Emergence of mammalian-adaptive PB2 mutations enhances polymerase activity and pathogenicity of cattle-derived H5N1 influenza A virus

Domain: biology
Published: 2025-12-20
Editorial outcome: minor_revision

Abstract
Highly pathogenic avian influenza H5N1 poses an increasing public health risk, particularly following its spillover into dairy cows and associated human infections in the U.S. since March 2024. Here, we systematically identified critical PB2 mutations emerged during avian-to-cattle transmission and subsequent adaptation in cattle, notably PB2 M631L, which conferred pathogenicity in mice comparable to the well-characterized PB2 E627K mutation. Retrospective analysis reveals that PB2 631L also circulated in avian and human H5N1 strains during the 2013–2014 outbreaks in Cambodia and Vietnam. Additional adaptive mutations include established markers (E627K, Q591R, D701N), and novel variants (I647V, G685R, K736R). These mutations enhance polymerase activity by improving the utilization of both bovine and human ANP32A proteins, thereby increasing viral fitness and pathogenicity in mammals. The convergence of these adaptations highlights the elevated zoonotic risk of cattle-adapted H5N1 viruses and underscores the urgent need for heightened surveillance across avian and mammalian hosts.

Introduction
The potential for a pandemic triggered by the avian influenza H5N1 virus has been a major concern for decades1–4. Since its first detection in Hong Kong in 1997, H5N1 virus infections have been reported in over 954 individuals, with 464 fatalities, resulting in a mortality rate of 48.6%5. Since 2021, H5N1 clade 2.3.4.4b viruses have spread extensively among bird populations6. In March 2024, spillover events involving this clade virus were detected in dairy cows in the United States7–9. The virus subsequently spread to hundreds of farms, resulting in infections in various mammalian species10,11, including humans12–14, marking an unprecedented situation in the history of H5N1 outbreaks. Since the onset of the 2024 H5N1 outbreak, 70 cases of human infection have been reported in the United States15.

Infection by the avian influenza virus in mammalian cells is typically constrained by several factors16–19, including receptor preference20, virion stability21, and the activity of the viral polymerase22. The influenza polymerase, a heterotrimeric complex composed of PB2, PB1, and PA proteins, is responsible for viral RNA transcription and replication23. The restriction of avian influenza polymerase activity in mammalian cells can be significantly alleviated by adaptive mutations in the polymerase22. The PB2 E627K mutation is the most frequently identified adaptation site in viruses that have undergone interspecies transmission from avian to mammal hosts24,25. In human seasonal influenza viruses, PB2 residue 627 is predominantly a lysine, a hallmark of mammalian adaption. However, an exception was observed in 2009 pandemic H1N1 strain, which retains the avian-type glutamic acid at residue 627 but harbored a PB2 Q591R mutation, located spatially close to residue 627 within the polymerase structure26. Additional mammalian-adaptive mutations include PB2 D701N, D740N, T271A, and E627V27–30. In addition, it has also been reported that mutations in PB1 and PA proteins enhance the activity of avian influenza polymerase in mammalian cells22,31–33.

The host restriction of avian influenza virus polymerase activity is primarily linked to host species-specific differences in acidic nuclear phosphoprotein 32 protein A (ANP32A)34. Unlike mammalian ANP32A, avian ANP32A contains a 33-amino acid insertion that significantly enhances its support for polymerase activity from viral strains of various origins34,35. Functional and structural studies have shown that host ANP32 proteins play a critical role in forming viral RNA replication platform in conjunction with the viral polymerase36–38. Due to the inability of mammalian ANP32 proteins to effectively support avian influenza virus polymerase, adaptive mutations in the avian viral polymerase during cross-species transmissions have evolved to enhance its capacity to utilize mammalian ANP32 proteins for efficient viral RNA replication39,40.

In this study, we conducted a systematic investigation into the role of the H5N1 viral polymerase in facilitating cross-species transmissions. A series of critical mutations including PB2 M631L mutation within the PB2 protein were identified to enhance polymerase activity in mammalian cells while concurrently leading to increased pathogenicity in mice. We also found that the key mutation PB2 M631L had previously co-occurred in avian and human H5N1 infections during the 2013–2014 Cambodia/Vietnam H5N1 outbreaks. These findings highlight a persistent underestimation of H5N1 avian influenza virus adaptability and underscore the urgent need to strengthen surveillance and comprehensive pathogenicity assessments.

Results
Systematic identification of key mutations enhancing polymerase activity in mammalian cells during avian-to-cattle transmission of H5N1 virus in 2024

To investigate the contribution of the viral polymerase protein of the H5N1 clade 2.3.4.4b virus to cross-species transmission from birds to dairy cows, we selected a representative H5N1 virus strain, A/dairy cow/Ohio/078707-24-12/2024 (cowOH12-H5N1), isolated from a dairy cow (Fig. 1A). This strain was chosen because its PB2 protein sequence was the most prevalent among cattle isolates, with 417 identical sequences found among 1210 total sequences analyzed as of October 30, 2024 (Fig. S1A). For comparison, we selected an H5N1 clade 2.3.4.4b virus, A/chicken/Maine/24-007946-001/2024 (chMA01-H5N1), which was isolated from chicken in the United States in March 2024 and is significantly evolutionarily different from the cattle isolates (Figs. 1A and S1B).

To evaluate the activity of the viral polymerase in mammalian cells, we constructed viral mini-replicon systems for the both cow and chicken H5N1 viruses in human 293T cells. Green fluorescent protein (GFP) was used as a reporter (Fig. 1B), driven by an RNA polymerase I (Pol I) promoter that expressed a viral-like RNA encoding the GFP, flanked by the non-coding regions of the influenza A virus nucleoprotein (NP) segment. The results demonstrated that the H5N1 viral polymerase from cow isolate was highly active in mammalian cells, whereas the chicken H5N1 viral polymerase exhibited significantly restricted activity. To identify the contributions of the polymerase subunits and NP responsible for this difference, we performed one-to-one replacements of the polymerase subunits and NP. Reciprocal exchange of the PB2 segments between the two viruses resulted in a significant reversal of polymerase activity, while replacement of the PB1 segment has only a slight effect (Fig. 1B). Exchanges of the PA and NP segments has no measurable impact. Quantitative assays using firefly luciferase as a reporter confirmed that the PB2 protein from the cow H5N1 virus plays a predominant role in enhancing polymerase activity in mammalian cells (Fig. 1C).

We next monitored the kinetics of three viral RNA species accumulation for two viral polymerases using a primer extension assay. Overall, the levels of mRNA, cRNA, and vRNA synthesized by the cow H5N1 polymerase were significantly higher than those synthesized by the chicken H5N1 polymerase at all time points post-transfection (Fig. 1D). Consistent with above findings, the replacement of chicken H5N1 PB2 with cow H5N1 PB2 was sufficient to enable the chicken H5N1 polymerase to efficiently accumulate all three viral RNA species in mammalian cells (Fig. 1D). These results indicate that the PB2 protein is the primary determinant of the enhanced activity of the cow H5N1 polymerase in mammalian cells.

To further elucidate the differences between the two polymerases responsible for enhanced activity in mammalian cells, we compared the amino acid sequences of the PB2 proteins from the cow and chicken H5N1 viruses. Our analysis revealed a total of 13 distinct amino acids differences between the two PB2 proteins (Figs. 1E and S1C). We then individually introduced these cow-specific PB2 amino acids into the chicken PB2 protein and assessed the resulting changes in polymerase activity. As shown in Fig. 1F, the PB2 M631L substitution significantly enhanced the chicken H5N1 polymerase activity by 23.3-fold. Additionally, substitutions such as PB2 T58A, A129T, V495I and V649I also increased polymerase activity by 1.8- to 9.0-fold (Fig. 1F). While individual mutations improved polymerase activity, none achieved the activity levels of the cow H5N1 polymerase. Therefore, we tested the combination of these amino acid substitutions. Notably, the combination of PB2 A129T, V495I, or V649I with M631L was sufficient to elevate the chicken H5N1 polymerase activity to match that of the cow H5N1 polymerase (Fig. 1G). It is important to note that the PB2 segment of the cattle H5N1 virus was recently identified as being derived from reassortment with North American low pathogenic avian influenza viruses1,9. Here, our functional data further demonstrate that the specific amino acids acquired through this event are the primary drivers of increased polymerase activity observed in the avian-to-cattle cross-species transmission of the H5N1 virus, with the PB2 M631L mutation playing a major role.

The PB2 M631L mutation previously co-occurred in both avian and human H5N1 virus isolates during outbreaks in Cambodia and Vietnam between 2013 to 2014

Given the significant role of the PB2 M631L mutation in cross-species transmission, we analyzed its occurrence across all H5N1 viruses available on GISAID database. Surprisingly, in addition to the recent detection of PB2 631L in human infections linked to cattle in the United States in 2024, this variant was also present in human H5N1 cases in Cambodia and Vietnam during 2013–2014 (Figs. 2A and S2A). Further genomic surveillance revealed that PB2 631L was concurrently circulating in both avian and human H5N1 viruses from those regions with highest frequency observed between 2013 and 2014 (Figs. 2A, and S2A, B). Notably, the PB2 M631L mutation first emerged in an avian influenza virus as early as 2005 in Thailand (Figs. 2A and S2B). These findings suggest that PB2 M631L emerged earlier than previously recognized and may have contributed to past, underappreciated zoonotic events. This underscores the importance of retrospective analysis of archived viral genomes to better understand the early emergence of mammalian-adaptative mutations in H5N1 viruses.

According to records from the World Health Organization (WHO), Cambodia and Vietnam have reported 74 and 130 cases of human H5N1 infections, respectively, since 2003 (Fig. S2C). Specifically, Cambodia documented 26 and 9 human infections in 2013 and 2014, respectively, accounting for 47% of its total cases5. To investigate potential viral factors underlying this spike, we collected and analyzed all available PB2 sequences from H5N1 viruses isolated from both humans and avian sources in Cambodia and Vietnam. Our analysis revealed that all human H5N1 viruses isolated during 2013–2014 harbored the PB2 M631L mutation (Fig. 2B). In contrast, the well-characterized PB2 E627K mutation, though present in roughly half of the other human H5N1 infections reported in the region over the years, was absent in the 2013–2014 human cases that harbored PB2 631L (Fig. 2B). Among H5N1 viruses from avian viruses from avian sources, primarily chickens and ducks, PB2 E627K was detected at low frequencies, while PB2 M631L was presence in 20–50% of local avian isolates during 2013–2015 (Fig. 2B). These findings suggest a strong temporal and geographic association between the increased prevalence of the PB2 M631L variant in poultry and the high incidence of human infections during this period. This supports the hypothesis that PB2 M631L contributed to enhanced zoonotic potential and highlights the importance of ongoing molecular surveillance in animal reservoirs.

We further conducted a phylogenetic analysis of PB2 amino acid sequences from H5N1 viruses isolated in Cambodia and Vietnam during 2013–2014 (Fig. S2D). The analysis indicated that all viruses carrying the PB2 631L clustered within a single monophyletic clade, which was clearly distinct from PB2 631M-containing viruses, located at the base of the phylogenetic tree. The tree topology showed that human isolates were embedded within avian viruses carrying the PB2 631L mutation, supporting the hypothesis of poultry-to-human interspecies transmission during this period. Notably, H5N1 viruses isolated from a civet cat in Cambodia and a tiger in Vietnam also clustered within the PB2 631L clade, suggesting that this variant was capable of infecting a broader range of mammalian hosts. These findings indicate that the PB2 M631L mutation may have facilitated expanded host tropism and contributed to multiple interspecies transmission events, including spillover into both humans and nonhuman mammals.

