Pathogenicity and Transmissibility of Clade 2.3.4.4h H5N6 Avian Influenza Viruses in Mammals

Simple Summary In the past decade, the spread of H5N6 avian influenza viruses (AIVs) in birds and infection in humans has attracted increasing global attention, and these viruses have the potential to become a pandemic threat to global health. In this study, the HA genes of the HB1907 and HB1905 AIVs were clustered in the 2.3.4.4h clade, and the HA genes of both strains exhibited highly pathogenic avian influenza virus (HPAIV) characteristics. The HB1905 strain in this study has a binding preference for avian-like (α-2,3) receptors only, whereas the HB1907 strain has a binding preference for both avian-like (α-2,3) and human-like (α-2,6) receptors. Compared with the HB1905 strain, the HB1907 strain showed better replication ability in MDCK cells in the early stage of infection. At the same time, the HB1907 strain showed advantages in the pathogenicity of mice and the transmission ability of direct contact between guinea pigs. These results further suggest that epidemiological surveillance and the related studies of H5N6 AIVs are essential for public health safety and the healthy and sustainable development of the livestock industry. Abstract Avian influenza viruses (AIVs) have the potential for cross-species transmission and pandemics. In recent years, clade 2.3.4.4 H5N6 AIVs are prevalent in domestic poultry, posing a threat to the domestic poultry industry and public health. In this study, two strains of H5N6 AIVs were isolated from chickens in Hebei, China, in 2019: A/chicken/Hebei/HB1907/2019(H5N6) and A/chicken/Hebei/HB1905/2019(H5N6). Phylogenetic analysis showed that both viral HA genes clustered in the 2.3.4.4h clade. Receptor binding analysis showed that the HB1905 strain preferentially binds to α-2,3-linked sialic acid (SA) receptors, while the HB1907 strain preferentially binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors. During early infection, the HB1907 strain is highly replicable in MDCK cells, more so than the HB1905 strain. Pathogenicity assays in mice showed that both viruses could replicate in the lungs without prior adaptation, with HB1907 being more highly pathogenic in mice than the HB1905 strain. Significantly, both the HB1905 and HB1907 strains can be transmitted through direct contact among guinea pigs, but the transmission efficiency of the HB1907 strain through contact between guinea pigs is much greater than that of the HB1905 strain. These results strengthen the need for ongoing surveillance and early warning of H5N6 AIVs in poultry.


Introduction
Highly pathogenic avian influenza (HPAI) H5N1 was first isolated in Guangdong Province, China, and subsequently reported worldwide [1,2]. The genetic evolution of H5 subtype avian influenza viruses (AIVs) has led to the differentiation and production of 10 different viral clades (0-9) and several subclades [3,4]. In 2014, a new clade of H5Nx

Viruses
A/chicken/Hebei/HB1905/2019(H5N6) (abbreviated as HB1905) (GenBank: MZ61892 2-MZ618924) and A/chicken/Hebei/HB1907/2019(H5N6) (abbreviated as HB1907) (Gen-Bank: MZ618947-MZ618949) were isolated from two free-range broiler farms in Hebei Province, China, in March 2019. The sampling design covered the entire areas of the two free-range broiler farms [18]. We collected samples from all diseased broilers [19]. The clinical sample collection and transportation were performed according to the Chinese Guidelines for the Technical Specifications for the Collection, Preservation and Transport of Highly Pathogenic Avian Influenza Samples. Typical influenza symptoms were observed in infected broilers, including clinical symptoms of neurological disease and diarrhea, and these clinical findings were consistent with previous reports [18,20]. All infected broilers died within 24 h after onset, and autopsy showed gastrointestinal bleeding, severe pneumonia and encephalitis. The incidence rate of chicken farms with the HB1905 strain isolated was as high as 80% (224/279), and the incidence rate of chicken farms with the HB1907 strain isolated was 95% (832/875). We collected oropharyngeal swabs and anal swabs from diseased broilers to obtain isolates. In brief, swabs were collected in 1 mL of phosphate-buffered saline (PBS). The supernatant was filtered using a 0.22 µm filter and inoculated into specific-pathogen-free (SPF) chicken embryos (Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd., Beijing, China). The virus was isolated and purified from SPF chicken embryos. Following incubation at 37 • C for 48 h, allantoic fluid was harvested Animals 2022, 12, 3079 3 of 14 and stored at −80 • C. All viruses were then passed three times by limiting dilution in SPF chicken embryos. Nucleic acid was extracted from allantoic fluid, and the detection of viral RNA was performed with real-time RT-PCR assay, as previously reported [21]. The results supported H5 positivity.

