This study aimed to analyze the biological characteristics of the newly isolated H8N4 subtype avian influenza virus (AIV), so as to provide the basis for avian influenza surveillance and comprehensive prevention and control.

The whole-genome sequencing of two strains of H8N4 subtype avian influenza virus DK/HuN/S11158/2024 (DK/S11158) and DK/HuN/S11301/2025 (DK/S11301) isolated from duck farms in Hunan Province were determined. Reference strain sequences with the highest homology to each segment were downloaded from the GISAID database. The phylogenetic trees for each gene segment of the isolated strains and reference strains were constructed using MEGA 7.0 software. The key amino acid residue variations in different encoded proteins of the two H8N4 subtype AIVs were analyzed. The differences in viral receptor-binding characteristics were assessed using bio-layer interferometry. SPF chickens and BALB/c mice were infected with a virus dose of 10⁶ EID₅₀ to evaluate the infection and pathogenicity of the two H8N4 subtype viruses in poultry and mammals.

BLAST comparison results showed that for the DK/S11158 strain, the PB1, PA, and NA gene segments had the highest nucleotide homology with corresponding segments from AIVs of South Korean waterfowl origin; the PB2, HA, and NS gene segments had the highest homology with those from Japanese wild bird-origin AIVs; the M gene segment had the highest homology with a duck-origin AIV from Iran; the NP gene segment had the highest homology with a wild bird-origin AIV from China. For the DK/S11301 strain, the PB2, PB1, PA, NP, and NS gene segments showed the highest nucleotide homology with corresponding segments from Japanese wild bird and waterfowl-origin AIVs; the HA and NA gene segments had the highest homology with those from North American waterfowl-origin AIVs; the M gene segment had the highest homology with a South Korean wild bird-origin AIV. The amino acid sequence at the HA protein cleavage site of both H8N4 subtype AIVs was PSIEPK↓GLF, containing only a single basic amino acid, which was consistent with the molecular characteristics of low pathogenic avian influenza virus (LPAIV). Several mammalian adaptation mutation sites were identified, including 89V, 358E, 389R in the PB2 protein, 3V, 622G in the PB1 protein, 37A, 409S in the PA protein, and 42S, 106M in the NS1 protein. Assessment of viral receptor-binding characteristics using bio-layer interferometry revealed that both viruses could bind to both α-2,3-sialic acid and α-2,6-sialic acid receptors, demonstrating dual receptor-binding capability. Results from the infectivity experiment in chickens showed that no virus was detected in any organs, oropharyngeal swabs, or cloacal swabs of chickens in the DK/S11158 and DK/S11301 infection groups. No virus was detected in the oropharyngeal or cloacal swabs of chickens in the contact transmission groups either. Serological antibody level testing 14 days post-infection showed seroconversion in only one chicken from the DK/S11158 infection group. After intranasal infection of BALB/c mice with 10⁶ EID₅₀/50μL of virus, the viral titer in the nasal turbinates and lungs of the DK/S11158 group were 5.58 log₁₀EID₅₀/mL and 2.75 log₁₀EID₅₀/mL, respectively. The viral titers in the nasal turbinates and lungs of the DK/S11301 group were 6.25 log₁₀EID₅₀/mL and 4.08 log₁₀EID₅₀/mL, respectively, accompanied by a certain degree of weight loss.

The above results indicate that the two H8N4 subtype AIVs were novel recombinant viruses with significant genetic diversity and posed a potential risk of infecting mammals and humans. This study provided important data support for the surveillance and comprehensive prevention and control of avian influenza.

H6 avian influenza virus (AIV) is widely prevalent in southern China, which is one of the most common subtypes of AIVs circulating in poultry in China. H6N1 AIVs frequently undergo gene reassortment events with other wild bird-origin virus, which can be a donor to provide internal gene segments to highly pathogenic AIVs, which may lead to the emergence of novel virus and threaten human health.

The aim of this study was to investigate the evolution and biological characteristics of H6N1 AIVs in China, so as to provide valuable data for the prevention and control of avian influenza in China.

From 2019 to 2022, cloacal and throat swabs were collected from live poultry markets and breeding farms across 25 provinces or autonomous regions in China. 7 H6N1 viruses were isolated by inoculating into chicken embryos. The complete genome sequences were determined, followed by analyzing their phylogenetic relationships, receptor binding properties, as well as replication in SPF chickens and BALB/c mice.

The phylogenetic analysis revealed that the genes of the 7 H6N1 AIVs were highly homologous to those of wild bird-origin viruses from North America and Southeast Asia, indicating a complex genetic origin with significant genetic diversity. According to the Bayesian evolution analysis, the HA gene of H6 subtype AIVs had many times of intercontinental transmission in the history, and the Eurasian lineage strains also had a long time of circulation in North America. The HA gene of one strain of the virus was highly homologous to that of the North American strain. Based on the results of Bayesian evolutionary analysis, it was hypothesized that the virus was introduced to China via wild birds after undergoing complex genetic reassortment in wild birds. Analysis of specific amino acid sites revealed that the cleavage site of HA protein was PQIETR↓GLF, which was the signature of low pathogenic AIVs. In addition, one other virus had a Y52H mutation in the NP protein, which was critical in BTN3A3 evasion. Receptor binding analysis demonstrated that some of the H6N1 AIVs bound to both avian-type receptor and human-type receptor, however, their affinity towards human receptors was weaker compared with avian receptors. Infectivity experiments on SPF chickens indicated that flocks was still able to shed virus up through oropharyngeal and cloacal routes after infection with A/chicken/Jiangxi/S40445/2019(H6N1) and that the virus could be transmitted via contact within flocks. Only a few chickens infected with A/duck/Jiangxi/S10941/2019(H6N1) shed virus through their oropharyngeal tract, and the virus could not be transmitted via contact among chickens. The infectivity experiments conducted on mice showed that H6N1 subtype AIVs could replicate in the respiratory organs of infected mice without prior adaptation, but display low pathogenicity levels in mice.

Most of the genes of H6N1 subtype AIV isolated in China from 2019 to 2022 were derived from wild bird-origin viruses, and those migratory birds could introduce the viruses into China via the East Asia-Australasian migratory flyway. Some of the H6 AIVs bound to human-type receptors and replicate in the respiratory organs of mice suggested that H6N1 viruses posed a potential threat to human health.