To investigate whether additional potential adaptive mutations emerged following H5N1 virus infection in humans, we analyzed variations in the PB2 protein from viral sequences obtained from human cases in Cambodia and Vietnam during the 2013–2014 outbreak (Fig. S2E). Despite the limited dataset of only 24 PB2 sequences, we observed a relatively high degree of amino acid variation. For example, the PB2 T521A mutation was present in 10 sequences, alongside other substitutions such as V356I, N456D, T524A, and D740N. Importantly, PB2 D740N has been previously characterized as a mammalian-adaptive mutation29, indicating that these variations may have contributed to enhanced polymerase activity and replication efficiency of avian H5N1 viruses in human cells. These mutations were rarely observed in contemporaneous avian H5N1 viruses, confirming their role in host adaptation (Fig. S2F). Although direct correlations between individual clinical outcomes and specific mutations cannot be established with current data, WHO reports indicated that the 2013–2014 outbreak resulted in 21 deaths among 39 confirmed human infections in Cambodia and Vietnam, corresponding to a mortality rate exceeding 50%5. These findings suggest the high pathogenic potential of H5N1 viruses harboring PB2 631L, particularly when accompanied by additional adaptive mutations that may arise during human infections, and reinforce the importance of genomic surveillance during zoonotic outbreaks.

PB2 M631L mutation confers high virulence in mice, comparable to the canonical mammalian adaptive PB2 E627K mutation

Previous studies have shown that the PB2 M631L mutation enhances polymerase activity in mammalian cells, suggesting a potential role in mammalian adaptation similar to that of the well-characterized PB2 E627K mutation41–44. However, the in vivo pathogenic effects of PB2 M631L remain poorly characterized. To address this, we employed reverse genetics to generate a 2+6 reassortant virus by combining the hemagglutinin (HA) and neuraminidase (NA) segments from the laboratory-adapted A/WSN/1933 (H1N1) virus with the remaining six segments from A/dairy cow/Ohio/078707-24-12/2024 (H5N1). This strategy enabled safe handling of the virus under biosafety level 2 conditions and allowed us to evaluate the impact of polymerase mutations on viral infectivity. We generated all possible recombinant viruses carrying single or combined mutations at PB2 positions 627 and 631. Groups of female BALB/c mice were intranasally inoculated with 2000 PFU of each virus, and body weight and survival were monitored daily over a 14-day period. On day 5 post-infection, lung tissues were harvested for viral titration, histopathological examination, and immunohistochemistry (IHC) analysis.

The results demonstrated that the wild-type PB2 of the cattle-derived H5N1 virus (PB2 627E+631L) enabled efficient infection in mice, leading to mortality between days 7 and 10 post-infection (Fig. 2C, D). In contrast, introducing a reversion mutation at position 631 (PB2 627E+631M) markedly attenuated viral pathogenicity, as evidenced by minimal weight loss and 100% survival. Strikingly, the addition of the E627K mutation on the L631M background (PB2 627K+631M) restored high pathogenicity, causing rapid mortality within 6–7 days. The combination of E627K and M631L (PB2 627K+631L) further augmented virulence, with mice succumbing as early as days 4–6. Consistently, viral titer assays revealed that both PB2 631L and E627K mutations independently conferred elevated viral loads in lung tissues, and their combination produced the highest titers (Fig. 2E). In contrast, the L631M mutation significantly diminished viral replication. Histopathological and immunohistochemical (IHC) analyses of lung tissues corroborated these findings: extensive tissue damage and high viral antigen expression were observed in mice infected with viruses harboring 627K and/or 631L, whereas limited pathology and viral antigen were detected in lungs infected by the PB2 627E+631M variant (Fig. 2F). The WSN virus served as a positive control and confirmed successful infection in all experimental settings. Together, these results provide compelling evidence that PB2 M631L, akin to E627K, significantly enhances mammalian adaptation and contributes to increased pathogenicity of the H5N1 virus in a mammalian host.

Ongoing H5N1 outbreaks in cattle and other mammals have facilitated the emergence of additional mammalian-adapted mutations in PB2

Between March 2024 and June 2025, more than 1073 cattle herds in the United States were confirmed to be infected with H5N1 virus15. This unprecedented scale of cross-species transmission over a relatively short timeframe raises serious concerns about the virus acquiring adaptive mutations that enhance its replication in mammals. To assess this, we analyzed a total of 3028 PB2 sequences from cattle-derived H5N1 viruses available as of March 31, 2025. This analysis identified 172 amino acid substitutions, with several positions showing notably high variation frequencies (Figs. 3A and S3).

Of particular concern, mammalian-adaptive mutations such as E627K and Q591R were found to emerge on the genetic background of the PB2 631L variant, with E627K showing a marked increase in prevalence during 2025 (Figs. 3B and S3). The well-characterized PB2 D701N mutation was also detected, predominately in a distinct genotype D1.1 virus associated with recent H5N1 2.3.4.4b clade infections in dairy cows45. Notably, the PB2 D740N mutation, previously reported in human H5N1 infections in Cambodia and Vietnam (2013–2014), was identified in multiple cattle-derived viral sequences (Figs. 3B and S2E). In addition, PB2 A58T and K670R have shown significant accumulation since September 2024 (Figs. 3B and S3). Collectively, these findings suggest that PB2 mutations emerging in H5N1 viruses circulating among cattle may enhance viral fitness and adaptation in mammalian hosts.

To assess the functional consequences of newly emerged PB2 mutations, 28 amino acid substitutions with either high variation frequencies or potential mammalian-adaptive significance were introduced individually into the PB2 gene of the cow OH12-H5N1 strain and performed by viral mini-replicon system (Fig. 3C). The results showed that multiple PB2 mutations significantly enhanced polymerase activity. As expected, the PB2 E627K mutation led to the most substantial increase, a 19.2-fold enhancement, consistent with its previously observed role in promoting virulence and pathogenicity in mammalian models (Fig. 2C–F). Several other mutations also conferred moderate but notable increases in polymerase activity, such as PB2 R589K, Q591R, I647V, and D701N elevated polymerase activity by 2.2 to 4.4-fold, while PB2 D740N, previously associated with human infections in Cambodia and Vietnam, increased activity by 2.9-fold (Fig. 3C). Taken together, these findings indicate that the widespread and sustained transmission of H5N1 viruses in cattle populations is fostering the occurrence of mammalian-adaptive mutations in PB2. These mutations significantly enhance polymerase activity in mammalian cells, potentially contributing to increased viral infectivity and pathogenicity in mammalian hosts.

In addition to widespread infections in cattle, H5N1 infections have also been reported in other mammals, including humans, cats, red foxes and raccoons, as documented in recent surveillance data5,15. To further investigate potential mammalian-adaptive mutations, we obtained and analyzed PB2 sequences from viruses isolated from nonhuman mammals, particularly those closely related to the cattle-derived H5N1 lineage (Fig. S4). Among these, we identified that the PB2 K736R mutation in viruses isolated from infected cats enhanced polymerase activity by 2.8-fold (Fig. 3D), indicating that this mutation may contribute to mammalian adaptation of the virus. We also observed that the PB2 V596A mutation led to a significant reduction in polymerase activity, although the reason for this effect is currently unknown.

We further analyzed PB2 protein variations in H5N1 viruses associated with human infections, focusing on strains belonging to the B3.13 genotype that originated from dairy cow transmission. Notably, the first human H5N1 isolate (huTX37-H5N1), obtained in late March 202414, contained the mammalian-adaptive PB2 E627K mutation, along with avian-like signatures 631M and 362E41 (Fig. S5). In addition to huTX37-H5N1, all other human H5N1 PB2 sequences harbored the PB2 631L substitution, while none contained E627K substitution (Fig. S5). Functional analysis showed that E627K significantly enhanced polymerase activity by 19.2-fold in the cow OH12-H5N1 mini-replicon system (Fig. 3C), and G362E increased activity by 4.1-fold (Fig. 3E). In contrast, L631M reduced polymerase activity, but the combination of E627K and L631M retained high activity (Fig. 3E). Among additional human PB2 sequences analyzed, all retained the 631L mutation, yet none carried the E627K mutation, reinforcing prior evidence that PB2 M631L plays a significant role in mammalian adaptation. Furthermore, four other mutations (A58T, E249G, I562V, K670R) detected in human isolates were also found in cattle-derived viruses, but did not significantly alter polymerase activity in our assays (Fig. 3E). The expression levels of all PB2 mutant proteins were confirmed to be unaffected by the mutations (Fig. S6A). Overall, these findings suggest that the cow H5N1 virus possesses an inherently robust polymerase function in human cells.

The adaptive mutations in PB2 arising from H5N1 virus transmission in cattle and other mammals facilitate the virus polymerase to utilize both cow and human ANP32 proteins

The host ANP32 protein family is essential for influenza virus RNA synthesis34,46. Specifically, chicken ANP32A (chANP32A) supports the polymerase activity of both avian and mammal-adapted influenza viruses, while human ANP32A (huANP32A) and ANP32B (huANP32B) primarily support only mammal-adapted strains39,40. In contrast, avian ANP32B, ANP32E and human ANP32E do not support influenza virus polymerase activity due to inherent functional limitations47. Phylogenetic analysis indicated that cow ANP32A and ANP32B are closely related to their human counterparts, implying a comparable role in supporting influenza virus replication (Fig. 4A). To test this, we constructed mini-replicons in 293T ANP32A, ANP32B, and ANP32E triple-knockout cells (293T TKO48) complemented with ANP32 proteins from different species (Figs. 4B and S6B). The results indicated that chANP32A effectively supported cow H5N1 polymerase activity, while cow and human ANP32A and ANP32B supported replication to a significantly lesser extent. Among cow ANP32 proteins, ANP32A provided stronger support than ANP32B, mirroring the pattern observed with human ANP32 proteins. Increasing ANP32 expression levels enhanced polymerase activity in all cases (Fig. 4B).

Structural analysis of the complex of influenza virus polymerase and ANP32 protein revealed that most of these adaptive mutations are located at key functional interfaces, including the region adjacent to the ANP32 binding site and the dimerization interface of the viral polymerase complex (Fig. 4C). Consistent with previous studies, these mutations enhance the positive charge in the PB2 627 domain and nuclear localization signal (NLS) domain, thereby strengthening electrostatic interactions with the negatively charged mammalian ANP32 proteins (Fig. 4D)36–38. To assess the functional impact of these mutations, we tested polymerase activity in 293T TKO cells complemented with either avian or mammalian ANP32 proteins (Fig. 4E–G). The avian-specific PB2 L631M mutation only modestly reduced activity with chicken ANP32A but severely impaired polymerase activity with cow and human ANP32A, confirming its role as a mammalian-restrictive mutation (Fig. 4E–G). In contrast, other adaptive mutations had minimal effects on polymerase activity with chicken ANP32A, but significantly enhanced activity when cow or human ANP32A was present (Fig. 4E–G). Taken together, these findings demonstrate that H5N1 viruses circulating in cattle have acquired adaptive PB2 mutations that enhance the polymerase’s ability to utilize mammalian ANP32 proteins, thereby facilitating efficient replication in bovine hosts and increasing the risk of zoonotic transmission to humans.

Mammalian-adaptive PB2 mutations increase the pathogenicity of cattle-derived H5N1 virus in mice

Given the emergence of multiple PB2 mutations in cattle-derived H5N1 viruses that enhance polymerase activity in mammalian cells, these genetic adaptations may also elevate viral virulence, posing a growing public health concern. To systematically evaluate their impact on pathogenicity, we used reverse genetics to generate recombinant viruses containing the HA and NA segments from A/WSN/33 (H1N1) and the six internal gene segments from the cow OH12-H5N1 virus, each engineered with specific PB2 mutations of interest. Female BALB/c mice were intranasally inoculated with these reassortant viruses, and pathogenicity was assessed by monitoring body weight loss, viral titers in lung tissue, and histopathological changes, following the same protocols as in earlier experiment.