Viral Genome Sequencing and Analysis
The QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD, USA) was used to extract viral genomic RNA from allantoic fluid according to the manufacturer's instructions. The PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used to transcribe viral genomic RNA into cDNA. PCR amplification was performed using primers specific to AIV as previously reported [22]. PCR products were purified using the TaKaRa MiniBEST DNA Fragment Kit Ver.4.0 (TaKaRa, Dalian, China). A BigDye™ Terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) was used for sequencing. The SEQMAN program was used to analyze sequencing data (DNASTAR, Madison, WI, USA). From NCBI GenBank, reference sequences for the HA, NA and PB2 genes were retrieved. With Cluster W, the downloaded sequences were aligned and compared to the strains used in this work. The MEGA 7.0.21 program (Sinauer Associates, Inc., Sunderland, MA, USA) was used to perform a phylogenetic analysis based on the maximum likelihood (ML) with a bootstrap value of 1000. Figtree (v1.4.2, http://tree.bio.ed.ac.uk/software/figtree/) (accessed on 26 June 2022). was used to visualize the phylogenetic tree.

Cell Growth Curves
Madin-Darby canine kidney (MDCK) cells were used for growth evaluation according to a previous study [24,25]. The two strains (HB1905 and HB1907) infected cells with an MOI of 0.01 (10 5 cells). One hour after inoculation, the cells were washed twice with PBS, and fresh medium supplemented with 1 µg/mL tosyl phenylalanyl chloromethyl ketone (TPCK) and trypsin (Sigma Aldrich, St. Louis, MO, USA) was added. Cell supernatants were collected every 12-h post-infection (hpi) until the end of 60 hpi. All collected cell supernatants were inoculated into SPF chicken embryos, and EID 50 values were calculated. The experiments were performed in triplicate.

Mouse Study
The method for the mouse study was performed according to previous studies [26,27]. Forty-eight 6 weeks old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Fifteen BALB/c mice were randomly separated into three groups (n = 5 per group) and anesthetized with isoflurane. Two groups were inoculated intranasally with 50 µL of HB1905 or HB1907 at 10 6 EID 50 . Control mice were inoculated intranasally with an equal volume of PBS. The weight loss and survival rates of the BALB/c mice in the three groups were monitored daily for 14 days. Mice that lost >20% of their body weight were euthanized. Thirty-three BALB/c mice were randomly separated into three groups (three for control and fifteen per group for inoculation). Two inoculated groups were anesthetized with isoflurane and intranasally inoculated with HB1905 or HB1907 virus at 10 6 EID 50 , while the mice of the control group were intranasally inoculated with an equal volume of PBS. Three mice per inoculated group were euthanized at 1, 3, 5 and 7 days post-infection (dpi) to determine the viral load in the lungs. The lung samples were homogenized in 1 mL of PBS using a tissue lyser (Qiagen, Germany). Samples were clarified at 8000 rpm at 4 • C for 10 min. The above-clarified lung homogenates were inoculated into SPF chicken eggs, and the EID 50 was determined by hemadsorption. At 5 dpi, the lungs of three BALB/c mice per group from the three groups were removed and fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for pathological examination.

Guinea Pig Study
Referring to previous studies [27][28][29], 18 female guinea pigs (300-350 g) were used in the transmission test. Three guinea pigs in each group were inoculated intranasally with HB1905 or HB1907 of 10 6 EID 50 at 200 µL. The next day, three uninfected and three infected guinea pigs were placed on the same side of the individually ventilated cages (IVC) for direct contact transmission. Three uninfected guinea pigs in each group were placed on the opposite side of the infected guinea pig cage at a contact distance of 5 cm, which allows air to flow in the cage but prevents contact between guinea pigs. In addition, the guinea pigs were housed in individually ventilated cages (IVC) and provided with sterile water, bedding and feed. The wind speed was set to 0.4 m/s [30]. Nasal washes were collected every two days, samples were inoculated into SPF chicken eggs for viral titer determination and sera were collected to determine seroconversion at 21 dpi.

Statistical Analysis
Statistically significant differences were determined using one-way analysis of variance (ANOVA) for parametric parameters and the Mann-Whitney test for nonparametric parameters with the GraphPad Prism 8 software (San Diego, CA, USA). All assays were performed in at least three independent experiments. The error bars represent the standard deviation.

