Avian influenza viruses (AIVs) belong to the Alphainfluenzavirus genus (http://ictv.global) of Orthomyxoviridae family [1]. These AIVs are subtyped based on their combination of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins and there are 16 and 9 different subtypes, respectively [2]. These AIVs circulate in wild aquatic birds, which are considered the natural reservoir [2]. According to the classification of AIV pathogenicity by the World Organization for Animal Health, avian influenza is divided into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) [3]. With cross-over from wild birds into poultry, viruses of the H5 and H7 subtypes can evolve to become highly pathogenic, causing severe disease, devastating outbreaks and up to 100% mortality in chickens [4]. Most AIVs are classified as LPAI, causing minimal clinical signs or asymptomatic infection in birds, while some subtypes, such as H9N2, can cause obvious clinical symptoms such as respiratory disease and reduced egg production, which can lead to relatively large economic losses [5].

Influenza viruses of the H3 subtype can infect a variety of hosts, such as humans, pigs, horses, dogs, cats, seals, monkeys and birds including poultry and wild birds [6]. There have been three human cases of H3N8 subtype AIV infection in China since 2022, resulting in one death [7,8]. These cases, although sporadic, demonstrate that this subtype of AIV can be transmitted to mammalian hosts through an interspecific barrier and have raised concerns whether H3N8 AIVs will become a future major public health threat [9].

Since 2023, several large-scale egg farms in Jiangsu province in China have experienced cases of drops in food and water intake, with affected chickens showing respiratory signs including swelling of the sinuses and discharge from the eyes, nares, mouth, severe dyspnea and reduced egg production, with lower mortality rates. The trachea, lungs, liver, and intestines of dead chickens were collected and homogenized to extract DNA and RNA. The egg drop syndrome virus, AIV, Newcastle disease virus, infectious bronchitis virus, infectious bursitis virus and infectious laryngotracheitis virus were tested by real-time polymerase chain reaction methods (national or industry standards), with only AIV being positive. The H3N3 subtype was further identified using HA and NA specific primers [10] and sequencing. The viruses were isolated by inoculation into 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs with homogenate and named A/chicken/Jiangsu/NT322/2023(H3N3) (NT322/H3N3) and A/chicken/Jiangsu/NT308/2023(H3N3) (NT308/H3N3), respectively. Throat and cloacal swabs of chickens from a nearby live poultry market were also sampled and one strain A/chicken/Jiangsu/J1247/2023(H3N3) was isolated.

To investigate the origins of these H3N3 isolates, their genomes were sequenced using Sanger sequencing and phylogenetic analysis was performed. The phylogenetic trees for each gene segment were generated by using the neighbor-joining method in the MEGA 11 package. The bootstrap value was calculated with 1000 replicates. The eight gene segment sequences of the three strains shared 99.5 to 100% nucleotide identity among them, suggesting they were highly homologous. The HA genes of these viruses were highly homologous to those of H3N8 that caused the three human cases and shared 97.5 to 99.2% nucleotide identity between them (Figure 1(A)). Their NA genes were genetically close to those from H10N3 circulating in poultry in China and including the human case in 2021) in China (Figure 1(B)). The internal genes were genetically associated with the H9N2 circulating in chickens in China. The internal gene constellation was more similar to that of the human H10N3 isolate than that of the human H3N8 isolates, sharing 95.9 to 98.7% nucleotide homology with A/Jiangsu/428/2021(H10N3), except PA and M genes which were highly homologous with human H3N8 isolates at 98.8% and 99.5%, respectively.

Figure 1. Phylogenetic trees of hemagglutinin (A) and neuraminidase (B) genes of the novel three H3N3 AIVs isolated from chickens in China, 2023. The trees were generated by neighbor-joining method with the MAGE 11 software. The viruses from this study were labeled in black circles, the H3N8 and H10N3 AIVs that caused human infections in China were marked in red. Scale bars indicate branch length based on number of nucleotide substitutions per site. (C) Replication of H3N3 and H9N2 viruses in chickens. The SPF chickens were inoculated with the representative viruses; organ samples were collected, and the viruses were titrated in eggs at 3?dpi. The dashed line in each panel indicates the lower limit of detection. (D) Shedding of H3N3 and H9N2 viruses in chickens. The SPF chickens were inoculated with the representative viruses; Oropharyngeal and cloacal swabs were collected from chickens at the indicated times, and viruses were titrated in eggs. OP: oropharyngeal swab; CL: cloacal swab. The dashed line in each panel indicates the lower limit of detection. (E) Histological lesions in the liver (a), kidney (b), spleen (c), thymus (d), lung (e), and bursa of Fabricius (f) of H3N3 infected chickens. Chickens were intranasally inoculated with 106 EID50 virus. Pathogenicity of the H3N3 virus in mice. Five-week-old BALB/c mice were infected intranasally with 106 EID50 virus. Percentage of bodyweight change of mice infected with NT322/H3N3 virus (F). The viral titers of the lungs and nasal turbinates of the infected mice collected at 3?dpi were measured in eggs (G).