Consistent with previous findings, the PB2 L631M mutation significantly attenuated viral pathogenicity in mice, while the PB2 E627K mutation markedly enhanced virulence (Fig. 5A). Several established mammalian-adaptive mutations, including Q591R, D701N, and D740N, led to more rapid weight loss and earlier mortality compared to the wild-type virus, confirming their role in increased pathogenicity (Fig. 5A). In addition, newly identified mutations from this study, such as I647V, G685R, and K736R, also substantially elevated pathogenicity. Notably, I647V induced rapid weight loss and resulted in complete mortality by day 5 post-infection, closely resembling the impact of the E627K mutation. The G362E mutation caused moderately accelerated weight loss, while K670R showed a pathogenic profile similar to the wild-type virus (Fig. 5A). Histopathological analyses revealed extensive lung damage in mice infected with all PB2 mutants, except for L631M, indicating severe pulmonary involvement (Fig. 5B). Moreover, viral titers in lung tissues were elevated in all mutants except G362E and K670R, further supporting their enhanced replication capacity in vivo (Fig. 5C). Taken together, these results demonstrate that both known and newly emerging PB2 mutations in cattle-derived H5N1 viruses contribute to increased viral replication and pathogenicity in mammals, thereby heightening the risk of zoonotic transmission and severe disease.

Methods
Ethics statement and biosafety
All animal experiments were performed according to the protocols approved by the Animal Use and Care Committee of the Institute of Biophysics, Chinese Academy of Sciences (ABSL-2-2025068). Before experimentation, animals were housed in individually ventilated cages under specific pathogen-free conditions, with a room temperature maintained between 20 °C and 24 °C, a 12 h/12 h light/dark cycle, and 40-70% humidity. Food and water were provided ad libitum. Humane endpoints for euthanasia included a body weight loss of 25% or more with no possibility of recovery. The rescue and infection of the recombinant viruses were performed under Biosafety Level 2 (BSL-2) containment at the Institute of Microbiology, Chinese Academy of Sciences. All experiments were approved by the Institutional Biosafety Committee of the Institute of Microbiology, Chinese Academy of Sciences.

Cells
Human embryonic kidney HEK 293T (ATCC, CRL-3216) cells and Madin-Darby canine kidney (MDCK) (ATCC, CCL-34) cells were purchased from the American Type Culture Collection and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, C11995500CP) supplemented with 10% fetal bovine serum (FBS; Gibco, 10437–028), and 1% penicillin-streptomycin (Pen-Strep; Gibco, 15140-122). Human embryonic kidney 293T ANP32A, ANP32B, and ANP32E triple-knockout cells (293T TKO48) were kindly provided by Prof. Xiaojun Wang (State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin, China) and were cultured under the same conditions as those described above for 293T cells. Cells were cultured in humidified incubators at 37 °C with 5% CO2.

Mice experiments
Six-week-old female BALB/c mice were purchased from HuaFuKang Bioscience Co., Ltd (Beijing, China) and were kept in individually ventilated cages under specific pathogen-free conditions, with room temperature maintained between 20 °C and 24 °C on a 12 h dark/12 h light cycle and 40–70% humidity. After acclimating for at least 3 days, mice were randomly allocated into different groups. For infection, mice were randomly allocated into different groups. Mice were anesthetized via intraperitoneal injection of 200 μL of 1.25% tribromoethanol and subsequently inoculated intranasally with 2000 PFU of each virus in 30 μL PBS. Mice were monitored for 2 weeks post-infection, with daily assessment of weight loss and other signs of disease. Any mouse losing ≥25% of its initial body weight was euthanized. To determined viral titers of lungs, three mice per group were euthanized on day 5 post-infection, and viral titers in lung tissues were determined by plaque assays. For histology, tissues were fixed in 4% buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Tissue sections were also immunostained for viral nucleoprotein (NP) with a monoclonal primary antibody (Millipore, MAB8251).

Plasmids
The RNP reconstitution system plasmids (pcDNA-PB2, pcDNA-PB1, pcDNA-PA, pcDNA-NP) of influenza A/chicken/Maine/24-007946-001/2024 (chMA01-H5N1) and A/dairy cow/Ohio/078707-24-12/2024 (cowOH12-H5N1) viruses and the pCAGGS plasmids expressing cow-ANP32A (GenPept accession no. NP_001181948.1), cow ANP32B (GenPept accession no. NP_001030246.1) were generated by gene synthesis (Azenta Life Sciences, China). pPOLI-NP/NCR-GFP was derived from the pPOLI-WSN/NP plasmid by subcloning the enhanced green fluorescent protein (eGFP) gene in place of the original NP ORF. pPOLI-Firefly was generated by replacing the firefly luciferase gene into the ORF region of the pPOLI-WSN/NP plasmid. The reverse genetics vectors of cowOH12-H5N1 were in vitro subcloned to pHW2000 (pHW2000-cowOH12-H5N1 PB2, PB1, PA, NP, M and NS). pHW2000-WSN/HA and pHW2000-WSN/NA were stored in our laboratory54. All mutant plasmids were generated by site-directed PCR mutagenesis (NEB, E0554S) and were identified by DNA sequencing.

Generation of recombinant viruses
Recombinant H5N1 viruses (the wild-type and PB2 mutants) were generated using a previously described eight-plasmid rescue system55. Briefly, 293T cells were co-transfected with pHW2000-H5N1-PB2 (or PB2 mutant), PB1, PA, NP, M, NS, and pHW2000-WSN-HA, NA plasmids. At 48 h post-transfection, the supernatant was harvested and the rescued viruses were plaque purified. The purified viruses were subsequently passaged for two generations on MDCK cells, and viral genomes were sequenced. Viral titers were determined by a standard plaque assay in MDCK cells.

Plaque assay
Confluent monolayers of MDCK cells (2.3 × 105 cells/well, 12-well plate) were seeded the day prior to infection. For each virus assayed, 10-fold serial dilutions (10-1 to 10-6) were prepared. For infection, cells were washed twice with PBS and 300 μL of each viral dilution were added. Viral absorption was allowed to occur for 1 h at room temperature and gently shaken every 15 min. After viral absorption, cells were overlaid with media with a final concentration of 1% low-melting agarose (Sigma-Aldrich, A9414). Infection was allowed to proceed for 3 days at 37 °C with 5% CO2. Subsequently, cells were fixed with 4% paraformaldehyde at 4 °C overnight. The supernatant was then removed and cells stained for 10 min with 1% crystal violet in PBS. Crystal violet was removed and cells carefully washed with water. Plaques were counted and PFU/ml were calculated.

Western blot
Cell pellets were lysed with RIPA Lysis Buffer (Beyotime, P0013B) for 25 min at room temperature, and then the debris were removed by centrifugation at 13,800 g for 15 min. The lysates were denatured with reducing SDS loading buffer and boiled at 95 °C for 5 min. Protein samples fractionated by SDS-PAGE were then transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST and incubated with primary antibodies, followed by secondary antibodies. Protein expression levels were visualized using an enhanced chemiluminescence detection kit (NCM Biotech). Mouse monoclonal anti-β-actin (TransGene, HC201-01) and mouse monoclonal anti-Flag (TransGene, HT201-01) were purchased from TransGen Biotech Co., LTD (Beijing). Rabbit polyclonal anti-PB2 was stored in our laboratory56. Goat anti-rabbit IgG (H&L)-HRP conjugated (Easybio, BE0101-100) and goat anti-mouse IgG (H&L)-HRP conjugated (Easybio, BE0102-100) were purchased from Boao Yijie Technology Co., LTD (Beijing).

Mini-replicon GFP reporter assay
HEK 293T cells in 24-well plates were transfected with a mixture of plasmids pcDNA-PB2 (50 ng), pcDNA-PB1 (50 ng), pcDNA-PA (25 ng), and pcDNA-NP (100 ng) together with pPOLI-NP/NCR-GFP (25 ng) using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s instructions. The plasmids of chicken H5N1 and cow H5N1 polymerase subunits (PB2, PB1, PA) and NP were reciprocally exchanged on a one-to-one basis. After transfection for 24 h, the expression level of GFP was detected by fluorescence microscopy.

Mini-replicon luciferase reporter assay
To examine the effect of the PB2 mutations on viral polymerase activity, HEK 293T cells in 24-well plates were co-transfected with plasmids pcDNA-PB2 (50 ng), pcDNA-PB1 (50 ng), pcDNA-PA (25 ng), pcDNA-NP (100 ng), pPOLI-Firefly (50 ng), and a Renilla luciferase expression plasmid (5 ng) as an internal control for the dual-luciferase assay. To determine the effect of ANP32 proteins on viral polymerase activity, 293T TKO cells were co-transfected with the viral RNP reconstitution system plasmids together with a pCAGGS-ANP32 (10 ng) expression plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. After transfection for 24 h, cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, E1910) and a GLOMAX 96 Microplate luminometer (Promega). The relative polymerase activity was calculated by dividing the Firefly luciferase signal by the Renilla luciferase signal, followed by normalization. All experiments were independently replicated at least three times.

Primer extension analysis
Approximately 106 293T cells were co-transfected with 0.5 μg of protein expression plasmids encoding pcDNA-PB2, pcDNA-PB1, pcDNA-PA, pcDNA-NP and 0.5 μg of the vRNA expression plasmids pPOLI-WSN/NA using Lipofectamine 2000 and Opti-MEM (Gibco, 31985070) according to the manufacturer’s instructions. At 12, 24, 36, and 48 h post-transfection, total RNA was extracted using TRIzol reagent. The steady-state levels of mRNA, cRNA and vRNA were analyzed by primer extension with 32P-labeled specific primers for negative- or positive-sense RNA of NA. The primers used for the detection of the NA-specific RNA species and internal control 5S rRNA were described previously57. The primerextension products for 5S rRNA, vRNA, cRNA, and mRNA were approximately 100 nt, 130 nt, 160 nt, and 170-175 nt in length, respectively, consistent with previous reports58,59. These extension products were analyzed by 6% PAGE gel containing 7 M urea in Tris-borate-EDTA (TBE) buffer and detected by autoradiography.

Bioinformatics analysis
Sequences from H5N1 clade 2.3.4.4b viruses were retrieved from the GISAID database (http://gisaid.org/) for avian, dairy cows, humans or other mammals. Sequences were aligned using MAFFT (v7.526). Specific residues of interest were summarized along with sequence metadata using a custom Python script and plotted using the R program (v4.4.1). The custom code has been deposited on GitHub (https://github.com/laiyuerong/H5N1).

Phylogenetic analysis
PB2 amino acid sequences of H5N1 viruses isolated from human, mammalian, and avian infections in Cambodia and Vietnam during 2013 and 2014 were obtained from the GISAID database. The dataset included 138 sequences derived from avian viruses (of which 92 strains carried the 631M residue and 44 strains carried 631L), 24 from human viruses (all of which possessed the 631L), one from tiger virus (carried the 631L), and one from civet cat virus (carried the 631L). The sequences were aligned using MAFFT (v7.526) and a maximum likelihood phylogenetic tree was constructed using MEGA (v7.0.20) software with 1000 bootstrap replications. The analysis revealed that all sequences with the M631L mutation clustered together, while the 92 sequences carrying 631M were located at the base of the phylogenetic tree. For clearer visualization, 15 representative sequences with 631M were selected and analyzed together with all 631L mutant sequences to generate a final maximum likelihood tree using MEGA (v7.0.20).

