Article https://doi.org/10.1038/s41467-023-38415-7 Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America Ahmed Kandeil 1,2,9, Christopher Patton 1,3,9, Jeremy C. Jones 1,9, Trushar Jeevan1,9, Walter N. Harrington 1,9, Sanja Trifkovic 1, Jon P. Seiler1, Thomas Fabrizio 1, Karlie Woodard1, Jasmine C. Turner1, Jeri-Carol Crumpton1, Lance Miller1, Adam Rubrum1, Jennifer DeBeauchamp1, Charles J. Russell 1, ElenaA.Govorkova 1, Peter Vogel 4,Mia Kim-Torchetti5, Yohannes Berhane6,7, David Stallknecht8, Rebecca Poulson8, Lisa Kercher 1 & Richard J.Webby 1,3 Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly fol- lowed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically andphenotypically diverse, with many causing severe disease with dramatic neurologic involve- ment in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous systemwarrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants. The detection of highly pathogenic A/Goose/Guangdong/1/1996 (GsGD) A(H5N1) influenza viruses in Southern China in 1996 preceded two and a half decades of virus evolution characterized by geographic expansion and contraction and causing significant economic losses and the depopulation of billions of poultry1. Over time, viruses of the GsGD H5-lineage have evolved into several transient, phylogenetically distincthemagglutinin (HA) clades2. Genotype turnover drivenby virus reassortment has generally occurred only sporadically, but such events occurredwith increased frequency amongclade 2.3.4.4 yielding A(H5Nx) viruses possessing different neuraminidases (NA), most commonly N1, N6, or N8. Although geographic spread of GsGD H5- lineages is dynamic, clade 2.3.4.4b viruses have consistently expanded since 2020. In the 4 months prior to February 2022, 30 countries or territories across Asia, Europe, and Africa reported the detection of viruses of this clade in birds3. Most of these 2.3.4.4b outbreaks had been caused by A(H5N1) viruses, with the noticeable exception of the Received: 20 October 2022 Accepted: 27 April 2023 Check for updates 1Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA. 2Center of Scientific Excellence for Influenza Viruses, National Research Centre, Giza 12622, Egypt. 3Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38105, USA. 4Comparative Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA. 5National Veterinary Services Laboratories, Animal and Plant Health Inspection Service (APHIS), US Department of Agriculture (USDA), Ames, IA 50011, USA. 6National Centre for Foreign Animal Disease, Winnipeg, MB R3E 3M4, Canada. 7Department of Animal Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. 8Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA. 9These authors contributed equally: Ahmed Kandeil, Christopher Patton, Jeremy C. Jones, Trushar Jeevan, Walter N. Harrington. e-mail: richard.webby@stjude.org Nature Communications | (2023) 14:3082 1 12 34 56 78 9 0 () :,; 12 34 56 78 9 0 () :,; http://orcid.org/0000-0003-3253-6961 http://orcid.org/0000-0003-3253-6961 http://orcid.org/0000-0003-3253-6961 http://orcid.org/0000-0003-3253-6961 http://orcid.org/0000-0003-3253-6961 http://orcid.org/0000-0002-3685-4337 http://orcid.org/0000-0002-3685-4337 http://orcid.org/0000-0002-3685-4337 http://orcid.org/0000-0002-3685-4337 http://orcid.org/0000-0002-3685-4337 http://orcid.org/0000-0002-9980-2112 http://orcid.org/0000-0002-9980-2112 http://orcid.org/0000-0002-9980-2112 http://orcid.org/0000-0002-9980-2112 http://orcid.org/0000-0002-9980-2112 http://orcid.org/0000-0003-3314-584X http://orcid.org/0000-0003-3314-584X http://orcid.org/0000-0003-3314-584X http://orcid.org/0000-0003-3314-584X http://orcid.org/0000-0003-3314-584X http://orcid.org/0000-0002-0710-9514 http://orcid.org/0000-0002-0710-9514 http://orcid.org/0000-0002-0710-9514 http://orcid.org/0000-0002-0710-9514 http://orcid.org/0000-0002-0710-9514 http://orcid.org/0000-0002-8960-0728 http://orcid.org/0000-0002-8960-0728 http://orcid.org/0000-0002-8960-0728 http://orcid.org/0000-0002-8960-0728 http://orcid.org/0000-0002-8960-0728 http://orcid.org/0000-0001-5683-3990 http://orcid.org/0000-0001-5683-3990 http://orcid.org/0000-0001-5683-3990 http://orcid.org/0000-0001-5683-3990 http://orcid.org/0000-0001-5683-3990 http://orcid.org/0000-0001-9067-5682 http://orcid.org/0000-0001-9067-5682 http://orcid.org/0000-0001-9067-5682 http://orcid.org/0000-0001-9067-5682 http://orcid.org/0000-0001-9067-5682 http://orcid.org/0000-0002-7535-0545 http://orcid.org/0000-0002-7535-0545 http://orcid.org/0000-0002-7535-0545 http://orcid.org/0000-0002-7535-0545 http://orcid.org/0000-0002-7535-0545 http://orcid.org/0000-0001-6300-0452 http://orcid.org/0000-0001-6300-0452 http://orcid.org/0000-0001-6300-0452 http://orcid.org/0000-0001-6300-0452 http://orcid.org/0000-0001-6300-0452 http://orcid.org/0000-0002-4397-7132 http://orcid.org/0000-0002-4397-7132 http://orcid.org/0000-0002-4397-7132 http://orcid.org/0000-0002-4397-7132 http://orcid.org/0000-0002-4397-7132 http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-38415-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-38415-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-38415-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-38415-7&domain=pdf mailto:richard.webby@stjude.org outbreaks of A(H5N6) in China which have been associated with human infections. Of note, A(H5N6) viruses of clade 2.3.4.4h had been the previously predominant clade in poultry in China and had also caused human infections4. In December 2021, A(H5N1) viruses were detected in poultry and a gull in Eastern Canada. The virus responsible was closely related to 2.3.4.4b viruses identified in Europe in the spring of that year5. Thiswas only the second time that a GsGD H5-lineage virus was detected in birds in the Americas; the first such occurrence was in 2014, when the infected birds had crossed the Bering Strait and entered the Pacific flyway. After the initial 2021 detection in Newfoundland, infected wild birds were reported in the U.S. south atlantic states6 and in several other U.S. regions soon thereafter. The purpose of this study was to understand the course of the genetic and phenotypic evolution of the 2.3.4.4b viruses as they spread throughout North America (NAm). We demonstrate that westward intercontinental 2.3.4.4b expansion resulted in reassortment with NAm wild bird viruses yielding reassor- tants with diverse ribonucleoprotein gene mixtures. The resulting viruses have distinct in vitro phenotypes including increased virus replication rates and pH of activation, but most concerningly, they cause severe disease outcomes with dramatic neurologic involvement in mammalian animal models. Results Virus pathogenesis and transmission in chickens and ferrets We first conducted a risk assessment to determine the pathogenic and transmission properties of two early isolates in birds andmammals; A/ American wigeon/South Carolina/22-000345-001/2021 (Wigeon/SC/ 21) and A/bald eagle/Florida/W22-134-OP/2022 (Eagle/FL/22) were collected in December 2021 and February 2022, respectively (Table 1). White leghorn chickens and ferrets were initially used, both of which are susceptible to a variety of influenza viruses and are critical animal models among influenza risk assessment pipelines (IRAT, TIPRA)7, 8. Both chicken-to-chicken and chicken-to-ferret transmission were examined. Naïve chickens were placed in the same cage as inoculated birds, and ferrets were housed in a separate cage 12 inches away from inoculated chickens (Supplementary Fig 1A). Both viruses replicated robustly in inoculated chickens but transmitted inefficiently to naïve contact chickens (Supplementary Fig. 1B). Chickens (n = 3 per virus) directly inoculated with either Wigeon/SC/21 or Eagle/FL/22 died within 48 h post-inoculation (hpi). Two of twelve contact chickens paired with Wigeon/SC/21 inoculated chickens shed virus and met euthanasia endpoints at 4 or 7 dpi, while two additional non-shedding contacts died at 4 or 