The HB1907 Virus Showed Better Replication in MDCK Cells
The growth curve in MDCK cell lines shows the viral replication abilities. The titers of the HB1905 and HB1907 viruses were highest at 36 hpi, 10 5.50 EID 50 /mL, and 10 6.17 EID 50 /mL, respectively ( Figure 4). However, HB1907 was significantly higher than HB1905 at 12 hpi (p < 0.001) and 24 hpi (p < 0.01), which is an indication that the ability of the HB1907 virus to replicate was significantly higher than that of the HB1905 virus at the early stage of infection ( Figure 4).

The HB1907 Virus Showed Better Replication in MDCK Cells
The growth curve in MDCK cell lines shows the viral replication abilities. The titers of the HB1905 and HB1907 viruses were highest at 36 hpi, 10 5.50 EID50/mL, and 10 6.17 EID50/mL, respectively ( Figure 4). However, HB1907 was significantly higher than HB1905 at 12 hpi (p < 0.001) and 24 hpi (p < 0.01), which is an indication that the ability of the HB1907 virus to replicate was significantly higher than that of the HB1905 virus at the early stage of infection ( Figure 4).

The HB1907 Strain Showed a Higher Pathogenicity
There was a gradual decrease in weight in mice in the HB1905 group, before it began to rise at 5 dpi, while the weight of mice in the HB1907 group declined to approximately 76% at 9 dpi ( Figure 5A). The survival rate of mice in the HB1905 group was 80%, but the   (HB1905 and HB1907) viruses. In the control group, HB777(H5N1) only preferred to bind avian-like (α-2,3) receptors, while CA04 (H1N1) only preferred to bind human-like (α-2,6) receptors. HB1905 prefers to bind avian-like (α-2,3) receptors. The HB1907 strain showed similar binding preferences with avian influenza virus receptors (α-2,3) and human influenza virus receptors (α-2,6). In each group, three separate experiments were carried out.

The HB1907 Virus Showed Better Replication in MDCK Cells
The growth curve in MDCK cell lines shows the viral replication abilities. The titers of the HB1905 and HB1907 viruses were highest at 36 hpi, 10 5.50 EID50/mL, and 10 6.17 EID50/mL, respectively ( Figure 4). However, HB1907 was significantly higher than HB1905 at 12 hpi (p < 0.001) and 24 hpi (p < 0.01), which is an indication that the ability of the HB1907 virus to replicate was significantly higher than that of the HB1905 virus at the early stage of infection ( Figure 4).

The HB1907 Strain Showed a Higher Pathogenicity
There was a gradual decrease in weight in mice in the HB1905 group, before it began to rise at 5 dpi, while the weight of mice in the HB1907 group declined to approximately 76% at 9 dpi ( Figure 5A). The survival rate of mice in the HB1905 group was 80%, but the

The HB1907 Strain Showed a Higher Pathogenicity
There was a gradual decrease in weight in mice in the HB1905 group, before it began to rise at 5 dpi, while the weight of mice in the HB1907 group declined to approximately 76% at 9 dpi ( Figure 5A). The survival rate of mice in the HB1905 group was 80%, but the survival rate of mice in the HB1907 group was 20%. Death occurred at 6 dpi in the HB1905-infected group and 3, 5 and 9 dpi in the HB1907-infected group ( Figure 5B). Based on the data above, we conclude that HB1907 showed greater pathogenicity than HB1905 in mice (p < 0.001). At 1, 3, 5 and 7 dpi, both viruses could be detected in the lungs of all the mice with titers ranging from 10 1.33 to 10 6.33 EID 50 /mL ( Figure 5C). The viral titers of HB1907 were higher than those of HB1905 at 1, 3, 5 and 7 dpi (p < 0.01). The highest viral load was observed at 5 dpi. The titer of HB1907 was approximately 10 6.33 EID 50 /mL at 5 dpi, which was 148-fold higher than that of HB1905. survival rate of mice in the HB1907 group was 20%. Death occurred at 6 dpi in the HB1905infected group and 3, 5 and 9 dpi in the HB1907-infected group ( Figure 5B). Based on the data above, we conclude that HB1907 showed greater pathogenicity than HB1905 in mice (p < 0.001). Images were acquired using a ×20 magnification objective. Alveolar wall thickening (arrow green), lymphocyte infiltration (arrow red), epithelial cell necrosis (arrow yellow), inflammatory cell infiltration; (arrow blue), acidophilic protein-like exudation. Three independent experiments were performed in each group (** p < 0.01, *** p < 0.001). At 1, 3, 5 and 7 dpi, both viruses could be detected in the lungs of all the mice with titers ranging from 10 1 . 33 to 10 6 . 33 EID50/mL ( Figure 5C). The viral titers of HB1907 were higher than those of HB1905 at 1, 3, 5 and 7 dpi (p < 0.01). The highest viral load was observed at 5 dpi. The titer of HB1907 was approximately 10 6 . 33 EID50/mL at 5 dpi, which was 148-fold higher than that of HB1905.
As shown in Figure 5D-F, mice infected with HB1905 and HB1907 showed significant lung lesions. Pathological results were scored in each part of each lung: 0-no pathological changes; 1-lesion area ≤10%; 2-lesion area 10-50%; 3-lesion area ≥50%. When pulmonary edema and/or alveolar hemorrhage were scanned, the score was increased by 1 point. According to the above criteria, the pathogenicity of the HB1907 virus in mice was higher than that of the HB1905 virus ( Figure 5G).