Statistics
GraphPad Prism software (v8) was used for statistical analysis. One-way analysis of variance (ANOVA) with Dunnett’s correction and two-way ANOVA with Dunnett’s multiple comparisons test were used for comparisons.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors examine the consequences for viral polymerase activity of PB2 mutations observed recently in H5N1 viruses.\nResults are complemented by basic bioinformatic analysis of the prevalence of these mutations in different hosts and during\ndifferent eras of H5N1 circulation.\nKnowledge gained is as follows. The examination of PB2 mutations observed across mammalian isolates of 2.3.4.4b H5N1\nadvances understanding of the sites in this protein that augment viral polymerase activity in mammalian cells. The\nexperiments with ANP32A/B from chickens, cattle and humans are also valuable and show that cattle ANP32A behaves\nsimilarly to human ANP32A in supporting the replication of different PB2 variant polymerases. This suggests that cow\nANP32 proteins impose similar selective pressure on PB2 as human ANP32 proteins do, such that cattle-adapted viruses\nare likely to have increased zoonotic potential.\nThe work addresses an important topic and delivers some novel information but carries several major weaknesses. The first\nrelates to limited conceptual novelty: The role of PB2 in mammalian adaptation of IAV has been well understood for >15\nyears. That PB2 M631L of the cattle lineage confers the needed improvement of pol activity in mammalian cells was shown\nin 2017 and confirmed recently (refs 37, 41, 42). The second major weakness relates to the rudimentary handling of viral\nsequence data to evaluate the population level dynamics of PB2 631L variant viruses. These issues feature in several of the\ncomments itemized below.\nSpecific comments:\n1. The claim that the PB2 M631L mutation is strongly associated with human infection is not well supported. A rigorous test\nof quantitative enrichment that removes confounders arising due to circumstances of exposure is needed.\na. In Cambodia and VietNam, what proportion of human cases carried M vs. L? What proportion of avian sequences carried\nM vs. L? Do these proportions differ significantly? Was there any ecological pattern in the affected avian species that\nresulted in greater human exposure to L viruses?\nb. In the US, it is obvious that the prevalence of 631L in human cases would stem from human exposure to dairy cattle and\nchickens infected with B3.13 viruses in which this mutation was fixed. The association of 631L with human infection\ntherefore cannot be interpreted as causative.\nc. No methodological information or statistical support is provided for the phylogenetic tree shown in Figure 2E. It is not even\nclear which viral gene is examined. This tree is consequently meaningless and does not support the inference that PB2\n631L promoted interspecies transmission in Cambodia and VietNam in 2013 and 2014.\n2. Figure 2 and throughout: the classification \"avian\" misses the important distinction between wild and domestic birds. In\nthe US in 2024, many poultry outbreaks were seeded from cattle and therefore caused by 631L viruses. These outbreaks\nwere furthermore contained and are therefore evolutionary dead ends. This is a very different situation (in both cause and\nimplications) from 631L increasing in prevalence in wild birds. This distinction needs to be made.\n3. Figure 3 shows that aa changes observed in the PB2 of H5N1 viruses isolated from various mammalian hosts increase\npol activity in human cells. This is valuable information, but interpretation of these findings needs to be presented more\ncarefully.\na. Figure 3 / line 207-209. \"...transmission of the H5N1 virus in cattle herds drive the accumulation of mammalian adaptive\nmutations...\" This conclusion is not rigorously supported. Are variants in PB2 seen at higher frequency in the cattle lineage\ncompared to related H5N1 viruses circulating in wild birds? Have these mutations actually accumulated, or have they been\nobserved transiently?\nb. \"These mutations may contribute to severe disease or even death observed in infected cats\". Line 222. This statement is\nunsupported. Were the mutations referred to associated with severe outcomes in cats? If yes, include that information. If not,\nremove the statement.\n4. Fig 4B needs a western blot showing expression levels of the ANP32 proteins to validate the assumption that increasing\nng of plasmids transfected led to increased protein concentration equivalently across the different ANP32 variants. Similarly,\nFig 4 E-J need to be accompanied by Western blots showing that expression of the different PB2 variants was equivalent.\n5. Discussion, first paragraph inappropriately claims that sequential accumulation of PB2 mutations enabled cross species\ntransmission. The data presented to do not document sequential accumulation of mutations nor investigate the role of these\nmutations in cross species transmission. Indeed, most of the mutations examined happened once H5N1 was established in\ncows and have not been associated with cross species transmission.\n6. Line 146 and elsewhere, it is not clear what is meant by 'clustered cross-species human infections'. Were there\ntransmission clusters? Independent cases clustered in time? Viral strains clustered phylogenetically?\n7. Fig 2f: Were there no human cases with 631M in 2013-2014 in Cambodia and Vietnam? In 2024 in USA?\n8. Fig 2f: This schematic should include an arrow from birds to humans in 2024, reflecting the transmission from poultry to\nhumans in the US.\n9. The titles above Fig 3C, 3E and 3G & Fig 4D, 4E and 4F are inaccurate. These mutations were observed in cows,\nnonhuman mammals or humans. That does not make them adaptive to these species.\n10. Fig 5 omits cattle -to-poultry transmission in the U.S. and the likely role of cattle as an intermediary host in the TX37\nhuman infection.\n11. Line 31: remove \"global\". Pandemics are global by definition.", "reviewer_2": "The recent outbreak of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus has raised concerns about spillover and\nadaptation to humans. In this study, the authors identified amino acid mutations in PB2 that enhanced polymerase activity in\nthe recent H5N1 outbreak. Among these, the PB2-631L mutation was also detected in human H5N1 cases in Cambodia and\nVietnam during the 2013–2014 outbreaks.\nThis is a well-conducted and well-presented study; however, the enhancement of polymerase activity by PB2-631L has\nalready been reported (Gu et al., Nature, 2024). Additionally, several specific points require further clarification.\nMajor Point:\n1. The significance of the PB2-M631L mutation should be thoroughly investigated using the mutant virus. Examining viral\nreplication and pathogenesis in both cell culture and animal models would be of great interest to readers and would\nstrengthen the authors' claims regarding human pathogenesis (Lines 174–177).\nMinor Points:\n1. Figure 2 – The authors identified a high prevalence of PB2-631L in avian H5N1 viruses in North America (2022–2024).\nGiven that the recent outbreak of clade 2.3.4.4b viruses originated in Europe, the authors should analyze the prevalence of\nPB2-631L in European strains as well.\n2. Figure 2 – It is unclear whether the human H5N1 viruses from Cambodia and Vietnam carried the mammalian-adaptive\nPB2-627K mutation.\n3. Line 181 – The reference to Fig. 2G should be corrected to Fig. 2F.\n4. Lines 205–207 – Since the authors did not evaluate polymerase activity with the combination of these mutations, this\nsentence should be removed.\n5. Figure 3D – Did raccoons and foxes become infected through spillover from cows? The authors should provide a\nreference to support this claim. If no such evidence exists, this figure should be removed to avoid confusion.\n6. Figure 3E – The V596A mutation significantly suppressed polymerase activity. The authors should comment on this\nobservation.\n7. Lines 212–223 – The authors should provide data on the prevalence of PB2-627K mutations in the mammalian species\nmentioned.\n8. Line 280 – \"PB2 L631M\" should be corrected to \"PB2 M631L\".\n9. Line 285 – \"Fig. 4F-4J\" should be corrected to \"Fig. 4F-G, 4I-J\".", "reviewer_3": "In this manuscript, the authors systematically examined the role of the H5N1 virus polymerase in facilitating interspecies\ntransmission from avian host to cattle in the United States during 2024. It was previously reported that PB2 M631L mutation\nenhances polymerase activity in mammalian cells and plays a key role in the interspecies transmission of the H5N1 virus\nfrom avian to cow. The authors of this manuscript identified additional PB2 mutations that exhibit a synergistic effect when\ncombined with M631L. They also discovered that the PB2 M631L mutation in avian H5N1 viruses is strongly associated with\nclustered human H5N1 infections during the 2013-2014 Cambodia/Vietnam outbreaks, as well as the 2024 U.S. cross-\nspecies transmissions. The majority of adaptive PB2 mutations localized at the interface adjacent to the ANP32 protein and\nat the dimerization interface of two viral polymerases, which aligns with previous reports. It is important to understand the\nmutations responsible for the H5N1 outbreak in cattle. However, limited studies have been done in this study (only\nmeasuring polymerase activity using a reporter gene). Also, the outcome of the experiments (specific interaction with\nspecies specific ANP32 through PB2 mutations) are similar to what is already well established in the field. Unfortunately,\nthere is not much new data other than the identification of PB2 mutations contributing to mammalian host adaptation.\nLine 53-54: “While adaptations in the PB1 and PA proteins have also been documented, their contributions to enhancing\npolymerase activity are comparatively weaker20,29” – This statement is not accurate since mutations in PA have been\nreported to be a major contributor of enhanced activity of 2009 pH1N1 (PMCID: PMC3126589, PMCID: PMC9576711,\nPMCID: PMC7232137, PMID: 25210166).\nLine 84-85: “This strain exhibited the highest amino acid similarity in the PB2 protein among all cattle isolates, with 417\nidentical sequences out of 1848 total sequences (Fig. 1A). – Figure 1A does not show this data of sequence homology.\nLine 181: Fig. 2G should be Fig. 2F.", "reviewer_4": "The manuscript by Zhang et al. entitled \"Mammalian-adapted mutations in influenza A (H5N1) virus polymerase:\nimplications for cross-species transmission surveillance\" examines amino acid substitutions in the PB2 of H5N1 viruses\nisolated from mammals, particularly cattle, and humans, and their influence on polymerase activity in human 293T cells.\nMany of the amino acid substitutions studied are located in a domain of the PB2 protein (called the 627 domain) that is\nresponsible for interaction with the host factor ANP32. Accordingly, some of these amino acid substitutions confer increased\npolymerase activity in the presence of human or bovine ANP32A/B. Due to the continued circulation of H5N1 viruses in\nbirds and the increasing transmission of these viruses to various mammalian species, including humans, risk assessment of\nthese H5N1 isolates and detection of the emergence of mammalian adaptation is critical. Although the study by Zhang et al.\npresents some interesting results, such as the detailed investigation of individual PB2 exchanges and their influence on\npolymerase activity, the study presented here is primarily a screening approach that suffers from some important and (partly)\nunstated limitations. First, all experiments in this study are based on the well-established minireplicon reporter system, but\nno live virus infection data were performed. This is important because it is unclear how a 23-fold increase in polymerase\nactivity (as observed with the M631L substitution, Fig. 1F) translates into viral replication in mammalian cells and/or to what\nextent viral replication is enhanced by the other substitutions in PB2 (see Fig. 1E). Second, the synergistic effects of i) these\nPB2 substitutions remain largely unaddressed, with the exception of those shown in Figures 1G and 3G, and ii)\ncombinatorial effects of other polymerase proteins (e.g., PA segment) are not mentioned. Third, and most importantly, many of\nthe PB2 substitutions studied here have been previously described by others and the significance of these amino acid\nexchanges for ANP32 usage and the enhanced PB2-ANP32 interaction has been mechanistically\ninvestigated (Idoko-Akoh et al, 2023 Nature Communications; Staller, Carrique et al, 2024\nNature Communications).\nSome more specific comments:\n1. All polymerase reconstitution assays lack confirmation of equal protein levels.\n2. How were the \"relative polymerase activities\" calculated? Were they calculated relative to Renilla luciferase and further\nnormalized?