10 dpi. Three of twelve contact chickens paired with Eagle/FL/22 inoculated chickens shed virus and met euthanasia endpoints 3- 4 days post-inoculation (dpi); virus was transiently detected in a fourth bird which survived (Supplementary Fig. 1B). No aerosol transmission was detected from infected chickens to naïve ferrets (Supplementary Fig. 1C), suggesting that the risk of transmis- sion at the avian-mammalian interface remains low. We next assessed ferret-to-ferret contact transmission by placing inoculated ferrets in the same cage as naive ferrets. Neither virus transmitted from inocu- lated ferrets to naïve direct-contact ferrets (Fig. 1A, andSupplementary Table 4). Surprisingly, there were dramatic differences in disease severity among directly inoculated ferrets between the two viruses. Consistent with a recent report9, Wigeon/SC/21 infection resulted in mild disease and the ferrets survived (Fig. 1B). Only one of three Wigeon/SC/21 inoculated ferrets exhibited weight loss (Fig. 1C), and upper respiratory tract virus shedding was detected until 5 or 7 (n = 1 ferret) dpi (Fig. 1D). In contrast, Eagle/FL/22 inoculation resulted in rapid weight loss (Fig. 1C), lethargy, and severe neurologic symptoms, including ataxia and hindlimb paralysis (Supplementary Table 4). By 7 dpi, all Eagle/FL/22 inoculated ferrets reached humane end- points (Fig. 1B). The differential pathogenicity of the two viruses was reflected in the higher mean nasal wash titers at 1, 3, and 5 dpi for Eagle/FL/22 (4.2 to 5.2 log10 TCID50/mL) than forWigeon/SC/21 (1.7 to 3.4 log10 TCID50/ mL) (P < 0.0001) (Fig. 1D). Eagle/FL/22 inoculated ferrets also had higher viral loads in turbinate, tracheal, and lung samples (P ≤0.001), and virus was observed in brain tissues at 3 dpi (mean titer, 6.2 log10 TCID50/g) and 5 dpi (mean titer, 8.2 log10 TCID50/g) (Fig. 1E). In con- trast,Wigeon/SC/21 inoculation led to virus beingdetectedprimarily in turbinate and tracheal tissues, trace amounts in the lung at 5 dpi only, and no virus detected in brain tissues (Fig. 1E). Genotypic and antigenic analyses of clade 2.3.4.4b viruses To begin to understand molecular determinants contributing to the higher virulence of Eagle/FL/22, sequence analysis was performed on A(H5N1) wild bird viruses from this outbreak, which included Wigeon/ SC/21 and Eagle/FL/22. These data revealed that reassortment event(s) among H5 clade 2.3.4.4b viruses and NAm wild bird influenza viruses had occurred soon after introduction of the A(H5N1) viruses into NAm (Fig. 2). Whereas Wigeon/SC/21 had the same genotype as the first viruses detected in Canada and those detected in Europe in the spring of 2021, Eagle/FL/22 had undergone reassortment, and acquired NAm wild bird-lineage polymerase basic 2 (PB2), polymerase basic 1 (PB1), and nucleoprotein (NP) genes (Fig. 2A). In total, we identified four different viral genotypes among the 58 A(H5N1) viruses sequenced in this study (Fig. 2B). All viruses main- tained the parental Eurasian-origin (EA) HA, NA, matrix (M), and non- structural (NS) gene segments, but they had different combinations of polymerase and NP gene segments of either EA or NAm origin. In all cases except for PB2, for which two phylogenetically distinct genes were detected (Fig. 2A), the NAm gene segments were monophyletic, suggesting that a single or minimal number of reassortment events had occurred, with the resulting viruses spreading geographically. The NAm-lineage proteins of A(H5N1) viruses had no markers associated with increased virulence in mammalian hosts10, 11, and all viruses remained antigenically homogeneous, as determined by hemaggluti- nation inhibition (HAI) assays (Supplementary Table 1). Pathotyping of additional clade 2.3.4.4b viruses in ferrets To further assess if virulence was linked to acquisition of NAm gene segments, we used the ferret model to pathotype four additional A(H5N1) viruses of genotypic diversity represented later in the out- break period (Fig. 3A). These viruses were A/Fancy Chicken/ Table 1 | North American HPAI A(H5N1) clade 2.3.4.4b viruses used in this study Groupa Virus Genotypeb Abbreviation 1 A/American wigeon/South Carolina/22-000345- 001/2021 EA Wigeon/SC/21 A/Bald eagle/Florida/W22- 134-OP/2022 EA/Nam (PB1, PB2, NP) Eagle/FL/22 2 A/Fancy Chicken/New- foundland/FAV-0033/2021 EA Ck/NL/21 A/Bald eagle/North Car- olina/W22-140/2022 EA/NAm (PB2, NP) Eagle/NC/22 A/Red-shouldered hawk/ North Carolina/W22- 121/2022 EA/NAm (PB1, PB2, NP) Hawk/NC/22 A/Lesser scaup/Georgia/ W22-145E/2022 EA/NAm (PB1, PB2, PA, NP) Scaup/GA/22 aArbitrary numbering relating to the original ferret inoculation experiments described in Fig. 1 (Group 1), and Fig. 3 (Group 2). bThe gene segments acquired from avian influenza viruses from NAmwild birds are indicated in parentheses. EA Eurasian-origin, NAm North American-origin, PB1 polymerase basic 1 gene, PB2 polymerase basic 2 gene, PA polymerase acidic gene, NP nucleoprotein gene. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 2 0150 0 50 100 Days post-inoculation S ur vi va l ( % ) Eagle/FL/22 Wigeon/SC/21 Intranasal inoculation Add contact ferrets 24hrs Necropsy d3 Necropsy d5 Days post-inoculation 1 3 5 7 10 0 = Inoculated ferret = Contact ferret = Necropsy ferret = Nasal wash sample = Wigeon/SC/21 = Eagle/FL/22 Eagle/FL/22 (3 dpi) Eagle/FL/22 (5 dpi) Wigeon/SC/21 (3 dpi) Wigeon/SC/21 (5 dpi) /g ) T C ID 50 Nasal turbinate Trachea Lung Brain 0 2 4 6 8 10 V iru s tit er ( lo g 1 0 ✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ /m L) T C ID 50 1 3 5 7 10 0 2 4 6 Days post-inoculation V iru s tit er ( lo g 10 ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ Wigeon/SC/21 Eagle/FL/22 A B C ED 1 2 3 4 5 6 7 8 80 100 120 Days post-inoculation W ei gh t c ha ng e (% ± S E ) Eagle/FL/22 Wigeon/SC/21 ** Fig. 1 | Pathogenicity of North AmericanHPAI Influenza A(H5N1) clade 2.3.4.4b Wigeon/SC/21 and Eagle/FL/22 viruses in ferrets. A Experimental design of ferret pathogenesis and transmission. At 0dpi, ferrets (n=9 per virus) were inoculated with 106 EID50 units of A(H5N1) virus. Three inoculated ferrets were individually co- housed with 3 naïve contact ferrets beginning 1 dpi. Clinical course of infection was monitored, and nasal wash samples were taken at indicated time points from both inoculated and contact ferrets. The remaining inoculated ferrets were euthanized at 3 dpi and 5dpi (n= 3 per time point per virus) for viral titration in tissues. B Survival and C weight changes of inoculated ferrets (n= 3 per virus). Ferret weights every ≈48h were used to calculate percentage of weight change from the initial mean weight at 0dpi. Ferret weight values are the average ± SE for each group. P values for weight change were calculated using an unpaired t-test. **P<0.01. D Infectious viral titers from nasal washes (n= 3–9 ferrets per virus, mean virus titer [log10 TCID50/mL] ±SD) and E infectious viral titers from tissues (n= 3 ferrets per virus, mean virus titer [log10 TCID50 per g of wet tissue]). Symbols represent each individual animal’s titer. Dashed lines indicate the lower limit of virus titer detection (1.0 log10 TCID50/mL). P values for viral titers were calculated using two-way ANOVA with Tukey’s multiple- comparison post hoc test. ***P<0.001, ****P <0.0001. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 3 Newfoundland/FAV-0033/2021 (Ck/NL/21; native EA constellation; with the same genotype as Wigeon/SC/21); A/Red-shouldered hawk/ North Carolina/W22-121/2022 (Hawk/NC/22; with the same genotype as Eagle/FL/22); A/Lesser scaup/Georgia/W22-145E/2022 (Scaup/GA/ 22), with PB2, PB1, polymerase acidic (PA), and NP of NAm wild bird- lineage; and A/Bald eagle/North Carolina/W22-140/2022 (Eagle/NC/ 22), with PB2 and NP of NAmwild bird-lineage (Table 1). In general, the number of NAm gene segments acquired was strongly associated with disease severity in ferrets including degree of weight loss and the number of animals that ultimately succumbed to disease (Fig. 3B, C), with the least number of clinical signs andmortality observed with Ck/ NL/21, which has no NAm genes, and the most significant signs and highmortality observed with Scaup/GA/22, which possesses four NAm genes (Fig. 3B, C, Supplementary Figs. 2, 3, and Supplementary Table 4). This trend extended to viral replication in tissues. Peak mean nasal wash titers were 2.9, 3.8, 5.4, and 6.0 log10 TCID50/mL for Ck/NL/ 21, Eagle/NC/22, Hawk/NC/22, and Scaup/GA/22 viruses, respectively (Fig. 3D). All EA/NAm viruses were detected in multiple organs con- firming systemic viral spread, while the native constellation virus Ck/ NL/21 had very low or trace amounts of virus outside the respiratory tract (Fig. 3E). Additionally, infection with the EA/NAmviruses resulted in higher temperature increases than Ck/NL/21 along with respiratory and neurologic symptoms (Supplementary Table 4). Pathology Virulence trends were also reflected in the histopathologic examina- tion of the upper respiratory tract (URT), lower respiratory tract (LRT), and extrapulmonary tissues of ferrets infectedwith viruses of different genotypes. The most virulent virus, Scaup/GA/22, produced severe necrotizing lesions throughout the URT (Supplementary Fig. 3A, B) and LRT (Supplementary Fig. 4A, B) that correlated with robust viral antigen staining. Histopathology was less severe in ferrets infected with Hawk/NC/22, which produced fewer necrotizing lesions, limited multifocal clusters of neuroepithelium inURT (Supplementary Fig. 3C, D) and well demarcated LRT lesions (Supplementary Fig. 4C, D), cor- responding to less extensive viral antigen staining. Abundant antigen stainingwas observed throughout the central nervous system (CNS) of ferrets inoculated with Scaup/GA/22 (Supplementary Fig. 5A, B), whereas antigen staining for Hawk/NC/22 was limited to the olfactory bulb and olfactory cortex of inoculated ferrets (Supplementary Fig. 5C, D). Eagle/NC/22 produced even less severe histopathology, with only a few small foci of virus-positive cells being observed in the URT olfac- tory neuroepithelium and no lesions or virus antigen staining extending into the LRT (Supplementary Fig. 3E, F, and Supplementary Fig. 4E, F). Finally, in ferrets inoculated with Ck/NL/21, both the URT and LRT respiratory epithelia were devoid of lesions or cells staining for viral antigen, except for a single small focus of virus antigen- positive cells in the olfactory neuroepithelium (Supplementary Fig. 3G, H, and Supplementary Fig. 4G, H). Virus Pathogenesis in the mouse model To investigate whether the pathogenicity and virulence in ferrets was mirrored in other influenzamammalianmodels, we inoculated BALB/c mice with each virus. Viruses that caused 100% lethality in ferrets and those that had acquired increasing numbers of NAm gene segments, including Scaup/GA/22, Eagle/FL/22, and Hawk/NC/22, had the lowest 50% lethal doses (LD50s) in mice (i.e., less virus was required for lethality) at 3.8, 2.2, and 4.6 log10 EID50/mL, respectively. Additionally, A B # Isolates PB2 PB1 PA HA NP NA MP NS 16 Wigeon/SC/21 Ck/NL/21 Eagle/FL/22 Hawk/NC/22 Eagle/NC/22 Scaup/GA/22 9 Virus Genotype 12 21 A/Bald eagle/FL/W22-134-OP/2022* A/Lesser scaup/GA/W22-143/2022 A/Bald eagle/FL/W22-134-CL/2022 A/Bald eagle/NC/W22-140/2022* A/Lesser scaup/FL/W22-129A/2022 A/Lesser scaup/GA/W22-145B/2022 A/Lesser scaup/GA/W22-145D/2022 A/Lesser scaup/GA/W22-145A/2022 A/Bald eagle/FL/W22-142/2022 A/Lesser scaup/GA/W22-145C/2022 A/Lesser scaup/GA/W22-145E/2022* A/Red-shouldered hawk/NC/W22-121/2022* A/Lesser scaup/MD-LC-EESC-024/2022 A/American wigeon/SC/22-000345-001/2021* Eurasian Lineage North American Lineage Fig. 2 | Genotypic diversity among North American HPAI Influenza A(H5N1) clade 2.3.4.4b viruses. A Genotypic diversity of 58A(H5N1) viruses (some sequences generated directly from clinical material). A list of viruses used in this study and their GenBank or GISAID accession numbers are provided in Supple- mentary Tables 3 and 4, respectively. The colors denote common genotypes, with the orange color representing genes of Eurasian lineage and blue representing genes of NAm lineage (the different shades of blue represent phylogenetically distinct NAm genes). B Representative tangle plot showing the association of A(H5N1) virus HA genes (left side) with PB2 genes (right side) of Eurasian and NAm lineages. Nodes on the PB2 tree represent A(H5N1) viruses and representatives of other subtypes. Asterisks (*) indicate viruses that were used in subsequent experiments. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 4 0150 0 50 100 Days post-inoculation S ur vi va l ( % ) Ck/NL/21 Eagle/NC/22 Scaup/GA/22 Hawk/NC/22 B C A 0 1 2 3 4 5 6 7 8 9 10 80 100 120 Days post-inoculation W ei gh t c ha ng e (% ± S E ) Hawk/NC/22 Ck/NL/21 Eagle/NC/22 Scaup/GA/22 = Inoculated ferret = Necropsy ferret = Nasal wash sample = Serum sample = Ck/NL/21 = Eagle/NC/22 = Hawk/NC/22 = Scaup/GA/22 All surviving ferrets Pathology (n=3)Virus titration (n=3) Days post-inoculation 5 0 1 3 7 10 25 Intranasal inoculation Days post-inoculation T C ID /m L) V iru s tit er ( lo g 1 0 50 Ck/NL/21 Eagle/NC/22 Hawk/NC/22 Scaup/GA/22 1 3 5 0 2 4 6 8 T C ID V iru s tit er ( lo g 1 0 50 /g ) 0 2 4 6 8 10 Nasal turbinate Trachea Lung Brain Intestine Ck/NL/21 Scaup/GA/22 Eagle/NC/22 Hawk/NC/22 D E Fig. 3 | Impact of different detected genotypes of North American HPAI Influ- enzaA(H5N1) clade 2.3.4.4bvirusesonpathogenicity in ferrets. A Experimental design of ferret pathogenesis and transmission. At 0 dpi, ferrets (n = 9 per virus) were inoculated with 106 EID50 units of A(H5N1) virus. Clinical course of infection was monitored, and nasal wash samples were taken at the indicated time points. Ferrets (n = 3 per virus per analysis) were euthanized at 5 dpi for viral titration in tissues and pathology (Supplementary Figs. 3–5). B Survival and C weight changes of inoculated ferrets (n = 3 per virus). Ferret weights every ≈48 h were used to calculate percentage of weight change from the initial mean weight at 0 dpi. Ferret weight values are the average ± SE for each group. D Infectious viral titers from nasal washes (n = 6 ferrets per virus, except for Scaup/GA/22 at 5 dpi, for which n = 1, mean virus titer [log10 TCID50/ mL] ±SD) and E infectious viral titers from tissues (n = 3 ferrets per virus per time point, mean virus titer [log10 TCID50 per g of wet tissue]). Symbols represent each individual animal’s titer. Dashed lines indicate the lower limit of virus titer detection (1.0 log10 TCID50/mL). P values were calculated using two-way ANOVA with Tukey’s multiple-comparison post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 5 these viruses induced neurologic symptoms in mice, along with weight loss and virus replication in the lungs and brains. Viruses causing moderate or no lethality in ferrets, including Eagle/NC/2, Ck/ NL/21, and Wigeon/SC/21, had the highest mouse LD50s at >6 log10 EID50/mL (Supplementary Figs. 6 and 7). Phenotypic properties of clade 2.3.4.4b viruses Several influenza virus characteristics are known to contribute towards mammalian infection and spread, including receptor binding proper- ties, pHofHA activation, andpolymerase activity12–17.Wigeon/SC/21 and Eagle/FL/22 bound strongly to sialic acid receptors with a 3′SLN linkage to the underlying sugar (preferred by avian viruses), but poorly to 6′ SLN-linked sialic acid receptors (preferred by human viruses) (Fig. 4A, B). The pH of HA activation and inactivation segregates with host adaptation17, and the pH of HA activation for A(H5N1) viruses in avian species is 5.6–6.012. Consistent with this, bothWigeon/SC/21 and Eagle/ FL/22 had a relatively high pHofHAactivation of 5.8 by syncytiumassay (Fig. 4C and Supplementary Fig. 7). These values were slightly higher than the pH of HA activation of the control human virus A/California/ 04/2009 (CA/04) (H1N1)pdm09 (pH 5.6) and substantially higher than the pH of HA activation of