Direct Contact Transmission for HB1907 Virus Is Higher
Guinea pig models have been widely used to assess the transmissibility of influenza viruses [33]. Guinea pig nasal wash and serum samples were harvested to detect virus titers and HI antibody titers. The transmission efficiency of the influenza virus was evaluated by detecting the virus-positive rate of guinea pig nasal wash in different transmission groups. As shown in Figure 6A, in the HB1905 contact group, only one of the three guinea pigs replicated the virus at 2 (10 1.20 EID50/mL) and 4 (10 1.95 EID50/mL) dpi with a direct-contact transmission efficiency of 33.3%. As shown in Figure 6B, the titers of 2 dpi As shown in Figure 5D-F, mice infected with HB1905 and HB1907 showed significant lung lesions. Pathological results were scored in each part of each lung: 0-no pathological changes; 1-lesion area ≤ 10%; 2-lesion area 10-50%; 3-lesion area ≥ 50%. When pulmonary edema and/or alveolar hemorrhage were scanned, the score was increased by 1 point. According to the above criteria, the pathogenicity of the HB1907 virus in mice was higher than that of the HB1905 virus ( Figure 5G).

Direct Contact Transmission for HB1907 Virus Is Higher
Guinea pig models have been widely used to assess the transmissibility of influenza viruses [33]. Guinea pig nasal wash and serum samples were harvested to detect virus titers and HI antibody titers. The transmission efficiency of the influenza virus was evaluated by detecting the virus-positive rate of guinea pig nasal wash in different transmission groups. As shown in Figure 6A, in the HB1905 contact group, only one of the three guinea pigs replicated the virus at 2 (10 1.20 EID 50 /mL) and 4 (10 1.95 EID 50 /mL) dpi with a direct-contact transmission efficiency of 33.3%. As shown in Figure 6B, the titers of 2 dpi nasal washes of guinea pigs in the HB1907 direct contact transmission group were 10 1.45 EID 50 /mL, 10 2.20 EID 50 /mL and 10 0.95 EID 50 /mL, respectively. The titers of nasal washes at 4 dpi were 10 1.95 EID 50 /mL, 10 2.45 EID 50 /mL and 10 1.95 EID 50 /mL, respectively. The titers of nasal washes at 6 dpi were 10 2.20 EID 50 /mL, 10 1.45 EID 50 /mL and 10 1.20 EID 50 /mL, respectively. The three guinea pigs in the contact transmission group of HB1907 virus all shed detectable viruses with 100% efficiency for direct contraction. These results indicate that both HB1905 and HB1907 can be transmitted through direct contact, but the transmission efficiency of HB1907 was much higher than that of HB1905. Figure 6C-D show the HI antibody titers in guinea pig serum. Serum samples from the two strains of the virus-infected groups were positive, and serum samples from guinea pigs in the aerosol transmission group were negative. Serum samples from one of three guinea pigs that had direct contact with guinea pigs infected with HB1905 were positive, while serum samples from three guinea pigs that had direct contact with guinea pigs infected with HB1907 were all positive.
ble viruses with 100% efficiency for direct contraction. These results indicate that both HB1905 and HB1907 can be transmitted through direct contact, but the transmission efficiency of HB1907 was much higher than that of HB1905. Figure 6C-D show the HI antibody titers in guinea pig serum. Serum samples from the two strains of the virus-infected groups were positive, and serum samples from guinea pigs in the aerosol transmission group were negative. Serum samples from one of three guinea pigs that had direct contact with guinea pigs infected with HB1905 were positive, while serum samples from three guinea pigs that had direct contact with guinea pigs infected with HB1907 were all positive. Figure 6. Transmission of H5N6 subtype avian influenza isolates (HB1905 and HB1907) in guinea pigs. (A) The X-axis shows guinea pigs infected with HB1905, exposed to direct contact, and transmitted by aerosols. On the Y-axis, influenza virus titers in guinea pig nasal rinse are shown. (B) On the X-axis, guinea pigs infected with HB1907, exposed to direct contact group, and transmitted by aerosols are shown. On the Y-axis, influenza virus titers in guinea pig nasal rinse are shown. (C) The X-axis shows guinea pigs infected with HB1905 group, with direct contact group, and with the aerosol transmission group, and the Y-axis shows HI antibody titers of different guinea pigs. (D) The X-axis shows guinea pigs infected with HB1907, directly exposed group and aerosol transmitted group, and the Y-axis shows HI antibody titers of different guinea pigs. Each color bar represents an individual guinea pig. The horizontal dashed line represents the lower limit of assay detection.

