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During routine surveillance from November 8 to 19, 2024, a total of 15 clinically symptomatic black swans were identified in the Chongming Natural Reserve, Shanghai, exhibiting neurological signs including lethargy and head tremors.
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Virological analysis confirmed that all 15 diseased black swans found in the Chongming Nature Reserve were infected with HPAI H5N1 virus. Whole-genome sequencing revealed 96.88%–100.0% nucleotide identity among the eight gene segments of these viruses, which were abbreviated as CM-H5N1 viruses. Phylogenetic analysis revealed that the polymerase basic 2 (
PB2) and polymerase basic 1 (
PB1) genes of CM-H5N1 shared the highest sequence identity with those of wild bird-origin H5N1 viruses (98.72% and 99.28%), while the remaining six genes showed the highest sequence identity with avian-origin H5N1 viruses detected in South Korea and Japan during 2022-2023 (98.74%–99.53%) (Supplementary Table S1). The hemagglutinin (HA) cleavage site of these CM-H5N1 viruses possesses multiple basic amino acid sequences (PLREKRRKRGL), which is typical for HPAI viruses. Compared to the Re-14 vaccine strain, several mutations were observed in these CM-H5N1 strains at antigenic sites A (A144V) and B (N158D, N193K) (Supplementary Table S2); whether these mutations can cause antigenic drift requires further evaluation.
If current commercial vaccines are insufficient in providing comprehensive protection, there may be a risk of the virus spilling over from avian species to humans.
To elucidate the evolution of CM-H5N1 isolates and estimate the time to the most recent common ancestor (tMRCA), the time-scaled maximum clade credibility (MCC) trees for all eight gene segments were constructed using BEAST version 1.10.4 (
Fig. 1A, Supplementary Fig. S1, Table S3). An uncorrelated relaxed clock model was used, and the MCMC chain was set to 200 million states, with a 10% burn-in. The coalescent-based Bayesian phylogenetics is a popular and robust method to compare the tMRCA of each segment, inferring possible reassortment (
Vijaykrishna et al. 2015). The phylogenetic analysis of HA showed that the CM-H5N1 viruses belonged to clade 2.3.4.4b viruses and were all classified in the G10/G14 group (
Cui et al., 2022), distinct from EA1–EA5 group. Both the
HA and neuraminidase (
NA) genes clustered with H5N1 viruses isolated from domestic poultry that primarily circulated in Japan, South Korea during the winter of 2022–2023, and were genetically distinct from H5N1 viruses that previously identified in China. The
PB2 and
PB1 genes were closely related to H5N1 viruses detected from wild birds in East-Asia, while the structural topology of the other four genes was similar to that of the
HA genes. These results showed that the CM-H5N1 viruses likely originated from reassortment events involving avian and wild bird sources. The tMRCA analysis indicated that the possible reassortment event may have occurred in the 2021, with the virus emerging during the last wintering season (Supplementary Fig. S2).
Additionally, extended branch lengths observed in the HA and NA phylogenetic trees also supported the hypothesis that the virus has been circulating undetected in wild bird populations for an extended period.