most human-adapted influenza viruses14. In contrast, Wigeon/SC/21 and Eagle/FL/22 had virus inactivation pH values of 4.4, substantially lower than that of the CA/04 (H1N1)pdm09 virus (pH 5.4) (Fig. 4C). A virus inactivation pH that is lower than the HA activation pH is rare but has been observed with some swine H1 and H3 isolates and a bat A(H9N2) isolate18. The polymerase activities of Wigeon/SC/21 and Eagle/FL/22 were not statistically different when measured in the reporter gene assaywith transiently expressedproteins (Fig. 4D). However, we detected differences in the replication rates of whole virus in non-differentiated Calu-3 cells (Fig. 4E) and primary dif- ferentiated human airway cultures (Fig. 4F). At 48 hpi, cell cultures inoculated with viruses that had the most NAm gene segments (Scaup/ GA/22, Eagle/FL/22, and Hawk/NC/22) had the highest viral loads, fol- lowed by those inoculatedwith Eagle/NC/22, and then those inoculated with viruses containing no NAmgene segments (Ck/NL/21 andWigeon/ SC/21). Additionally, at 72 hpi, differentiated human airway cultures infected with Scaup/GA/22 and Eagle/FL/22 exhibited higher viral loads as compared to a representative seasonally circulating rg-A/Texas/71/ 2017 (H3N2) virus (Fig. 4F) (P≤0.001). In contrast,Widgeon/SC/21 titers trended lower, while CK/NL/21 titers were statistically lower than rg-A/ Texas/71/2017 (H3N2) (P=0.001). Protective antibody immunity to influenza virus targets the HA and NA and is an important consideration when assessing the risk posed by zoonotic influenza viruses16. Using a set of 48 human serum samples obtained fromblooddonors aged 18 to 46 years, we examined the presence of such cross-reactive antibodies. As expected, there were generally no significant neutralizing titers (serum dilution >1:20) to H5 HA protein (Fig. 4G). In contrast, an enzyme-linked lectin assay (ELLA) targeting antibodies to the N1 protein revealed the geometric mean titers (GMTs) of antibodies against both Wigeon/SC/21 and Eagle/FL/22 to be equivalent to those of antibodies against the seaso- nal CA/04 (H1N1)pdm09 NA in this serum panel (Fig. 4H). The NA proteins of the A(H5N1) viruses of clade 2.3.4.4b and those of A(H1N1) pdm09 viruses have 89.6% amino acid identity, with considerable conservation at some antigenic sites19, 20. Antiviral susceptibility of clade 2.3.4.4b viruses To evaluate whether the available antiviral therapies would be effec- tive against the A(H5N1) clade 2.3.4.4b viruses, we examined the phenotypic susceptibility of these viruses to antivirals from two U.S. FDA–approved classes: the NA inhibitors (NAIs) oseltamivir and zanamivir and the active metabolite of the PA endonuclease inhibitor baloxavir marboxil. All six viruses tested (both reassortant and native constellations) had IC50 or EC50 values comparable to those of drug- susceptible human A(H1N1)pdm09 influenza reference viruses (Table 2). Additionally, genotypic M gene analysis revealed the absence of amino acid changes associatedwith reduced susceptibility to the adamantane class of drugs (Table 2). Discussion With the spread of A(H5N1) viruses throughout the United States and the detection of an infection in a human21, the increased viru- lence of the reassortant viruses is of considerable concern. Except for pockets of endemic clade activity in South and Southeast Asia, clade 2.3.4.4b viruses have predominated over other A(H5Nx) clades over the past 18 months. Clade 2.3.4.4b viruses have become entrenched in Asia, in Europe, and probably in parts of Africa. The WHO update (covering September 2021 to February 2022) repor- ted 26 cases of 2.3.4.4b infection in humans, comprising 25 cases of A(H5N6) infection in China and one case of A(H5N1) in the United Kingdom, demonstrating the zoonotic transmission potential of these viruses3. From a public health perspective, the increased pathogenicity of the reassortant A(H5N1) viruses is of significant concern. However, this is tempered by the avian virus-like char- acteristics of the viruses with respect to their receptor binding preference and their pH of HA activation. Modification of these characteristics are likely required to enable sustained human-to- human transmission, although only a few amino acid changes among various influenza proteins are needed to switch these properties during adaptation in mammals22, 23. In addition to the representative viruses we assayed phenotypically, we did not identify any mutations previously associated with 6′SLN-linked sialic acid binding or alterations in pH of HA activation in the viruses we sequenced. To date, a single case of human A(H5N1) infection in an individual involved in poultry culling has been detected in North America during the current outbreak21. This individual underwent isolation and oseltamivir treatment and recovered after experiencing only minor symptoms. Publicly deposited sequence from samples collected from this case (albeit partial and missing PB2 and NP seg- ments) were of Eurasian origin. Zoonotic transmission is likely to continue if this virus remains present in North American wild birds, and vigilance must be maintained as these birds continue their migrations. While direct translation of animal model findings to humans must be done cautiously, future zoonotic infections with the reassortant A(H5Nx) viruses may well include severe cases. Con- sistent with this possibility is the recent identification of a severe reassortant virus infection of a child in Ecuador24. The A(H5Nx) clade 2.3.4.4 viruses originally identified in NAm in 2014–2015 ultimately disappeared from avian species for reasons that are unclear25, but previous experience is unlikely to predict future events in this situation. Like the currentNAmclade 2.3.4.4b viruses, the 2014–2015 viruses reassorted soon after detection on the continent, but this reassortment was not associated with changes in mammalian pathogenicity26. In contrast, our study with contemporary 2.3.4.4b viruses revealed that reassortants containing increasing numbers of NAm gene segments exhibited enhanced virulence with neurological involvement in mammalian models, including ferrets. However, these viruses retained avian-like receptor specificity, did not possess mole- cular markers of mammalian adaptation, or the ability to transmit between ferrets. Clade 2.3.4.4b viruses have thus far been associated with infections within a European mink farm27, spread among aquatic mammals in the Americas28–30, and reported identification from a variety of other NAm mammals including foxes, skunks, bobcats, mountain lions, and bears31. The increasing prevalence of 2.3.4.4b viruses are also changing the dynamics of disease in Europe, with the potential for transition from epizootic to enzootic status32. Our data highlight how quickly things can change in a natural system, and the potential for further A(H5Nx) reassortment and phenotypic diversifi- cation will only increase as the unprecedented global distribution of these viruses broadens. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 6 Methods Ethics statements and animal husbandry All animal studies including co-housing of chickens and ferrets in the same bioisolator were approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (IACUC, pro- tocol number 428) in accordance with the guidelines established by the Institute of Laboratory Animal Resources, approved by the Gov- erning Board of the US National Research Council, and carried out by ) (R ec ip ro ca l e nd po in t d ilu tio n, lo g 2 A 0 5 10 0.0 0.2 0.4 0.6 3'-SLN ( g/mL) O .D . ( 49 0 nm ) Eagle/FL/22 Wigeon/SC/21 CA/04 (H1N1)pdm09 B 0 5 10 0.0 0.2 0.4 0.6 6'-SLN ( g/mL) O .D . ( 49 0 nm ) Eagle/FL/22 Wigeon/SC/21 CA/04 (H1N1)pdm09 C 4.04.55.05.56.06.57.0 1 2 3 4 5 6 pH /m L) T C ID V iru s tit er ( lo g 1 0 50 CA/04 (H1N1)pdm09 Wigeon/SC/21 Eagle/FL/22 G H -N A a nt ib od y tit er ( IC 50 , l og 2) 0 5 10 15 E 1 12 24 36 48 72 0 2 4 6 8 10 Hours post-inoculation Wigeon/SC/21 Eagle/FL/22 Ck/NL/21 Eagle/NC/22 Hawk/NC/22 Scaup/GA/22 *** *** T C ID /m L) V iru s tit er ( lo g 1 0 50 F 1 24 36 48 72 0 2 4 6 8 10 Hours post-inoculation (3/3) (2/3) (1/3) (3/3) (1/3) (2/3) (3/3) **** *** **** Wigeon/SC/21 rgA/TX/17 (H3N2) Eagle/FL/22 Ck/NL/21 Eagle/NC/22 Hawk/NC/22 Scaup/GA/22 D 37.0 0 2×10 4 4×10 4 6×104 Temperature (°C) R el at iv e lu m in es ce nt un its Eagle/FL/22 Wigeon/SC/21ns 2 4 8 16 H I T ite r T C ID /m L) V iru s tit er ( lo g 1 0 50 Fig. 4 | Phenotypic properties of North AmericanHPAI Influenza A(H5N1) clade 2.3.4.4b viruses. A, B Solid-phase binding of A(H5N1) viruses to biotinylated sia- lylglycopolymersA3’-SialLacNAc-PAA-biotin (3’-SLN) orB6’-SialLacNAc-PAA-biotin (6’-SLN), representing galactose-linked sialic acids α2,3-SA (the avian virus pre- ferred receptor) and α2,6-SA (the human virus preferred receptor), respectively. The data are shown as the mean ± SD from duplicate wells and representing one of two independent experiments. C Kinetics of pH inactivation of the Wigeon/SC/21, Eagle/FL/22, and CA/04 (H1N1)pdm09 viruses at 37 °C. The data are shown as the mean ± SD from triplicate wells representing one of three independent experi- ments. D Minireplicon polymerase activities of Wigeon/SC/21 and Eagle/FL/22 at 37 °C. The data are shown as the mean± SD of 3-4 measurements over the hypo- thesized pH range for avian viruses, and 2measurements over previously described ranges for control virus CA/04 (H1N1)pdm09, and representing one of three independent experiments. NS = not significant as determined by paired, two-tailed t-test. E Viral replication kinetics in Calu-3 cells. Cells were inoculated at anMOI of 0.001 and incubated at 37 °C. The data are shown as themean ± SD from triplicate wells and representing one of two independent experiments. F Viral replication kinetics in primary differentiated human airway cultures. Cultures were inocu- lated at an MOI of 0.005 and incubated at 37 °C. The data are shown as the mean ± SD from triplicate culture inserts and representing one of two indepen- dent experiments. The number of inserts with viral replication out of the total is indicated in parentheses. Statistical significance (one-way ANOVA) was deter- mined by comparison to rg-A/Texas/71/2017 (H3N2) at 72hpi. G, H Neutralizing antibody levels in human serum samples (n = 48) against HA protein (asmeasured by HI assay, dotted line indicates limit of detection of 1:10 serum dilution) or NA protein (as measured by ELLA assay). Points joined by lines represent values for the same individual for the individual antigens tested. *P < 0.05, ***P < 0.001, ****P < 0.0001. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 7 trained personnel working in a United States Department of Agri- culture (USDA)-inspected Animal Biosafety Level 3+ animal facility in accordance with all regulations established by the Division of Agri- cultural Select Agents and Toxins (DASAT) at the USDA Animal and Plant Health Inspection Service (APHIS), as governed by the United States Federal Select Agent Program (FSAP) regulations (7 CFR Part 331, 9 CFR Part 121.3, 42 CFR Part 73.3). Animal holding rooms were on a 12 h light/12 h dark cycle, with dry-bulb temperatures set below each species lower critical temperature to minimize potential heat stress (71 °C ± 1 °C with alarm points beyond this value). Room humidity was set to 45%, with active humidification of each room allowing ≥40% humidity during winter months. Human sera were purchased from a commercial provider (BioIVT, Hicksville, NY)who played no role in our study. The specimenswere not collected specifically for our study, and we had no access to the subject identifiers linked to the specimens. This study utilized USDA-classified select agents and A(H5N1) viruses used herein are subject to the guidelines of, and compliance with, requirements discussed in Title 9 (CFR Parts 121 [Possession, Use, and Transfer of Select Agent Toxins] and 122 [Importation and Transpor- tation of Controlled Organisms and Vectors]). Cell culture Madin–Darby canine kidney (MDCK) cells (ATCC CCL-34) and African green monkey kidney (Vero) cells (ATCC CCL-81) were grown in cul- ture in Modified Eagle’s Medium (MEM) (CellGro) supplemented with 5% fetal bovine serum (FBS) (HyClone), 1mM L-glutamine, and 1× penicillin/streptomycin/amphotericin B (Gibco). Human embryonic kidney (HEK293T) cells (ATCC CRL-3519) were grown in culture in OptiMEM (Gibco) supplemented with 10% FBS and 1× penicillin/ streptomycin/amphotericin B. Human airway epithelial (Calu-3) cells (ATCC HTB-55) were grown in culture in MEM supplemented with 10% FBS, 1mM L-glutamine, 1mM sodium pyruvate, and 1× penicillin/ streptomycin/amphotericin B. All cells were maintained at 37 °C in 5% CO2. Primary differentiated human airway cultures were purchased from MatTek and maintained as discussed in the replication kinetics section. Influenza virus propagation and titration The A(H5N1) influenza viruses were isolated from wild birds in the allantoic cavities of 10-day-old embryonated chicken eggs (eggs) at 35 °C for up to 48 h. Seasonal influenza A(H1N1) or rg-A(H3N2) viruses were grown in MDCK cells at 37 °C for up to 72 h. Viral titers were determined by injecting 0.1mL of 10-fold dilutions of virus into the allantoic cavities of 10-day-old eggs and then calculating the 50% egg infectious dose (EID50) or by inoculating MDCK monolayers and then calculating the 50% tissue culture infectious dose (TCID50) by the method of Reed and Muench33. The lower limit of virus detection was 1.0 log10 TCID50/mL or 1.0 log10 EID50/mL. Replication kinetics Calu-3 cells (approximately 9 × 105 cells/well in 6-well plates) were washed with phosphate-buffered saline (PBS) and inoculated with virus (MOI of 0.001) in 2.0mL of infection medium (MEM, 1% bovine serum albumin [BSA, Sigma-Aldrich], 0.3–1μg/mL TPCK-trypsin [for non-A(H5N1) viruses], 1× penicillin/streptomycin/amphotericin B).One hour later, the inocula were removed, the monolayers were washed with PBS, and the supernatants were replaced with 3.0mL of infection medium. Primary differentiated human airway cultures (MatTek, AIR- 100) on trans-well inserts were cultured at an air-liquid interface using manufacturer providedmedium in the basal chambers andnomedium the apical chambers.Cells werewashed and inoculated (MOI of 0.005), with the addition of a lowpH2.0physiological salinewash to inactivate residual virus. No medium was added back to the apical chambers. At each timepoint, 200 µL of BEBM (Lonza) supplemented with 1% BSA was added to the apical chambers for 15min at 37 °C, then harvested. All cells or insert supernatants were sampled at 1 to 72 hpi as indicated, Table 2 | Susceptibility of North American HPAI A(H5N1) clade 2.3.4.4b to approved antiviral drugs Influenza A virus Subtype Susceptibility to antiviral drugs PA inhibitor (baloxavir)a NA inhibitorsc M2 inhibitor Mean EC50± SD (nM) Fold changeb Oseltamivir Zanamivir (amantadine)e Mean IC50 ± SD (nM) Fold changed Mean IC50 ± SD (nM) Fold changed Wigeon/SC/21 H5N1 0.16 ± 0.05 <1.0 1.25 ± 0.65 4.4 0.22 ± 0.03 1.1 S Eagle/FL/22 H5N1 0.22 ± 0.06 1.1 0.98 ±0.57 3.4 0.19 ± 0.04 1 S Ck/NL/21 H5N1 0.09 ± 0.09 <1.0 1.26 ± 0.62 4.4 0.20 ± 0.04 1 S Eagle/NC/22 H5N1 0.52 ± 0.04 2.7 1.07 ± 0.56 3.8 0.18 ± 0.05 <1.0 S Hawk/NC/22 H5N1 0.27 ± 0.03 1.4 1.34 ± 0.68 4.7 0.20 ± 0.04 1 S Scaup/GA/22 H5N1 0.17 ± 0.08 <1.0 1.13 ± 0.90 4 0.21 ± 0.04 1.1 S Reference influenza viruses rg-A/CA/04 (PA I38-WT) (H1N1) pdm09 0.19 ± 0.04 1 N/A N/A N/A N/A R (S31N) rg-A/CA/04 (PA I38T) (H1N1) pdm09 15.32 ± 3.13 80.6 N/A N/A N/A N/A R (S31N) A/Denmark/524/ 2009 (NA H275-WT) (H1N1) pdm09 N/A N/A 0.29 ± 0.65 1 0.20 ± 0.02 1 R (S31N) A/Denmark/528/ 2009 (NA H275Y) (H1N1) pdm09 N/A N/A 118.81 ± 0.65 416.4 0.24 ±0.05 1.2 R (S31N) aReduction in plaque formation number in virus-infected MDCK cells at 72 hpi or 96 hpi. Mean values (nM) ± SD from three independent dose-response curves are presented bFold change relative to the baloxavir susceptibility of reverse genetics (rg)-derived A/CA/04 (H1N1)pdm09 virus containing PA I38 (a baloxavir-susceptible genotype). A/CA/04 (H1N1)pdm09 containing PA I38T (a genotype with reduced susceptibility to baloxavir) is provided for comparison. cReduction in fluorescence signal from NA-cleaved MUNANA substrate. Mean values (nM) ± SD from four independent dose-response curves are presented. dFold change relative to the NAI susceptibility of A/Denmark/524/2009 (H1N1)pdm09 containing NA H275 (a genotype with normal inhibition by NAIs). A/Denmark/528/2009 (H1N1)pdm09 containing NA H275Y (a genotype for which inhibition by oseltamivir is highly reduced) is provided for comparison. eSusceptibility to adamantanes was based on the absence of substitutions at M2 residues known to mediate adamantane resistance (amino acids 26, 27, 30, 31, and/or 34). N/A not applicable to specific inhibitor analysis, S amantadine-susceptible virus, R amantadine-resistant virus. Amantadine resistance-associated mutations are shown in parentheses. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 8 and virus titers (log10 TCID50/mL) were determined in MDCK cells by the method of Reed and Muench33. Data are presented for one of two independent experiments conducted using triplicate wells/inserts for each time point and for each virus among each cell line. Receptor binding assay Flat-bottom, 96-well immune assay plates (ThermoFisher) were coated with 10 µg/mL fetuin (Sigma-Aldrich) at 4 °C overnight then washed three times with washing buffer (PBS with 0.01% Tween 80). Plates were blocked with PBS containing 1% BSA then incubated at 4 °C overnight with 32 HA units of the Wigeon/SC/21 A/(H5N1), Eagle/FL/ 22 A/(H5N1), or CA/04A/(H1N1)pdm09 viruses. Plates were incubated with biotinylated sialylglycopolymers: 3’-SialLacNAc-PAA-biotin (3’- SLN) (Glycotech) or 6’-SialLacNAc-PAA-biotin (6’-SLN) (Glycotech), serially diluted in the reactionbuffer (PBSwith0.02%Tween80, 0.02% BSA, and 5 µMoseltamivir carboxylate). After incubation for 2 h at 4 °C, the plates were washed and incubated for 1 h with horseradish peroxidase-conjugated streptavidin (Invitrogen; diluted 1:2000) in blocking solution. After washing, the plates were incubated with o-phenylenediamine dihydrochloride (Sigma-Aldrich) at room tem- perature for 10min. The reaction was stopped by adding 1 N H2SO4 (Fisher Chemicals), and the absorbance was measured at 490nm in a Synergy H1 microplate reader (BioTek). Syncytium assay Syncytium formation in vero cells was assayed as described34. Briefly, vero cells were seeded in a 12-well plate (1.5 × 105 cells/well) and incu- bated at 37 °C overnight. The cells were then washed with PBS, inoculated with each virus at anMOI of 3.0, and incubated at 37 °C for 1 h. The inocula were then removed and replaced with 1.0mL of infection medium. At 6 hpi [for the A(H5N1) viruses] or 24 hpi [for the CA/04 (H1N1)pdm09 virus], the supernatants were removed, the cells werewashedwith PBS, 1.0mLof infectionmedium supplementedwith 5 µg/mL of TPCK-treated trypsin was added, and the plates were incubated at 37 °C for 10min. The infection medium was then removed, and residual trypsin was neutralized with 1.0mL of MEM supplemented with 5% FBS. The supernatants were replaced with 0.5mL of PBS, the monolayers were examined visually to verify their confluence, then the PBS was replaced with an equal volume of pH- adjusted buffer (pH 5.5–6.0) for 5min. The pH buffer supernatants were then removed, and the cells were washedwith PBS and incubated in 1.0mL of MEM supplemented with 5% FBS at 37 °C for 3 h. The cells were fixed and stained using the Hema 3 Stat Pack (Fisher Scientific). Micrographs were obtained using a Nikon Eclipse TS100 microscope with a Zeiss Axiocam ERc 5 s camera at 100× total magnification. The HA activation pH was defined as the highest pH at which syncytium formation was observed. The resolution of this assay was 0.1 pH units. Mock-inoculated cells (treatedwith PBSonly) and cells inoculatedwith CA/04 (H1N1)pdm09viruswereused as negative andpositive controls, respectively. pH of inactivation The A(H5N1) influenza viruses were standardized to a concentration of 1.6 × 107 TCID50/mL and incubated with pH-adjusted buffer at 37 °C for 1 h. Samples were neutralized by adding infection medium. The infectious titersof neutralized samplesweredetermined inMDCKcells by TCID50 assay33. The resolution of this assay was 0.2 pH units. The CA/04 (H1N1)pdm09 virus was used as a positive control. Enzyme-linked lectin assay (ELLA) The presence of NA-specific antibodies was determined in ELLAs35. Tested human sera samples were purchased from a commercial ven- dor (BioIVT). The vendor had no role in the study, the samples were not collected specifically for this study, and no subject identifiers linked to the specimenswere available to the investigators. Briefly, flat- bottom, 96-well plates (Thermo Scientific) were coated with fetuin (Sigma-Aldrich) at 25 µg/mL in 0.1M PBS at 4 °C for 48 h. After block- ing, heat-inactivated sera (inactivated at 56 °C for 1 h) were serially diluted in Dulbecco PBS (DPBS) (Gibco) supplemented with 1% BSA and 0.5% Tween 20 and added to the plates. This was followed by the addition of a standardized reverse genetics (rg)–derivedH6Nx antigen (virus). The plates were incubated at 37 °C for 16–18 h andwashedwith PBS containing 0.05% Tween 20, then horseradish peroxidase- conjugated peanut agglutinin (Sigma-Aldrich) was added at 1μg/mL and the plates were incubated for 2 h at room temperature. The plates were then washed and 3,3’,5,5’-tetramethylbenzidine (Sigma-Aldrich) was added. The color reaction was stopped after 10min by adding 1 N H2SO4. The plates were read at 450 nm for 0.1 s in a Synergy H1 microplate reader. The NI titers were defined as the reciprocal of the last dilution that resulted in at least 50% inhibition. Phenotypic susceptibility to neuraminidase inhibitors The neuraminidase (NA) inhibitors oseltamivir carboxylate (oseltami- vir) and zanamivir were purchased from MedChem Express. NA sus- ceptibility was determined by fluorometric assay using the substrate 2′-(4-methylumberlliferyl)-α-D-N-acetylneuraminic acid (MUNANA) (Sigma-Aldrich) with additional modifications36, 37. Influenza viruses were standardized to equivalent NA activity and incubatedwith 10-fold dilutions of each NA inhibitor (at concentrations of 5 pM to 50 µM). The fluorescence signal of the released 4-methylumbelliferone was measured using a Synergy H1microplate reader at excitation/emission (Ex/Em) wavelengths of 360nM/460 nM. The 50% inhibitory con- centrations (IC50s)were estimated fromdose-responsecurvesby using the sigmoidal, four-parameter logistic non-linear regression equation (inGraphPadPrismv9). The results are representative of the combined values of at least three independent dose-response curves and include influenza A/Denmark/524/2009 (H1N1)pdm09 (NA H275, NAI suscep- tible), and A/Denmark/528/2009 (H1N1)pdm09 (NA H275Y, NAI reduced susceptibility) as reference viruses. Phenotypic susceptibility to endonuclease inhibitors The active metabolite of baloxavir marboxil, baloxavir acid (BXA), was purchased from MedChem Express. BXA susceptibility was deter- mined by plaque-reduction assay. MDCK cells (106 cells/well in 6-well plates) were inoculated with a virus volume previously determined to yield approximately 50–100 plaque-forming units (PFUs). At 1 hpi, the cells were washed and overlaid with MEM containing 0.45% immunodiffusion-grade agarose (MP Biomedical), 0.1% BSA, 1μg/mL TPCK-trypsin (for non-A(H5N1) viruses), and 10-fold dilutions of BXA (1 pM to 1μM). At 72 hpi, the overlays were removed, and the cell monolayers were stained and fixed with 1% crystal violet, 10% for- maldehyde. The number of PFU per well was calculated, and the 50% effective concentrations (EC50s) were determined by using the log (inhibitor) versus response logistic nonlinear regression equation (in