\n3. The statistics of the polymerase assays are misleading. The authors should use SD instead of SEM when comparing the\nvariability of individual data points in a data set.\n4. Figure 2A, B and lines 134-135: What was the prevalence of 631M in PB2 of H5N1 viruses during the same time period?\nWhat was the number of sequences used for this analysis (it is not described in the figure legends or the methods section)?\nIt is hard to follow the authors' argument. While 35 cases of H5N1 were reported in Cambodia and 4 in Vietnam in 2013-15\n(see WHO H5N1 Factsheet), there were 177 cases in Egypt and 10 cases in China during the same period. Was the M631L\nexchange also highly prevalent in these much larger outbreaks? What is the significance of this 631L substitution for\ntransmission of the virus to humans, and how does this fit with the statements in lines 174-181?\n5. Line 181: should refer to Figure 2F\n6. Figure 3B: Where is the M631L substitution shown?\n7. Lines 256-258, the sentence should read: \"To verify this, we constructed mini-replicons in ANP32A, ANP32B, and\nANP32E triple knockout 293T cells (293T TKO) complemented with ANP32A and ANP32B proteins from different species\".\n8. Figure 4B: Are the authors surprised to see that chANP32A, but not any of the mammalian ANP32 proteins tested,\nenhances the polymerase activity of the cow-adapted H5N1 polymerase at low concentrations? This seems counterintuitive\nto the reviewer.\n9. In Figure 4D, the authors should add information about the electrostatic surface shown. What does the blue and red color\nindicate?"}, "author_response": "Point-by-point responses\nReviewer #1 (Remarks to the Author):\nThe authors examine the consequences for viral polymerase activity of PB2 mutations\nobserved recently in H5N1 viruses. Results are complemented by basic bioinformatic\nanalysis of the prevalence of these mutations in different hosts and during different eras of\nH5N1 circulation.\nKnowledge gained is as follows. The examination of PB2 mutations observed across\nmammalian isolates of 2.3.4.4b H5N1 advances understanding of the sites in this protein\nthat augment viral polymerase activity in mammalian cells. The experiments with\nANP32A/B from chickens, cattle and humans are also valuable and show that cattle\nANP32A behaves similarly to human ANP32A in supporting the replication of different PB2\nvariant polymerases. This suggests that cow ANP32 proteins impose similar selective\npressure on PB2 as human ANP32 proteins do, such that cattle-adapted viruses are likely\nto have increased zoonotic potential.\nThe work addresses an important topic and delivers some novel information but carries\nseveral major weaknesses. The first relates to limited conceptual novelty: The role of PB2\nin mammalian adaptation of IAV has been well understood for >15 years. That PB2 M631L\nof the cattle lineage confers the needed improvement of pol activity in mammalian cells\nwas shown in 2017 and confirmed recently (refs 37, 41, 42). The second major weakness\nrelates to the rudimentary handling of viral sequence data to evaluate the population level\ndynamics of PB2 631L variant viruses. These issues feature in several of the comments\nitemized below.\nResponse: We thank the reviewer for recognizing that our work addresses an important\ntopic and provides novel information. Regarding the perceived weaknesses, we have\ncarefully considered the comments and conducted additional experiments to strengthen\nthe study's overall value and comprehensiveness.\nSpecific comments:\n1. The claim that the PB2 M631L mutation is strongly associated with human infection is\nnot well supported. A rigorous test of quantitative enrichment that removes confounders\narising due to circumstances of exposure is needed.\na. In Cambodia and VietNam, what proportion of human cases carried M vs. L? What\nproportion of avian sequences carried M vs. L? Do these proportions differ significantly?\nWas there any ecological pattern in the affected avian species that resulted in greater\nhuman exposure to L viruses?\nResponse: We thank the reviewer for this insightful comment. We have re-analyzed the\nprevalence of the PB2 M631L mutation (now presented in revised Fig. 2A-B). The updated\nfigures clearly illustrate the occurrence of PB2 631L in H5N1 viruses from human infections\nand avian species. Specifically, for Cambodia and Vietnam during 2013-2014, PB2 631L\nwas present in 100% of the human H5N1 infection cases. In avian infections during 2013-\n2015, PB2 631L was found in approximately 20-50% of viruses. Interestingly, the well-\ncharacterized PB2 E627K mutation, though present in roughly half of the other human\nH5N1 infections reported in the region over the years, was absent in the 2013-2014 human\ncases that harbored PB2 631L. This suggests that PB2 M631L may effectively substitute\nfor the mammalian adaptation function typically associated with E627K. Indeed, we have\nfurther supported this hypothesis through animal experiments (revised Fig. 2C-F).\nWe noted that the avian H5N1 virus sequences were predominantly isolated from chickens\nand ducks, which are the primary species monitored in avian influenza surveillance. Given\ntheir close proximity to human habitats, exposure to viruses from these species likely\nconstitutes a significant risk factor for human infection. However, due to limited data,\nestablishing a direct ecological pattern linking specific avian species to greater human\nexposure to PB2 631L-viruses remains challenging. We have added careful descriptions\nfor these results (lines 151-158).\nb. In the US, it is obvious that the prevalence of 631L in human cases would stem from\nhuman exposure to dairy cattle and chickens infected with B3.13 viruses in which this\nmutation was fixed. The association of 631L with human infection therefore cannot be\ninterpreted as causative.\nResponse: We fully agree with the reviewer's point. We have removed such expressions.\nc. No methodological information or statistical support is provided for the phylogenetic tree\nshown in Figure 2E. It is not even clear which viral gene is examined. This tree is\nconsequently meaningless and does not support the inference that PB2 631L promoted\ninterspecies transmission in Cambodia and VietNam in 2013 and 2014.\nResponse: We apologize for this oversight. The phylogenetic tree was constructed based\non PB2 amino acid sequences using the Maximum Likelihood method in MEGA software,\nwith 1000 bootstrap replications. This methodological detail has now been included (lines\n589-600). We agree that the phylogenetic tree alone cannot definitively prove that PB2\nM631L promoted interspecies transmission. However, the tree shows that viral sequences\ncontaining PB2 M631L, including one isolated from a civet cat in Cambodia and another\nfrom a tiger in Vietnam, cluster together. This pattern suggests a potential association\nbetween the PB2 M631L variant and cross-species transmission events from birds to\nmammals. We have revised the figure (now Fig. S2D) and tempered our conclusions\naccordingly, presenting this observation as suggestive rather than conclusive (lines 164-\n171).\n2. Figure 2 and throughout: the classification \"avian\" misses the important distinction\nbetween wild and domestic birds. In the US in 2024, many poultry outbreaks were seeded\nfrom cattle and therefore caused by 631L viruses. These outbreaks were furthermore\ncontained and are therefore evolutionary dead ends. This is a very different situation (in\nboth cause and implications) from 631L increasing in prevalence in wild birds. This\ndistinction needs to be made.\nResponse: We agree with the reviewer's important point regarding the distinction between\nwild and domestic birds. As mentioned above, our analysis for Vietnam and Cambodia\nshowed that the avian viruses were predominantly isolated from chickens and ducks, not\nonly during 2013-2014 but also in other years. This likely reflects the focus of local avian\ninfluenza surveillance on these domestic poultry species. However, due to insufficient data\nto definitively confirm that these domestic poultry were specifically infected with PB2\nM631L variant viruses distinct from those circulating in wild birds, we retained the broader\nclassification \"avian\" for consistency. While we cannot rule out the possibility of human-to-\npoultry transmission in Cambodia and Vietnam during 2013-2014, similar to the cattle-to-\npoultry transmission observed in 2024, it is noteworthy that avian infections with PB2\nM631L viruses were still detected in 2015 and 2017 in the absence of reported human\ncases. This supports the possibility of sustained, low-level circulation of PB2 M631L\nvariants within avian populations themselves during those periods. This information has\nnow been clarified in the manuscript (lines 151-158).\n3. Figure 3 shows that aa changes observed in the PB2 of H5N1 viruses isolated from\nvarious mammalian hosts increase pol activity in human cells. This is valuable information,\nbut interpretation of these findings needs to be presented more carefully.\na. Figure 3 / line 207-209. \"...transmission of the H5N1 virus in cattle herds drive the\naccumulation of mammalian adaptive mutations...\" This conclusion is not rigorously\nsupported. Are variants in PB2 seen at higher frequency in the cattle lineage compared to\nrelated H5N1 viruses circulating in wild birds? Have these mutations actually accumulated,\nor have they been observed transiently?\nResponse: We thank the reviewer for this careful comment. In this revision, we have\nadjusted our interpretation accordingly. We analyzed the temporal dynamics of mutations\ndetected in viruses from cattle (revised Fig. 3B). The data show that certain mutations,\nsuch as A58T and K670R, became significantly more prevalent after September 2024.\nSimilarly, D740N was predominantly observed between May and August 2024, and E627K\nshowed a marked increase in frequency by 2025. This pattern suggests enrichment of\nthese specific variants within the cattle population over time. Combined with our\nexperimental findings that mutations like E627K and D740N significantly enhance\npolymerase activity in mammalian cells (Fig. 3C) and increase pathogenicity in mice (Fig.\n5A-C), these observations provide valuable insights into potential viral adaptation\npathways, which is critical for risk assessment.\nb. \"These mutations may contribute to severe disease or even death observed in infected\ncats\". Line 222. This statement is unsupported. Were the mutations referred to associated\nwith severe outcomes in cats? If yes, include that information. If not, remove the statement.\nResponse: Thanks for pointing this out. The statement has been removed.\n4. Fig 4B needs a western blot showing expression levels of the ANP32 proteins to validate\nthe assumption that increasing ng of plasmids transfected led to increased protein\nconcentration equivalently across the different ANP32 variants. Similarly, Fig 4 E-J need to\nbe accompanied by Western blots showing that expression of the different PB2 variants\nwas equivalent.\nResponse: We thank the reviewer for this constructive suggestion. We have now\nperformed Western blot analyses to confirm equivalent expression levels of the transfected\nANP32 proteins and the different PB2 variants. These new data are included in the Fig. S6.\n5. Discussion, first paragraph inappropriately claims that sequential accumulation of PB2\nmutations enabled cross species transmission. The data presented to do not document\nsequential accumulation of mutations nor investigate the role of these mutations in cross\nspecies transmission. Indeed, most of the mutations examined happened once H5N1 was\nestablished in cows and have not been associated with cross species transmission.\nResponse: We thank the reviewer for this comment. We have thoroughly revised the\nDiscussion section to more accurately reflect our findings and avoid overstatement. The\nrevised discussion incorporates the new data from the pathogenicity assessment in mice\nand presents a more cautious interpretation of the potential role of these mutations (358-\n372).\n6. Line 146 and elsewhere, it is not clear what is meant by 'clustered cross-species human\ninfections'. Were there transmission clusters? Independent cases clustered in time? Viral\nstrains clustered phylogenetically?\nResponse: We apologize for the lack of clarity. We intended to convey that during 2013-\n2014, multiple human infections with H5N1 viruses carrying the PB2 M631L mutation\noccurred within a relatively short timeframe. The term \"clustered\" may have been\nmisleading, and we have now removed this description.\n7. Fig 2f: Were there no human cases with 631M in 2013-2014 in Cambodia and Vietnam?\nIn 2024 in USA?