KUNV replication in primary duck cells and hold potential for future antiviral drug screening. The KUNV reverse genetics system, alongside the in vitro and in vivo duck infection models, offers a valuable platform for identifying pathogenic factors relevant to KUNV infection in humans, horses, and avian species. Additionally, this system could be instrumental in advancing vaccine development and antiviral drug research against KUNV.