Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket

Cardenas, Matias; Seibert, Brittany; Cowan, Brianna; Caceres, C. Joaquin; Gay, L. Claire; Cargnin Faccin, Flavio; Perez, Daniel R.; Baker, Amy L.; Anderson, Tavis K.; Rajao, Daniela S.

doi:10.1038/s42003-024-06928-6

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Published: 01 October 2024

Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket

Communications Biology volume 7, Article number: 1230 (2024) Cite this article

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Abstract

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA’s role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca²⁺) binding in the low-affinity Ca²⁺-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca²⁺-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca²⁺ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca²⁺ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca²⁺ binding in the low-affinity calcium-binding pocket.

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Introduction

Interspecies transmission of Influenza A viruses (FLUAV) is common; however, sustained transmission within a new host species is rare. This is because the virus must undergo adaptive evolution to replicate, transmit, and become endemic in the new host population. FLUAV have two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The HA recognizes sialic acids (SAs) on the cell surface and triggers virus entry into the host cells^1,2. The NA shows an opposite activity, cleaving SAs to allow the virus to move through the respiratory mucus by preventing binding to decoy receptors and to promote virus release from infected cell^3,4.

Due to its central role in the infection process, the HA protein is considered the primary driver of adaptation to a new host species. This adaptation often involves the acquisition of mutations in the receptor binding site (RBS) that enhance affinity for host-specific SA conformations^5,6,7. Previous studies have demonstrated that adaptation of FLUAVs isolated from humans to pigs results in amino acid changes in the HA RBS, increasing affinity for swine airway epithelial cells⁸ and improving transmission efficiency compared to the original human virus⁹. However, other gene segments also contribute to the adaptation of human-origin FLUAVs to pigs, with the acquisition of a well-adapted internal gene segment constellation seemingly essential^10,11,12. Human FLUAVs introduced to pigs frequently lose their internal gene segments through reassortment, acquiring swine-adapted gene segments from endemic strains¹³. Interestingly, the surface gene segments (HA and NA) often persist in the swine population, albeit with significant genetic differences from the precursor strains^13,14, leading to the establishment of new lineages.

The NA protein is a critical factor in FLUAV adaptation across species, as it must maintain a functional balance with HA for successful infection¹⁵. Adaptive mutations in the HA RBS can disrupt this balance, often prompting compensatory mutations in the NA protein¹⁶. NA is a homotetrameric enzyme with hydrolase activity, with each monomer consisting of a transmembrane domain, a stalk domain, and a head domain containing the catalytic pocket^17,18,19. While the catalytic pocket is present in each monomer, optimal sialidase activity is only achieved in the tetrameric state^20,21,22. Calcium (Ca²⁺) is an essential cofactor for NA activity, and most NA subtypes possess a high-affinity Ca²⁺-binding pocket near the active site of each monomer^23,24,25. This binding event is thought to induce a conformational change in the catalytic site, facilitating proper SA binding^26,27. In addition to the high-affinity site, certain subtypes such as N1 and N2 NAs have a second, low-affinity Ca²⁺-binding pocket located within the symmetry axis of the tetramer^27,28. This second site is believed to contribute to tetramer stability and, indirectly, to NA activity, as NA activity is dependent on the tetrameric form^28,29,30. Notably, the crystal structure of the 2009 pandemic H1N1 virus NA revealed that the virus quickly changed its calcium preference in the symmetry axis after jumping from pigs to humans²⁸.

Despite these observations, the precise role of the second Ca²⁺-binding site in the adaptation of FLUAV between species remains poorly understood and requires further investigation. In this study, we investigated the adaptive mechanisms of human-derived FLUAV HA and NA gene segments during transmission in a swine host. Utilizing a reassortant virus carrying HA and NA segments from the human strain A/Victoria/361/2011 (hVIC/11) in a swine-origin FLUAV backbone. The rapid emergence of an adaptative mutation was observed in HA (A138S, H3 numbering—herein referred to as the hVIC/11-A138S strain) near the RBS that evolved after infection of pigs⁸. Subsequently, and after two serial transmission events in vivo in pigs, the emergence and fixation of a novel mutation in the NA, D113A, was observed. In silico structural analyses revealed that amino acid 113 in the N2 NA of hVIC/11 interacts with Ca²⁺ in the low-affinity Ca²⁺-binding pocket, a characteristic typically observed in human H3N2 strains. Remarkably, the D113A mutation abolished Ca²⁺ binding in this pocket, but concurrently enhanced NA thermostability and activity under Ca²⁺-depleted conditions. However, the A113 variant is uncommon in both human and swine H3N2 strains, and, notably, swine-origin H3N2 FLUAVs harboring this mutation are predicted to be unable to bind Ca²⁺ in the low-affinity pocket. This observation prompted further investigation into the molecular determinants of this differential binding behavior, which revealed an important role of amino acid at position 93 in NA that further influences Ca²⁺ binding in the low-affinity pocket and impacts replication and transmission efficiency in pigs. These findings highlight the intricate molecular determinants of FLUAV adaptation between hosts and underscore the critical role of Ca²⁺ binding in this process.