\nResponse: We apologize for the lack of clarity in the original figure. The revised figure (now\nFig. 2B) clearly shows that indeed, no human cases with PB2 631M were detected in\nCambodia and Vietnam during 2013-2014. Regarding the USA in 2024, human exposures\ninvolved viruses from multiple sources (cattle and various avian species), resulting in\nhuman infections caused by viruses with both 631M and 631L. The revised manuscript\nfocuses the discussion of Fig. 2 on the Cambodia/Vietnam context, where we believe the\npresentation is now clear.\n8. Fig 2f: This schematic should include an arrow from birds to humans in 2024, reflecting\nthe transmission from poultry to humans in the US.\nResponse: We thank the reviewer for this suggestion. Consistent with our focus on the\nCambodia/Vietnam scenario in this figure, and considering the inherent uncertainties in\nillustrating transmission routes schematically, we have decided to remove this particular\nschematic diagram from the revised manuscript to avoid potential misinterpretation.\n9. The titles above Fig 3C, 3E and 3G & Fig 4D, 4E and 4F are inaccurate. These mutations\nwere observed in cows, nonhuman mammals or humans. That does not make them\nadaptive to these species.\nResponse: We thank the reviewer for this suggestion. We have revised the terminology\nfrom \"adapted\" to \"related\" in the respective figure titles and corresponding text to more\naccurately reflect the nature of the observations.\n10. Fig 5 omits cattle -to-poultry transmission in the U.S. and the likely role of cattle as an\nintermediary host in the TX37 human infection.\nResponse: We agree with the reviewer. The schematic figure has been revised accordingly.\n11. Line 31: remove \"global\". Pandemics are global by definition.\nResponse: The word \"global\" has been removed.\nReviewer #2 (Remarks to the Author):\nThe recent outbreak of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus has\nraised concerns about spillover and adaptation to humans. In this study, the authors\nidentified amino acid mutations in PB2 that enhanced polymerase activity in the recent\nH5N1 outbreak. Among these, the PB2-631L mutation was also detected in human H5N1\ncases in Cambodia and Vietnam during the 2013–2014 outbreaks.\nThis is a well-conducted and well-presented study; however, the enhancement of\npolymerase activity by PB2-631L has already been reported (Gu et al., Nature, 2024).\nAdditionally, several specific points require further clarification.\nResponse: We sincerely thank the reviewer for describing our study as \"well-conducted\nand well-presented.\" We acknowledge that the enhancement of polymerase activity by\nPB2-631L has been previously reported1,2. In this revised version, we have significantly\nstrengthened the study by including in vivo pathogenicity assessments for the PB2-631L\nvirus, alongside several other mutant viruses. We believe these additions make the revised\nmanuscript more comprehensive and valuable.\nMajor Point:\n1. The significance of the PB2-M631L mutation should be thoroughly investigated using\nthe mutant virus. Examining viral replication and pathogenesis in both cell culture and\nanimal models would be of great interest to readers and would strengthen the authors'\nclaims regarding human pathogenesis (Lines 174–177).\nResponse: We thank the reviewer for this constructive suggestion. We have generated\nrecombinant viruses carrying the PB2-631M and 631L, the well-known adaptive mutation\nPB2-E627K, and a combined PB2-E627K/M631L mutation. Infection experiments in mice\nclearly demonstrate that PB2-M631L significantly enhances viral pathogenicity,\ncomparable to the effect of E627K. Furthermore, the combination of M631L and E627K\nresulted in even greater pathogenicity. These new results are now presented in Fig. 2C-F.\nMinor Points:\n1. Figure 2 – The authors identified a high prevalence of PB2-631L in avian H5N1 viruses\nin North America (2022–2024). Given that the recent outbreak of clade 2.3.4.4b viruses\noriginated in Europe, the authors should analyze the prevalence of PB2-631L in European\nstrains as well.\nResponse: We apologize for the lack of clarity in the original description. We have analyzed\nall available avian and human H5N1 virus sequences and found that PB2-631L was\npresent primarily in Southeast Asia and Africa (2005-2021) and in North America (2022-\n2024). Notably, PB2-631L has not been detected in European strains. We have clarified\nthis point in the revised text (lines 130-137).\n2. Figure 2 – It is unclear whether the human H5N1 viruses from Cambodia and Vietnam\ncarried the mammalian-adaptive PB2-627K mutation.\nResponse: We apologize for the lack of clarity. The revised Fig. 2B clearly show that during\nthe 2013-2014 period, all human H5N1 viruses from Cambodia and Vietnam carried PB2-\nM631L but not PB2-E627K. In contrast, in human infection cases from other time periods,\nPB2-E627K was present in approximately half of the viruses.\n3. Line 181 – The reference to Fig. 2G should be corrected to Fig. 2F.\nResponse: Thank you for pointing this out. We have corrected this reference and\nperformed a thorough check of all figure citations.\n4. Lines 205–207 – Since the authors did not evaluate polymerase activity with the\ncombination of these mutations, this sentence should be removed.\nResponse: We agree and have removed the sentence.\n5. Figure 3D – Did raccoons and foxes become infected through spillover from cows? The\nauthors should provide a reference to support this claim. If no such evidence exists, this\nfigure should be removed to avoid confusion.\nResponse: The reviewer makes a valid point. As direct evidence for cow-to-raccoon/fox\ntransmission is lacking, we have removed this panel to avoid potential confusion.\n6. Figure 3E – The V596A mutation significantly suppressed polymerase activity. The\nauthors should comment on this observation.\nResponse: We thank the reviewer for this careful observation. We confirmed that the PB2\nV596A mutation does not affect protein expression (Fig. S6). The mechanism underlying\nthe significant suppression of polymerase activity by this mutation remains unclear. We\nhave now commented on this observation in the Results section (lines 270-272).\n7. Lines 212–223 – The authors should provide data on the prevalence of PB2-627K\nmutations in the mammalian species mentioned.\nResponse: We thank the reviewer for this suggestion. We have now included an analysis\nof the prevalence of PB2-E627K, noting its emergence in 2025, often in viruses already\nharboring M631L (Fig. 3B).\n8. Line 280 – \"PB2 L631M\" should be corrected to \"PB2 M631L\".\nResponse: Thank you for catching this error. It has been corrected.\n9. Line 285 – \"Fig. 4F-4J\" should be corrected to \"Fig. 4F-G, 4I-J\".\nResponse: Thank you for pointing this out. We have corrected the figure citation.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors systematically examined the role of the H5N1 virus\npolymerase in facilitating interspecies transmission from avian host to cattle in the United\nStates during 2024. It was previously reported that PB2 M631L mutation enhances\npolymerase activity in mammalian cells and plays a key role in the interspecies\ntransmission of the H5N1 virus from avian to cow. The authors of this manuscript identified\nadditional PB2 mutations that exhibit a synergistic effect when combined with M631L. They\nalso discovered that the PB2 M631L mutation in avian H5N1 viruses is strongly associated\nwith clustered human H5N1 infections during the 2013-2014 Cambodia/Vietnam outbreaks,\nas well as the 2024 U.S. cross-species transmissions. The majority of adaptive PB2\nmutations localized at the interface adjacent to the ANP32 protein and at the dimerization\ninterface of two viral polymerases, which aligns with previous reports. It is important to\nunderstand the mutations responsible for the H5N1 outbreak in cattle. However, limited\nstudies have been done in this study (only measuring polymerase activity using a reporter\ngene). Also, the outcome of the experiments (specific interaction with species specific\nANP32 through PB2 mutations) are similar to what is already well established in the field.\nUnfortunately, there is not much new data other than the identification of PB2 mutations\ncontributing to mammalian host adaptation.\nResponse: We thank the reviewer for acknowledging the importance of identifying\nmutations responsible for the H5N1 outbreak in cattle. In response to the comment that the\nstudy was initially limited to measuring polymerase activity, we have substantially\nexpanded the scope of our work in the revised manuscript. Beyond the polymerase activity\nassays, we have now generated recombinant viruses for several key PB2 mutations\nidentified in our study. We evaluated their pathogenicity in mice. Using viruses with various\nmutations at PB2 positions 631 and 627, we clearly demonstrate that PB2 M631L, similar\nto E627K, plays a significant role in mammalian adaptation. Importantly, the combination\nof M631L and E627K led to enhanced pathogenicity. Furthermore, we rescued viruses\ncarrying other polymerase-enhancing mutations identified herein (e.g., I647V, G685R,\nK736R) and confirmed their ability to increase pathogenicity in mice. These findings are\ncrucial for risk assessment, as they validate the functional significance of both known and\nnewly identified adaptive mutations in a whole-virus context.\nLine 53-54: “While adaptations in the PB1 and PA proteins have also been documented,\ntheir contributions to enhancing polymerase activity are comparatively weaker20,29” – This\nstatement is not accurate since mutations in PA have been reported to be a major\ncontributor of enhanced activity of 2009 pH1N1 (PMCID: PMC3126589, PMCID:\nPMC9576711, PMCID: PMC7232137, PMID: 25210166).\nResponse: We thank the reviewer for providing these relevant references. We have revised\nthis statement to more accurately reflect the documented role of PA and PB1 mutations in\nenhancing polymerase activity and have cited the suggested literature (lines 51-52).\nLine 84-85: “This strain exhibited the highest amino acid similarity in the PB2 protein among\nall cattle isolates, with 417 identical sequences out of 1848 total sequences (Fig. 1A). –\nFigure 1A does not show this data of sequence homology.\nResponse: We apologize for this oversight. The data supporting the sequence homology\nanalysis are now correctly presented in Fig. S1A, and the text citation has been updated\naccordingly (lines 80-81).\nLine 181: Fig. 2G should be Fig. 2F.\nResponse: Thank you for noting this error. It has been corrected.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Zhang et al. entitled \"Mammalian-adapted mutations in influenza A\n(H5N1) virus polymerase: implications for cross-species transmission surveillance\"\nexamines amino acid substitutions in the PB2 of H5N1 viruses isolated from mammals,\nparticularly cattle, and humans, and their influence on polymerase activity in human 293T\ncells. Many of the amino acid substitutions studied are located in a domain of the PB2\nprotein (called the 627 domain) that is responsible for interaction with the host factor\nANP32. Accordingly, some of these amino acid substitutions confer increased polymerase\nactivity in the presence of human or bovine ANP32A/B. Due to the continued circulation of\nH5N1 viruses in birds and the increasing transmission of these viruses to various\nmammalian species, including humans, risk assessment of these H5N1 isolates and\ndetection of the emergence of mammalian adaptation is critical. Although the study by\nZhang et al. presents some interesting results, such as the detailed investigation of\nindividual PB2 exchanges and their influence on polymerase activity, the study presented\nhere is primarily a screening approach that suffers from some important and (partly)\nunstated limitations. First, all experiments in this study are based on the well-established\nminireplicon reporter system, but no live virus infection data were performed. This is\nimportant because it is unclear how a 23-fold increase in polymerase activity (as observed\nwith the M631L substitution, Fig. 1F) translates into viral replication in mammalian cells\nand/or to what extent viral replication is enhanced by the other substitutions in PB2 (see\nFig. 1E). Second, the synergistic effects of i) these PB2 substitutions remain largely\nunaddressed, with the exception of those shown in Figures 1G and 3G, and ii)\ncombinatorial effects of other polymerase proteins (e.g., PA segment) are not mentioned.\nThird, and most importantly, many of the PB2 substitutions studied here have been\npreviously described by others and the significance of these amino acid exchanges for\nANP32 usage and the enhanced PB2-ANP32 interaction has been mechanistically\ninvestigated (Idoko-Akoh et al, 2023 Nature Communications; Staller, Carrique et al, 2024\nNature Communications).