Expansion of cluster 3 Tembusu virus in Guangdong geese with hepatitis-splenomegaly syndrome and evidence of recombination.

Tembusu virus (TMUV) is a mosquito-borne flavivirus that causes substantial economic losses in the Asian poultry industry. While phylogenetic Cluster 2 has historically been dominant, recent reports signal the expansion of Cluster 3. In this study, we investigated TMUV infection in geese presenting with hepatitis-splenomegaly syndrome in Guangdong, China, a major waterfowl‑producing region in China. Among 50 clinical liver samples collected in late 2024, 16 (32 %) tested positive for TMUV by semi-nested RT-PCR. Complete genome sequencing of eight selected strains revealed that all belonged to the expanding Cluster 3 and shared high intra-clade identity. Phylogenetic analyses based on complete genomes and the envelope gene confirmed their placement within Cluster 3, a lineage genetically closer to mosquito-origin viruses. Notably, comprehensive recombination analysis identified a statistically supported recombination event in strain GDQY-298. Bootscan and phylogenetic incongruence tests indicated that GDQY-298 likely originated from recombination between two co-circulating Cluster 3 strains (GDQY-273 and GD/1650/2022). Our findings provide direct evidence for the ongoing displacement of Cluster 2 by Cluster 3 in Guangdong geese, underscoring the urgent need for subclade-specific surveillance and timely vaccine updates to counter the threat posed by evolving TMUV strains.