Results

hVIC/11-A138S infects the lower respiratory tract and increases affinity following transmission to contact animals

To evaluate adaptation to a new host, 3-week-old pigs (seeders) were inoculated intratracheally and intranasally with 3 × 10⁶ TCID₅₀/pig of either hVIC/11-A138S⁹ or the control swine-adapted virus sOH/04³¹. Two days post-inoculation (dpi), three naïve pigs (contact 1, C1) were introduced with the inoculated pigs (seeders, Fig. 1). At 5 dpi, seeders were euthanized, and three new naïve pigs (contact 2, C2) were introduced to the C1 pigs. This cycle was repeated for a total of four contact groups. Nasal swabs were collected throughout the study, and lung samples and bronchoalveolar lavage fluid (BALF) were collected during necropsies at 5 dpi.

**Fig. 1: hVIC/11-A138S replicates in pigs’ upper and lower respiratory tract with increasing lung involvement by the fourth round of sustained transmission.**

Virus titration of nasal swabs confirmed successful transmission of hVIC/11-A138S among all contacts, peaking at approximately 10⁵ TCID₅₀eq/mL by 3 dpi (Fig. 1a). The sOH/04 virus control was also detected in all contacts (Fig. 1b), albeit at higher titers (approximately 10⁶ TCID₅₀eq/mL). The hVIC/11-A138S virus infected the trachea of all seeders and contact pigs (Fig. 1c). Virus titers in the right cranial, left cranial, and right caudal lobes (Fig. 1d–f) increased significantly by contact 4 (approximately 10⁴ TCID₅₀eq/mL) compared to the seeder and/or C1 pigs. While no significant differences were observed in the left caudal lobe or BALF samples (Fig. 1g, h), although a trend towards higher titers was noted in contact 4 pigs’ BALF samples. Notably, the virus initially failed to infect the accessory lobe of seeders and C1 pigs (Fig. 1i), with titers below the limit of detection (<10¹ TCID₅₀eq/mL) but was detected in subsequent contacts from C2 onward (approximately 10³ TCID₅₀eq/mL). These findings suggest that hVIC/11-A138S replication efficiency in the lower respiratory tract increased with each transmission, ultimately infecting all lung lobes by the study’s conclusion.

Serial transmission of hVIC/11-A138S in pigs selected for a mutant with a single amino acid mutation in NA

After sequencing analysis of nasal swabs and BALF samples, only minor variants (present in less than 50% of sequences) were observed in the hVIC/11-A138S HA segment (Fig. 2a), and none of these variants became fixed or were transmitted. However, a mutation in the NA, D113A, was detected in contact 2 pigs as a major variant (present in more than 50% of sequences) that eventually became prevalent and was transmitted to all contact 3 and 4 pigs (Fig. 2b). No positive selection was observed in the HA segment (Supplementary Fig. 1a), while signs of positive selection were observed for the NA segment in contacts 1 and 2 sequences (Supplementary Fig. 1b). After acquisition of the D113A amino acid change, no positive selection was observed in this segment. No major changes were observed in the sOH/04 control virus surface gene segments (Supplementary Fig. 2). Using the NA crystal structure information that closely matches the sequence of hVIC/11 NA (A/Tanzania/205/2010, Supplementary Fig. 3) revealed that the amino acid at position 113 located within the low-affinity Ca²⁺-binding pocket of the NA is essential for Ca²⁺ binding (Fig. 2c). The polar side chain of aspartic acid (D) at this position is predicted to stabilize a water molecule via a hydrogen bond (one per monomer) in the symmetry axis (Fig. 2c, top panel), facilitating the coordination of a single Ca²⁺ ion. When D113 is mutated to alanine (A), this polar interaction is lost, likely impairing Ca²⁺ binding (Fig. 2c, bottom panel). Notably, temporal analysis of residue frequency reveals that D113 and A113 were similarly prevalent in swine H3N2 viruses until the early 2000s (Fig. 2d). However, D113 has since become predominant, reaching 100% frequency by 2022.