\nResponse: We thank the reviewer for recognizing the importance of our research topic and\nfinding the results interesting. We acknowledge the limitations pointed out and have\nconducted substantial additional experiments to address them, significantly strengthening\nthe study.\n(1) We have now generated live recombinant viruses carrying various PB2 mutations,\nincluding single mutants (e.g., M631L, E627K) and the double mutant (E627K/M631L). We\nevaluated their pathogenicity in mice. The results clearly demonstrate that PB2-M631L\nsignificantly enhances viral pathogenicity, similar to the well-known E627K mutation.\nCrucially, the combination of M631L and E627K led to even greater pathogenicity. This\nprovides direct in vivo evidence for the functional significance of the PB2-M631L mutation.\n(2) We agree that investigating synergistic effects is valuable. Based on the results in Fig.\n1G, combinations like A129T/M631L, V495I/M631L, or V649I/M631L already restored\npolymerase activity to the level of the wild-type cow PB2, indicating that mutations in PB2\nplay the predominant role in this context. Therefore, we focused this study on establishing\nthe primary role of PB2 mutations, which are historically critical for mammalian adaptation.\nThe potential combinatorial effects with mutations in other polymerase subunits (e.g., PA)\nrepresent an important area for future research.\n(3) We acknowledge that some mutations identified here (e.g., E627K, D701N) have been\npreviously reported regarding their mechanism involving ANP32 usage. Their re-\nemergence and identification in the currently circulating bovine H5N1 viruses underscore\nthe persistent adaptive pressure and the potential risk these viruses pose. More importantly,\nour study also identifies new adaptive mutations (e.g., I647V, G685R, K736R) which we\nhave now functionally validated to enhance pathogenicity in mice. This contributes\nsignificantly to risk assessment by highlighting novel markers.\nSome more specific comments:\n1. All polymerase reconstitution assays lack confirmation of equal protein levels.\nResponse: We thank the reviewer for this suggestion. We have now performed and\nincluded Western blot analyses confirming equivalent expression levels for all PB2 mutant\nproteins used in the polymerase assays. These data are now presented in Fig. S6A.\n2. How were the \"relative polymerase activities\" calculated? Were they calculated relative\nto Renilla luciferase and further normalized?\nResponse: We apologize for the lack of clarity in our initial description. The reviewer is\ncorrect. The relative polymerase activity was calculated by dividing the Firefly luciferase\nsignal by the Renilla luciferase signal (serving as an internal transfection control), followed\nby normalization. All experiments were independently repeated at least three times. We\nhave now provided a detailed description of this procedure in the Methods section (lines\n567-569).\n3. The statistics of the polymerase assays are misleading. The authors should use SD\ninstead of SEM when comparing the variability of individual data points in a data set.\nResponse: We thank the reviewer for this statistical insight. We have re-analyzed the data\nfor the polymerase activity assays and now present the standard deviation (SD) in the\nrelevant figures to better represent the variability of the data points.\n4. Figure 2A, B and lines 134-135: What was the prevalence of 631M in PB2 of H5N1\nviruses during the same time period? What was the number of sequences used for this\nanalysis (it is not described in the figure legends or the methods section)? It is hard to\nfollow the authors' argument. While 35 cases of H5N1 were reported in Cambodia and 4\nin Vietnam in 2013-15 (see WHO H5N1 Factsheet), there were 177 cases in Egypt and 10\ncases in China during the same period. Was the M631L exchange also highly prevalent in\nthese much larger outbreaks? What is the significance of this 631L substitution for\ntransmission of the virus to humans, and how does this fit with the statements in lines 174-\n181?\nResponse: We apologize for the initial lack of clarity. Our analysis was performed on all\navailable H5N1 virus sequences in the GISAID database (totaling over 30,000 sequences).\nOverall, PB2 631M is the predominant residue, found in >98% of all sequences; therefore,\nour figures specifically highlight the occurrence of the less common 631L variant. We\nspecifically checked human H5N1 cases from Egypt and China during 2013-2015 and\nconfirmed the absence of PB2 M631L in those outbreaks. This indicates that the PB2 631L\nvariant was indeed geographically clustered in Cambodia/Vietnam, possibly associated\nwith specific viral lineages. According to WHO records, Cambodia reported a relatively high\nnumber of human cases (26 in 2013, 9 in 2014). Given our experimental data\ndemonstrating that viruses with PB2 631L exhibit enhanced pathogenicity in mammals,\nwe hypothesize that this mutation may have been a contributing factor to the observed case\ncluster, serving as a potential risk marker. We have toned down the language to reflect this\nas a correlation and hypothesis rather than a definitive causation (lines 186-189).\n5. Line 181: should refer to Figure 2F\nResponse: Thank you for noting this error. This has been corrected.\n6. Figure 3B: Where is the M631L substitution shown?\nResponse: We apologize for the confusion. In the initial bovine H5N1 outbreaks (2024,\ngenotype B3.13), the PB2 M631L mutation was virtually fixed (present in nearly 100% of\nsequences) and thus was not explicitly plotted as a separate variant. However, from 2025\nonwards, infections in cattle with a different genotype (D1.1), which carries PB2 631M,\nemerged. We have now revised the figure to clearly distinguish between these two\ngenotypes and explicitly label the residues (631M vs. 631L), making this information clear\n(Fig. 3B).\n7. Lines 256-258, the sentence should read: \"To verify this, we constructed mini-replicons\nin ANP32A, ANP32B, and ANP32E triple knockout 293T cells (293T TKO) complemented\nwith ANP32A and ANP32B proteins from different species\".\nResponse: We thank the reviewer for the precise wording. The sentence has been\ncorrected as suggested.\n8. Figure 4B: Are the authors surprised to see that chANP32A, but not any of the\nmammalian ANP32 proteins tested, enhances the polymerase activity of the cow-adapted\nH5N1 polymerase at low concentrations? This seems counterintuitive to the reviewer.\nResponse: We thank the reviewer for this insightful comment. We first confirmed that all\ntransfected ANP32 proteins were expressed at equivalent levels (Fig. S6B). The robust\nenhancement by chicken ANP32A (chANP32A) is actually consistent with expectations.\nThe cow-adapted H5N1 polymerase is not fully adapted to mammalian ANP32 proteins;\nit retains strong compatibility with its original avian host factor, chANP32A. The dose-\nresponse effect, where activity increases with ANP32 plasmid concentration from 0.01 ng\nto 10 ng, is consistent with previous reports demonstrating that polymerase activity is\ntitratable with ANP32 levels3.\n9. In Figure 4D, the authors should add information about the electrostatic surface shown.\nWhat does the blue and red color indicate?\nResponse: Thank you for this suggestion. We have now added a description in the figure\nlegend: the electrostatic surface potential is shown in red (negative charge) and blue\n(positive charge) (lines 829-830).\nReference\n1. Gu, C. et al. A human isolate of bovine H5N1 is transmissible and lethal in animal\nmodels. Nature (2024).\n2. Idoko-Akoh, A. et al. Creating resistance to avian influenza infection through genome\nediting of the ANP32 gene family. Nature Communications 14 (2023).\n3. Zhang, H. L. et al. Fundamental contribution and host range determination of ANP32A\nand ANP32B in influenza A virus polymerase activity. Journal of Virology 93, e00174-\n00119 (2019)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript is substantially improved through the additional quantitative analyses and clarity around the prevalence of the M631L mutation in mammalian and avian hosts in the US and in VN/Cambodia. The new Figures and supplementary figures on these topics are informative and clear. The addition of mouse data to evaluate the implications of the PB2 mutations for pathogenesis is also an important improvement, not least because these experiments are performed with whole viruses rather than a mini-replicon system.\nA couple of minor issues:\n1. The differences between the cow and chicken PB2 segments selected as prototypes are the result of reassortment of the 2.3.4.4b lineage with LPAIV, not the result of an accumulation of mutations. This should be acknowledged in the text. The legend to Fig 1 should also be modified to remove \"indicating chicken-to-cow mutations\".\n2. It is not clear what the histogram in Supp Fig 1a is showing. Are the 417 sequences identical to that chosen for the experiment? If yes, add this to the legend.", "reviewer_3": "In this manuscript, the authors conducted an extensive study on the polymerase activity of H5N1 viruses isolated from dairy cow. They showed strong evidence showing that M631L mutation plays a major role in viral polymerase activity and pathogenicity. They also identified a couple additional adaptive mutations affecting the polymerase activity. Additional data from a series of rescued mutants and their pathogenicity in a mouse model are informative and important for public health. The authors responded to most of the comments from the reviewers and appropriately modified it by adding new data. The minor issue is that they strongly suggest that M631L mutation is a key determinant for viral pathogenicity in mammalian hosts. The PB2 631L variant emerged in human H5N1 infections during the 2013–2014 outbreaks in Cambodia and Vietnam. The case fatality rate during this period was notably high, reaching 50%. However, the human infection with the current H5N1 viruses generally caused mild illness, even though they both have PB2 631L mutation. Although the mouse experiment with the recombinant virus (with WSN HA and NA) showed a critical role of 631L in viral pathogenicity, this mutation by itself does not explain the H5N1 pathogenicity in humans. The authors need to discuss the difference of H5N1 pathogenicity in humans and possible factors determining the H5N1 virulence.", "reviewer_4": "In the revised version of their manuscript, \"Emergence of Mammalian-Adaptive PB2 Mutations Enhances Polymerase Activity and Pathogenicity of Cattle-Derived H5N1 Influenza A Virus,\" Zhang et al. address the functional relevance of PB2 substitutions in bovine-derived H5N1 viruses. First, they use the minireplicon system to characterize polymerase activity in vitro, followed by pathogenicity studies in BALB/c mice using reassorted 2+6 H1N1/WSN-bovine H5N1 viruses. The authors demonstrate that substitutions in the ANP32 interaction domain of PB2, specifically E627K and M631L. These substitutions enhance the viral virulence and pathogenicity in infected mice. However, because the influence of amino acid exchanges in viral PB2 on ANP32 usage in general, and in bovine PB2 in particular, has already been investigated (Idoko-Akoh et al., 2023, Nature Communications; Staller et al., 2024, Nature Communications; Bayoumi et al., 2025, Emering Microbes & Infections), this study does not add new insights into the mechanistic concept or risk assessment for emerging influenza A viruses. In addition, the authors' line of reasoning must be noted. They are correct that the adaptation of the viral polymerase, especially PB2, is an important factor for zoonotic influenza A viruses. However, adaptations in PB2 represent a very low interspecies barrier because substitutions often occur in index patients or shortly after infection in experimental mouse models (Kim et al, 2025, Virology Journal). Therefore, contrary to what the authors suggest (see line 456), these PB2 adaptations per se do not increase pandemic risk (because higher hurdles need to be overcome). A description of the adaptive hurdles for zoonotic influenza A viruses is available (Long et al., 2019, Nature Reviews Microbiology) and should be considered by the authors in their choice of words and discussion.\nFinally, the new mouse experiments shown in Figure 2 and Figure 5 raise the following questions:\nFigure 5: The authors use some of the same viruses as in Figure 2 and the same PFU but observe different time points for the human endpoints. How can the considerable differences of up to two days in the survival of infected mice be explained? Furthermore, how is it possible that seven infected mice result in a 50 percent survival rate? That is, 3.5 mice survive."