Discussion
HPAI H5N1 was first reported in 1996 on a goose farm in Guangdong, China, which caused a mortality rate of up to 40% [34]. Despite efforts to promote vaccines to control Figure 6. Transmission of H5N6 subtype avian influenza isolates (HB1905 and HB1907) in guinea pigs. (A) The X-axis shows guinea pigs infected with HB1905, exposed to direct contact, and transmitted by aerosols. On the Y-axis, influenza virus titers in guinea pig nasal rinse are shown. (B) On the X-axis, guinea pigs infected with HB1907, exposed to direct contact group, and transmitted by aerosols are shown. On the Y-axis, influenza virus titers in guinea pig nasal rinse are shown. (C) The X-axis shows guinea pigs infected with HB1905 group, with direct contact group, and with the aerosol transmission group, and the Y-axis shows HI antibody titers of different guinea pigs. (D) The X-axis shows guinea pigs infected with HB1907, directly exposed group and aerosol transmitted group, and the Y-axis shows HI antibody titers of different guinea pigs. Each color bar represents an individual guinea pig. The horizontal dashed line represents the lower limit of assay detection.

Discussion
HPAI H5N1 was first reported in 1996 on a goose farm in Guangdong, China, which caused a mortality rate of up to 40% [34]. Despite efforts to promote vaccines to control the H5 virus, unprecedented large-scale mutations of the virus have generated various genotypes or sublineages, causing huge economic losses in China [14,15,35,36]. Since 2013, an unprecedented outbreak of HPAI H5 clade 2.3.4.4 viruses has been reported among poultry and wild birds in China. The virus has multiple NA subtypes, including H5N2, H5N5, H5N6 and H5N8 strains, and the prevalence is increasing year by year [37,38]. Large poultry farms in northern China are not only potential victims of influenza outbreaks but also important sites for the natural mutation and cross-species spread of influenza viruses. Regular monitoring and the early warning of influenza virus on farms is very conducive to the prevention and control of new influenza outbreaks.
Phylogenetic analysis of HA genes showed that the two H5N6 strains isolated in this study belong to the clade 2.3.4.4h H5N6 AIVs, which is consistent with the clade of H5N6 strains prevailing in China during 2018-2020 reported in previous studies [39]. At the same time, the HA proteins of the two strains in this study had multiple basic amino acids at the cleavage site (RERRRKR↓G), which is a characteristic of HPAIV [17,39,40]. These indicated that the viruses were capable of causing high death rates in poultry [6,41]. The HA gene of HB1907 was closely related to the human strains (A/Jiangsu/1/2018 (H5N6) and A/Jiangsu Nanjing/1128/2020 (H5N6)), and the HA gene of HB1905 was closely related to the poultry strains (A/duck/Jiangxi/2.28NCNP25K3-OC/2018 (H5N6) and A/Goose/Guangdong/7.20DGCP010-C/2017 (H5N6)). Previous studies have shown that avian influenza strains could be more transmissible and reproducible in mammals with higher homology to human strains [42,43]. The NA genes of the two strains of viruses were clustered in the Eurasian lineage, which is consistent with many H5N6 AIV subtypes isolated in recent years [37]. Given the rapid spread of the H5N6 AIVs in avian species and the ability to generate new recombinant strains, increasing attention should be given to prevent the reassortment of novel AIVs to infect humans or other animal species.
Receptor-binding preference is considered to be one of the important factors affecting the pathogenicity and transmissibility of influenza A virus [44]. The acquisition of the binding capacity of human-like (α-2,6) receptors by influenza viruses is an essential step in adaptation to human hosts [45]. The HB1905 strain in this study showed a binding preference for only avian-like (α-2,3) receptors, yet it replicated in mammals. The deletion of 11 amino acids in the NA stalk regions of the two strains in this study indicates that they may have certain adaptability and pathogenicity in mammals [46]. Interestingly, some new amino acid substitutions have also been detected in this study in addition to the mutations that led to the preference for avian-like receptors (Q226 and G228), which may be the