GraphPad Prism v9). The results are representative of three indepen- dent dose-response curves and include rg–derived CA/04 A(H1N1) pdm09 viruses with the wild-type PA I38 (BXA sensitive) or the PA I38T substitution (reduced susceptibility to BXA) as reference viruses38, 39. Genotypic susceptibility to M2 protein inhibitors Susceptibility to M2 protein inhibitors (amantadine, rimantadine) was determined from theM2 splice product of the full-lengthM gene, with analysis of theM2 amino acid substitutions (residues 26, 27, 30, 31, and 34) mediating resistance. Generation of reverse-genetics (rg) viruses Influenza virus gene segments were amplified from viral RNA using gene-specific primers (Integrated DNA Technologies, Supplementary Table 5) and cloned into the influenza A virus rg dual-promoter expression vector pHW200040. To generate rg viruses, plasmids Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 9 encoding cDNAs of all eight genomic RNA segments were transfected into HEK293T cells by using Lipofectamine 3000 reagent (Thermo Fisher). At 48 h post-transfection, the cell supernatant was harvested, and 0.2mL of the supernatant was injected into 10-day-old embryo- nated chicken eggs to propagate the virus. Viruses used in ELLAs were generated with the HA gene of A/Teal/Hong Kong/w312/1997 (H6N1), all internal gene segmentswere fromA/PuertoRico/8/1934 (H1N1), and the NA gene was from either Wigeon/SC/21 (H5N1) or Eagle/FL/22 (H5N1). rg-A/H1N1 and rg-A/H3N2 viruses containing PA substitutions conferring baloxavir reduced susceptibility were generated in pre- vious studies38, 39. Minireplicon assay Influenza virus genes encoding PB1, PB2, PA, and NP were cloned into pHW2000 as described previously, propagated in Top 10 competent cells (Invitrogen), and purified with a HiSpeed Plasmid Maxi Kit (Qiagen). HEK293T cells (1.5 × 105 cells/well in a 48-well plate) were transfected (using Mirus TransIT-LT1 transfection reagent) with the virus plasmids, a pPolI-358 NP firefly luciferase reporter gene for influenza polymerase activity (kindly provided by Megan Shaw, Mount Sinai School of Medicine, New York, NY), and a pCMV-β- galactosidase plasmid for transfection control normalization. After 24 hpi at 37°C, themonolayers were lysed with 0.2mL of passive lysis buffer (Promega) and the supernatants were clarified by centrifuga- tion. The ratio of luciferase activity (as measured with the Luciferase Assay System [Promega]) to β-galactosidase activity was determined using 15 µL of clarified supernatant. Data shown are representative of one of three independent assays using at least triplicate measures for each sample. Sanger sequencing Viral RNA was extracted using a RNeasy Mini Kit (Qiagen), and cDNA was synthesized using a OneStep RT-RCR Kit (Qiagen). PCR amplifi- cation of influenza A virus gene segments was performed using pri- mers (sequences available upon request). PCR products were extracted from 1.5% agarose gel by using a QIAquick Gel Extraction Kit (Qiagen). Sanger sequencing was performed by the Hartwell Center at St. Jude Children’s Research Hospital on an ABI Prism capillary sequencer (Applied Biosystems) and assembled with DNASTAR Lasergene version 15.3.0, 422. Illumina sequencing Viral RNA was extracted using a RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the Superscript IV First-Strand Synthesis System (Invitrogen). The influenza A virus gene segmentswere amplified using modified universal primers in amulti-segment PCR as described41. PCR products were purified using Agencourt AMPure XP beads according to the manufacturer’s protocol (BeckmanCoulter). Libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer’s protocol and sequenced using a MiSeq Reagent Kit v2 (300 cycles) on a MiSeq System (Illumina). Sequencing readswere thenquality trimmedandassembledusingCLC Genomics Workbench (version 22.0.1). A total of 423 gene segment sequences obtained from53 isolates in this studyweredeposited in the Influenza Research Database and are available under GenBank acces- sion numbers presented in Supplementary Table 3. Phylogenetic analyses Sequences other than those found in this study were retrieved from the National Center for Biotechnology Information Influenza Virus Sequence Database and from the EpiFlu database of the Global Initia- tive on Sharing All Influenza Data (GISAID42). Sequences were then aligned, and ends were trimmed to equal lengths with BioEdit sequence alignment editor software (v.7.2.5). Similar sequences, including sequences obtained in this study, were removed, and phylogenetic relationships were inferred by the neighbor-joining method from 500 bootstrap values; topology was confirmed by the maximum likelihood method43; and evolutionary analyses were con- ducted with the MEGA 7 software44. Tanglegram Additional North American and Eurasian influenza HA and PB2 gene sequences isolated from May 2021 to May 2022 were obtained from the GISAID database (Supplementary Table 4). Redundant PB2 se- quences were eliminated by setting a sequence identity threshold of 97%, using CD-HIT (v.4.8.1)45. Twenty-five non-redundant PB2 sequences from the GISAID database were combined with 14 stu- died North American A(H5N1) sequences by using BioEdit (v.7.2.5). Using the default parameters of Augur (v.15.0.1)46, the sequences were aligned, and a phylogenetic tree was constructed. Furthermore, a PB2 gene time-resolved tree was generated using TreeTime (v.0.8.6)47, which is included in Augur. Using exclusively the 14 studied A(H5N1) sequences, an HA gene time-resolved tree was generated through the same Augur process. These data were visualized using Auspice (v.2.29.1)48. Virulence and transmission in ferrets Four-to-six-month-old outbred influenza-seronegative male ferrets (Triple F Farms, Sayre, PA, USA) were lightly anesthetized with iso- flurane and inoculated intranasally with 106 EID50 units of A(H5N1) virus diluted in 1.0mL of PBS. Ferrets (n = 3 to 6 per group) were monitored daily for clinical signs of infection. Characteristics mon- itored included body temperature, weight loss, relative inactivity indices49, ataxia, respiratory symptoms, stool consistency, and neu- ropathologic signs. Animals reaching the humane endpoint, according to an IACUC-approved clinical scoring system, were euthanized. Nasal washes were collected from all surviving ferrets at 1, 3, 5, 7, and 10 days post-infection (dpi). Ketamine was used to induce sneezing. At 3 and 5 dpi (in the initial phenotyping experiment) and 5 dpi (in the follow-up experiment), ferrets (n = 3 per group) were euthanized, and tissue samples were collected from the respiratory tract (nasal cavity, tra- chea, and lungs), the central nervous system (a combination of the brain stem, cerebellum, and frontal lobes), and the small intestine (not examined for Wigeon/SC/21 and Eagle/FL/22). Viral titers were deter- mined in MDCK cells by TCID50 assay. For transmission studies in the initial phenotyping experiment, three ferrets were inoculated intra- nasally with A(H5N1) virus as described above and housed individually in separate cages. Direct-contact transmission was assessed by placing anaïve ferret into the cageof the inoculated ferret at 1 dpi. Ferretswere monitoreddaily, andnasalwasheswere collected at 1, 3, 5, 7 and 10 dpi, starting just before the inoculated and contact ferrets were co-housed. At 25 days post contact, sera were collected from all surviving inocu- lated and contact ferrets and treatedwith receptor-destroying enzyme (Denko) as per manufacturer instructions. Degree of seroconversion was determined by hemagglutination inhibition (HAI) assay50, and discussed in detail in the Serological Testing section. Pathogenicity in mice Groups of 6-to-8-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were lightly anesthetized with isoflurane and inoculated intranasally with 10-fold serial dilutions of virus suspension containing 102 to 106 EID50 units of A(H5N1) virus in 30 µL of PBS. After virus