}, "author_response": "Point-by-point responses\nReviewer #1 (Remarks to the Author):\nThe manuscript is substantially improved through the additional quantitative analyses and\nclarity around the prevalence of the M631L mutation in mammalian and avian hosts in the\nUS and in VN/Cambodia. The new Figures and supplementary figures on these topics are\ninformative and clear. The addition of mouse data to evaluate the implications of the PB2\nmutations for pathogenesis is also an important improvement, not least because these\nexperiments are performed with whole viruses rather than a mini-replicon system.\nResponse: Thank you very much for your encouraging feedback on our manuscript. We\nappreciate your recognition of the enhancements made through the additional analyses\nand the inclusion of mouse data, which further strengthen our findings.\nA couple of minor issues:\n1. The differences between the cow and chicken PB2 segments selected as prototypes\nare the result of reassortment of the 2.3.4.4b lineage with LPAIV, not the result of an\naccumulation of mutations. This should be acknowledged in the text. The legend to Fig 1\nshould also be modified to remove \"indicating chicken-to-cow mutations\".\nResponse: We appreciate the reviewer’s advice very much and apologize for the oversight.\nWe have added a relevant description in the text: \"It is important to note that the PB2\nsegment of the cattle H5N1 virus was recently identified as being derived from\nreassortment with North American low pathogenic avian influenza viruses (lines 123-125)\".\nAdditionally, we have modified the legend for Figure 1E and removed the description\n\"indicating chicken-to-cow mutations\".\n2. It is not clear what the histogram in Supp Fig 1a is showing. Are the 417 sequences\nidentical to that chosen for the experiment? If yes, add this to the legend.\nResponse: We thank the reviewer for pointing this out. We have now clarified the legend\nfor Supplementary Figure 1A with the following more detailed description: \"Among all\nanalyzed sequences, the highest frequency was observed for an identical sequence cluster\nconsisting of 417 sequences. One representative strain from this dominant cluster,\nA/dairy cow/Ohio/078707-24-12/2024 (designated cowOH12-H5N1), was selected as\nthe prototype cow H5N1 virus for subsequent analyses in this study\".\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors conducted an extensive study on the polymerase activity of\nH5N1 viruses isolated from dairy cow. They showed strong evidence showing that M631L\nmutation plays a major role in viral polymerase activity and pathogenicity. They also\nidentified a couple additional adaptive mutations affecting the polymerase activity.\nAdditional data from a series of rescued mutants and their pathogenicity in a mouse model\nare informative and important for public health. The authors responded to most of the\ncomments from the reviewers and appropriately modified it by adding new data. The minor\nissue is that they strongly suggest that M631L mutation is a key determinant for viral\npathogenicity in mammalian hosts. The PB2 631L variant emerged in human H5N1\ninfections during the 2013–2014 outbreaks in Cambodia and Vietnam. The case fatality\nrate during this period was notably high, reaching 50%. However, the human infection with\nthe current H5N1 viruses generally caused mild illness, even though they both have PB2\n631L mutation. Although the mouse experiment with the recombinant virus (with WSN HA\nand NA) showed a critical role of 631L in viral pathogenicity, this mutation by itself does not\nexplain the H5N1 pathogenicity in humans. The authors need to discuss the difference of\nH5N1 pathogenicity in humans and possible factors determining the H5N1 virulence.\nResponse: We thank the reviewer for the positive feedback and for raising an insightful\npoint. We fully agree that the presence of the PB2 M631L mutation alone does not\nsufficiently account for the observed differences in pathogenicity of H5N1 viruses in\nhumans. Recent reports have indeed highlighted the wide spectrum of clinical\npresentations in human A(H5N1) cases, ranging from asymptomatic illness, conjunctivitis,\nand mild upper respiratory tract symptoms to lower respiratory tract disease and critical\nillness, including death1. However, it remains unclear why the recent cases in the United\nStates have generally exhibited milder clinical symptoms1.\nThe variability in clinical outcomes can be attributed to a multitude of factors2,3. We have\nnow expanded our discussion to emphasize that pathogenicity is a multifaceted issue in\nhumans, influenced not only by specific mutations like M631L but also by the overall viral\ngenotype, the primary route of infection (e.g., conjunctival versus respiratory), and the\ngenetic background resulting from reassortment events4. Additionally, early antiviral\ntreatment has been shown to impact disease severity. For instance, a significant proportion\nof patients identified in the recent U.S. cases exhibited mild conjunctivitis and received\noseltamivir, initiated a median of 2 days post-symptom onset, which may have played a\ncrucial role in the milder outcomes observed1. We have also underscored that mutations\nin other viral proteins, particularly the hemagglutinin (HA), play a critical role in influencing\ndisease severity and should not be overlooked when evaluating pathogenicity5,6. This\nadditional context has been incorporated into the revised manuscript (lines 387-395,\n467-469).\nReviewer #4 (Remarks to the Author):\nIn the revised version of their manuscript, \"Emergence of Mammalian-Adaptive PB2\nMutations Enhances Polymerase Activity and Pathogenicity of Cattle-Derived H5N1\nInfluenza A Virus,\" Zhang et al. address the functional relevance of PB2 substitutions in\nbovine-derived H5N1 viruses. First, they use the minireplicon system to characterize\npolymerase activity in vitro, followed by pathogenicity studies in BALB/c mice using\nreassorted 2+6 H1N1/WSN-bovine H5N1 viruses. The authors demonstrate that\nsubstitutions in the ANP32 interaction domain of PB2, specifically E627K and M631L.\nThese substitutions enhance the viral virulence and pathogenicity in infected mice.\nHowever, because the influence of amino acid exchanges in viral PB2 on ANP32 usage in\ngeneral, and in bovine PB2 in particular, has already been investigated (Idoko-Akoh et al.,\n2023, Nature Communications; Staller et al., 2024, Nature Communications; Bayoumi et\nal., 2025, Emering Microbes & Infections), this study does not add new insights into the\nmechanistic concept or risk assessment for emerging influenza A viruses. In addition, the\nauthors' line of reasoning must be noted. They are correct that the adaptation of the viral\npolymerase, especially PB2, is an important factor for zoonotic influenza A viruses.\nHowever, adaptations in PB2 represent a very low interspecies barrier because\nsubstitutions often occur in index patients or shortly after infection in experimental mouse\nmodels (Kim et al, 2025, Virology Journal). Therefore, contrary to what the authors suggest\n(see line 456), these PB2 adaptations per se do not increase pandemic risk (because\nhigher hurdles need to be overcome). A description of the adaptive hurdles for zoonotic\ninfluenza A viruses is available (Long et al., 2019, Nature Reviews Microbiology) and\nshould be considered by the authors in their choice of words and discussion.\nResponse: We sincerely thank the reviewer for their insightful comments and for\nhighlighting the relevant references. We fully acknowledge that the role of specific PB2\nmutations in ANP32 usage has been well documented in previous studies. In this work, we\nconducted a comprehensive functional study of the polymerase activity of the recently\noutbreaking cattle H5N1 viruses. Our results not only reinforce earlier key findings but,\nmore importantly, identify several additional adaptive mutations that affect the polymerase\nactivity. Furthermore, pathogenicity studies in mice using reassorted viruses provide critical\nand timely experimental evidence for public health risk assessment of these emerging\nstrains.\nWe appreciate the reviewer’s mention regarding the relatively low interspecies barrier\nassociated with PB2 adaptations. It is well recognized that due to the error-prone nature of\nthe viral polymerase, mutations that enhance viral replication and dissemination can arise\nand accumulate progressively7. Polymerase adaptations such as E627K often emerge\nearly during cross-species infection and significantly boost viral replication within individual\nhosts. Furthermore, additional adaptive changes—particularly in HA, such as those\nconferring binding to human-type α-2,6-linked sialic acid receptors or improving acid\nstability—constitute further critical hurdles for pandemic potential7. Notable examples\ninclude the HA Q226L substitution6, which shifts receptor preference toward humans, and\nthe Q226H variant observed in severe human cases alongside PB2 E627K5. The\nemergence of a pandemic virus hinges on the stepwise accumulation of such mutations7.\nIn response to the reviewer’s suggestion, we have incorporated a discussion of these\nadaptive hurdles into the revised manuscript, referencing the framework described by\nLong et al7（lines 463-469）. We have also adjusted our wording in the discussion to more\naccurately reflect that while PB2 adaptations enhance virulence in mammalian models,\nthey represent only one component of the multi-faceted adaptation process (lines 387-395).\nWe believe these revisions provide a more balanced interpretation of our findings within\nthe established context of influenza host adaptation and pandemic risk.\nFinally, the new mouse experiments shown in Figure 2 and Figure 5 raise the following\nquestions:\nFigure 5: The authors use some of the same viruses as in Figure 2 and the same PFU but\nobserve different time points for the human endpoints. How can the considerable\ndifferences of up to two days in the survival of infected mice be explained? Furthermore,\nhow is it possible that seven infected mice result in a 50 percent survival rate? That is, 3.5\nmice survive.\nResponse: We thank the reviewer for carefully examining the in vivo data. The survival\ncurves shown in Figure 2 and Figure 5 were obtained from independent animal cohorts,\neven when the same virus strain and PFU were used. The difference in survival time mainly\nstems from Mouse 2 in the Figure 2 cohort, which was infected with the wild-type PB2\nreassortant virus (627E+631L). Mouse 2 showed approximately 25% body-weight loss on\ndays 7-9 post-infection but did not exceed that threshold, and was humanely euthanized\non day 10 when the weight loss finally surpassed 25% (see Addendum Table 1 for detailed\nweight tracking). All other mice infected with the same virus (in both Figure 2 and Figure 5)\nreached humane endpoints or died by days 7–8. The complete body-weight trajectories for\nall animals have been included in the Source Data.\nAddendum Table 1. Body weight changes (%) in mice infected with wild-type PB2 reassortant virus\nWT virus (in Figure 2C) WT virus (in Figure 5A)\ndpi. Mouse1 Mouse2 Mouse3 Mouse1 Mouse2 Mouse3 Mouse4\n0 100 100 100 100 100 100 100\n1 96.26 96.68 96.48 94.52 99.44 98.01 94.03\n2 93.80 94.48 95.75 91.87 98.63 98.51 90.75\n3 89.45 94.29 89.20 87.15 93.76 94.79 82.96\n4 83.25 89.22 83.01 86.03 90.14 90.69 82.08\n5 82.52 84.01 81.43 83.21 88.95 87.03 80.80\n6 76.81 79.50 77.12 78.20 83.33 82.68 75.88\n7 died 76.87 died 73.07 77.53 76.60 71.19\n8 75.49 72.03 72.69\n9 75.05\n10 73.92\nRegarding the survival rate in Figure 5, we apologize for the confusing presentation. In the\nFigure 5 experiment, each group initially comprised seven infected mice. At 5 days post-\ninfection, three mice were randomly selected and euthanized for lung viral titration and\nhistopathological analysis, and the remaining four mice were monitored longitudinally for\nbody weight and survival. Thus, the survival curves in Figure 5 are based on a total of four\nmice (n = 4). We have now clarified this design in the revised figure legend to avoid any\nconfusion (lines 901-906).\nReference\n1. Garg, S. et al. Highly Pathogenic Avian Influenza A(H5N1) Virus Infections in Humans.\nNew Engl J Med (2024).\n2. Kupferschmidt, K. Why hasn't the bird flu pandemic started? Science 386, 1205-1206\n(2024).\n3. Kupferschmidt, K. How did cow flu start? Scientists still don't know. Science 387, 1342-\n1342 (2025).\n4. Peacock, T. et al. The global H5N1 influenza panzootic in mammals. Nature (2024).\n5. Jassem, A. N. Critical Illness in an Adolescent with Influenza A(H5N1) Virus Infection.\nN Engl J Med (2024).\n6. Lin, T. H. et al. A single mutation in bovine influenza H5N1 hemagglutinin switches\nspecificity to human receptors. Science 386, 1128-1134 (2024).\n7. Long, J. S., Mistry, B., Haslam, S. M. & Barclay, W. S. Host and viral determinants of\ninfluenza A virus species specificity. Nature Reviews Microbiology 17, 67-81 (2019)."}]

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