reason for the above phenomena. In future research, it will be interesting to explore the functions of these unexplored amino acid mutations and their interactions with other amino acids. The HB1907 strain has a binding preference for both avian-like (α-2,3) and human-like (α-2,6) receptors. The HA gene of influenza A plays an important role in its receptorbinding preference. The receptor-binding sites of HB1905 and HB1907 both contained Q226 and G228, indicating an avian α-2,3-SA receptor binding preference [47,48]. However, the S128P mutation in HB1907 strain HA may enhance its adaptation and infectivity in mammals [49,50]. There are other amino acid sites in the HA gene that are different between the two strains, and whether they affect receptor binding preference remains to be further studied.
The pathogenicity of HB1905 in mice was consistent with studies of swan-origin H5N6 AIV isolated in 2020 and H5N6 AIV isolated from wild duck feces in 2022 [6,17]. The pathogenicity of the HB1907 virus in mice was significantly higher than that of the HB1905 virus. Unlike HB1905, HB1907 has a 627K locus in the PB2 gene without mouse adaptation, which often indicates a potential risk of mammalian infection [51]. Whether other amino acid positions in the PB2 gene of both strains play a role in mammalian fitness and virulence remains to be further investigated. Given the rapid spread of H5N6 AIVs in avian species and the ability to generate new recombinant strains, increasing attention should be paid to the prevention of the reassortment of novel AIVs to infect humans or other animal species.
Previous studies have shown that clade 2.3.4.4 H5N6 AIVs can be transmitted by direct contact in guinea pigs or ferrets [26,[52][53][54]. Our study showed that both HB1905 and HB1907 viruses could be transmitted by direct contact, and the transmission efficiency of the HB1907 virus through contact was significantly higher than that of the HB1905 virus. This indicates that our results are consistent with previous studies that the clade 2.3.4.4 H5N6 AIVs pose a potential pandemic threat. Multiple parameters, such as receptor-binding ability, replication ability, and virus survival characteristics under various environmental circumstances, may influence the potential of AIVs to transmit between mammals [55]. This study had several other limitations. First, although we carried out histological evaluations, we did not perform immunohistochemistry staining in the lung. Second, the data from mutation tracking and pathogenicity evaluation may not be sufficient to understand the possible pandemic risk of this naturally evolved H5N6 virus. Finally, this study is only a preliminary characterization of two chicken-origin H5N6 AIVs in mammalian models. Further mechanistic studies are needed in the future. Many other biotechnologies, including bioinformatics, synthetic biology and prediction models, will need to be employed as efficient tools in the future to determine more about the link between viruses and their hosts [56].

Conclusions
In conclusion, the two strains of H5N6 AIVs from the same branch in this study showed differences in mammalian models. We speculate that this difference may be caused by differences in amino acid mutation sites. Further studies are needed to investigate these findings, along with the biological roles of amino acid mutation sites. At the same time, the HB1907 virus in this study is highly replicated in MDCK cells, is highly pathogenic to mice, and has good direct contact transmission ability between guinea pigs. These results further suggest our long-term epidemiological surveillance of H5N6 AIVs and related research. It is beneficial to human public health security and the healthy and sustainable development of animal husbandry. Research in mammalian models broadens our knowledge of chicken-origin H5N6 AIV clade 2.3.4.4h.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani12223079/s1, Table S1: Amino acid differences between two H5N6 influenza viruses; Table S2: Nucleotide similarity of HA gene of two H5N6 isolates.  Data Availability Statement: The study's original contributions are included in the article; further inquiries can be directed to the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.