inoculation, mice were weighed daily and monitored for mor- tality (death or loss of ≥25% of their body weight) and any clinical signs of infection for 14 dpi. The 50% mouse lethal dose (LD50) was calcu- lated after the 14-day observation period. For viral replication studies, BALB/cmice (n = 5 per group)were lightly anesthetizedwith isoflurane and inoculated intranasally with 104 EID50 units of each tested virus in 30 µLof PBS. At 5 dpi,micewere euthanized, and their lungs andbrains were collected and homogenized in 1mL of infection medium. Viral Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 10 titers in harvested organs were determined by TCID50 assay in MDCK cells. Transmission in chickens Six-week-old specific-pathogen-free (SPF) white leghorn chickens (Charles River Laboratories, CT, USA) (n = 3 chickens per virus) were inoculated with 106 EID50 units of Wigeon/SC/21 (H5N1) or Eagle/FL/22 (H5N1) virus by the natural routes (0.2mL intranasally, 0.1mL intrao- cularly, 0.1mL intraesophageally, and 0.1mL intratracheally). At 1 hpi, naïve chickens (n = 12 per virus) were co-housed with the donor chicken and all birds were monitored daily for disease signs. Oro- pharyngeal and cloacal specimenswere collected at 2, 4, and6 dpi, and the infectious viral titers were determined by EID50 assay. To evaluate aerosol transmission fromchickens to ferrets, naïve ferrets (n = 3)were housed in a ferret cage that was placed adjacent to a donor chicken cage (distance between cages was 12 inches) within the same bioiso- lator. Ferret nasal washes were collected at 2, 4, 6, 8, and 10 dpi, and infectious viral titers were determined by EID50 assay. All animals were monitored twice daily for disease signs. At 21 dpi, sera were collected from all ferrets to assess the degree of seroconversion by HAI assay as described below. Serologic testing Sera samples ferrets or chickens were treated with receptor- destroying enzyme II (Denka Seiken Co.) at 37 °C overnight, heat- inactivated at 56 °C for 45min. Hemagglutination inhibition (HI) titer was determined by incubating 2-fold serial dilutions of serum sample with 25 µl of 4 hemagglutinating (HA) units (HAU) in 96-well U-bot- tom plates (Corning). Sera and virus mixtures were incubated at room temperature for 45min prior to addition of a 0.5% solution of chicken red blood cells (Rockland Immunochemicals) in PBS and subsequent incubation at room temperature for 30min. The HI titers were recorded as the reciprocal of the highest serum dilution where there was complete inhibition of hemagglutination and reported as endpoint doubling dilution or Log2 as indicated in each figure or table. Pathology Ferret lungs and nasal mucosa were fixed via intratracheal/intranasal infusion with 10% neutral-buffered formalin (NBF) followed by con- tinued immersion in 10% NBF. Tissues were routinely processed and embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Serial sections were subjected to antigen retrieval for 30min at 98 °C before undergoing immunohistochemical labeling of viral anti- gen, using a primary goat polyclonal antibody (US Biological, Swampscott, MA) against influenza A/USSR/1977 (H1N1) virus at a dilution of 1:1000 and a secondary biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology) at a dilution of 1:200. Statistical analysis Data were analyzed using unpaired t-tests, two-way ANOVA with Tukey’s multiple-comparison post hoc test, and univariant log-rank analysis (survival curves) in GraphPad Prism v9. Replicates, group comparisons, and P values are listed in each figure legend. 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Laycock for the valuable editing of themanuscript, the staff of the Animal Resources Center for their excellent care of the research animals, and the Hartwell Center for Bioinformatics and Bio- technology at St. Jude Children’s Research Hospital for their help with the next-generation sequencing. Graphical Figs. 1, 3, and Supplemental Fig. 1 were created using BioRender.com. This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Depart- ment of Health and Human Services, under contracts HHSN272201400006C (R.W.) and 75N93021C00016 (R.W., D.S.), U.S. National Science Foundation (1911955, 2200310, R.W.) award, NIAID/NIH R01AI150745 (R.W.) grant, and by St. Jude Children’s Research Hospital and ALSAC (R.W.). Author contributions All authors actively participated in scientific discussion of the manu- script and provided comments, critiques, and/or approvals prior to submissions and revisions. R.W., L.K., R.P., D.S., and P.V. conceptualized the research project(s). A.K., C.P., J.J., T.J., R.W. conceived the meth- odologies. P.V., L.K., andW.H. provided data visualization. L.K., J.B., E.G., and R.W. administered the projects. A.K., C.P., J.J., T.J., W.H., S.T., J.P., T.F., K.W., J.T., J.C., L.M., A.R., J.D., L.K. acquired the data. R.W., L.K., E.G. wrote the original draft. R.W., L.K., E.G., J.J., C.P., M.T., P.V., W.H. reviewed and edited subsequent drafts. Funding was acquired by R.W. and D.S. Competing interests The authors declare no competing interests. Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 12 http://www.cdc.gov/media/releases/2022/s0428-avian-flu.html http://www.cdc.gov/media/releases/2022/s0428-avian-flu.html https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON434 https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON434 https://doi.org/10.1128/mSphere.00003-16 https://doi.org/10.1128/mSphere.00003-16 https://doi.org/10.2807/1560-7917.ES.2023.28.3.2300001 https://doi.org/10.1101/2023.03.03.531008 https://doi.org/10.1101/2023.03.03.531008 https://www.cidrap.umn.edu/avian-influenza-bird-flu/peru-confirms-h5n1-avian-flu-marine-mammals-part-southward-spread https://www.cidrap.umn.edu/avian-influenza-bird-flu/peru-confirms-h5n1-avian-flu-marine-mammals-part-southward-spread https://www.cidrap.umn.edu/avian-influenza-bird-flu/peru-confirms-h5n1-avian-flu-marine-mammals-part-southward-spread https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022/2022-hpai-mammals https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022/2022-hpai-mammals https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022/2022-hpai-mammals https://doi.org/10.1128/mBio.00430-18 https://doi.org/10.1128/mBio.00430-18 https://doi.org/10.2807/1560-7917.ES.2017.22.13.30494 https://doi.org/10.2807/1560-7917.ES.2017.22.13.30494 https://doi.org/10.21105/joss.02906 https://doi.org/10.21105/joss.02906 Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-38415-7. 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To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2023 Article https://doi.org/10.1038/s41467-023-38415-7 Nature Communications | (2023) 14:3082 13 https://doi.org/10.1038/s41467-023-38415-7 http://www.nature.com/reprints http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America Results Virus pathogenesis and transmission in chickens and ferrets Genotypic and antigenic analyses of clade 2.3.4.4b viruses Pathotyping of additional clade 2.3.4.4b viruses in ferrets Pathology Virus Pathogenesis in the mouse model Phenotypic properties of clade 2.3.4.4b viruses Antiviral susceptibility of clade 2.3.4.4b viruses Discussion Methods Ethics statements and animal husbandry Cell culture Influenza virus propagation and titration Replication kinetics Receptor binding assay Syncytium assay pH of inactivation Enzyme-linked lectin assay (ELLA) Phenotypic susceptibility to neuraminidase inhibitors Phenotypic susceptibility to endonuclease inhibitors Genotypic susceptibility to M2 protein inhibitors Generation of reverse-genetics (rg) viruses Minireplicon assay Sanger sequencing Illumina sequencing Phylogenetic analyses Tanglegram Virulence and transmission in ferrets Pathogenicity in mice Transmission in chickens Serologic testing Pathology Statistical analysis Reporting summary Data availability References Acknowledgements Author contributions Competing interests Additional information