**Fig. 2: In vivo transmission of hVIC/11-A138S selects for a single mutation in the NA segment.**

The adaptive N2 D113A mutation enhances NA stability under Ca²⁺- depleted conditions and reduces Ca²⁺ requirements for NA sialidase activity

Prior research with the N1 NA from the 2009 pandemic H1N1 virus demonstrated that disrupting Ca²⁺ binding in the low-affinity Ca²⁺-binding pocket impairs virus replication in vitro²⁸. To investigate this effect in viruses with the N2 NA D113A mutation, two recombinant viruses were produced carrying this mutation, with or without the HA A138S mutation (hVIC/11-A138S/D113A and hVIC/11-D113A, respectively). Replication kinetics of these viruses were evaluated in various human and swine cell lines (Fig. 3a) and compared to hVIC/11-A138S, hVIC/11, and sOH/04 viruses. The N2 NA D113A mutation did not affect virus replication in MDCK, PK15, A549, or differentiated human airway epithelial (HAE) cells (Fig. 3a). Neither hVIC/11 nor hVIC/11-D113A replicated in differentiated primary swine airway epithelial (SAE) cells, unlike hVIC/11-A138S and hVIC/11-A138S/D113A, confirming the advantage conferred by the HA A138S mutation for replication in swine cells⁹.

**Fig. 3: The NA D113A mutation provides no advantage for virus growth in SAE cells but increases NA thermostability and sialidase activity under Ca²⁺ -depleted conditions.**

Disruption of Ca²⁺ binding in the symmetry axis may negatively impact NA stability²⁸. To investigate this, the temperature at which 50% of NA activity is lost (T50) was established for NAs containing either D113 or A113 (Fig. 3b) in the absence or in the presence of physiological Ca²⁺ concentration (2 mM). The assay revealed that introducing the D113A mutation decreased the T50 of NA proteins at 2 mM Ca²⁺ (hVIC/11-D113A, 52.03 °C; hVIC/11-A138S/D113A, 51.19 °C) compared to control viruses with wild-type (WT) NA (hVIC/11, hVIC/11-A138S, and sOH/04), which exhibited T50 values averaging 5 °C higher (Fig. 3b, left panel; Fig. 3c). In the absence of Ca²⁺, all T50 values decreased compared to those at 2 mM Ca²⁺ (Fig. 3b, right panel). Remarkably, viruses carrying D113A, particularly hVIC/11-A138S/D113A (T50 44.44 °C), showed significantly higher values than the WT hVIC/11 (T50 39.51 °C) in the absence of Ca²⁺, behaving more like the sOH/04 strain (T50 43.40 °C) (Fig. 3c). These findings suggest that disrupting Ca²⁺ binding in the symmetry axis decreases NA thermostability at 2 mM Ca²⁺ but stabilizes the protein in the absence of Ca²⁺.

The sialidase activity in the absence of Ca²⁺as a percentage of the activity at 2 mM Ca²⁺ measured at 37 °C (Fig. 3d, Supplementary Fig. 4) revealed that mutant viruses containing the A113 mutation (hVIC/11-A138S/D113A and hVIC/11-D113A) maintained high levels of NA activity under Ca²⁺-depleted conditions similar to sOH/04 (39.14%). These mutants also exhibited enhanced activity compared to hVIC/11, which was nearly inactivated in the absence of Ca²⁺ (3.81%). Notably, the virus with both the A138S mutation in the HA and the D113A mutation in the NA retained the highest levels of sialidase activity in the absence of Ca²⁺, exceeding 50%.

Ca²⁺ binding within the low-affinity Ca²⁺ binding pocket is influenced by amino acid 93

Analysis of the predicted hVIC/11 and sOH/04 and NA structures (sOH/04 model derived from homology with A/Moscow/10/1999 (H3N2), Supplementary Fig. 5), suggests that the latter likely does not bind Ca²⁺ in this pocket despite having D113 (Fig. 4a). This model indicates altered interactions between sOH/04 NA and water molecules within the symmetry axis compared to hVIC/11, disrupting Ca²⁺ coordination. This is attributed to a reduced ϕ angle between G111 and G112 in sOH/04’s NA (159.3°) relative to hVIC/11’s NA (176.8°), which repositions the D113 side chain (Supplementary Fig. 6a). To verify this, the D113A mutation was introduced into sOH/04 NA (sOH/04-D113A). This did not alter NA activity at varying Ca²⁺ concentrations compared to WT sOH/04 NA (Fig. 4b) nor affected thermal stability at 2 mM or 0 mM Ca²⁺, further supporting the notion that sOH/04 NA does not bind Ca²⁺ in this pocket (Fig. 4c). Sequence alignment revealed an important difference at position 93, in which asparagine (N) is present in sOH/04 NA and glycine (G) in hVIC/11 NA (Supplementary Fig. 6b). Temporal analysis of human and swine H3N2 NA sequences (1992-2023) showed residue 93 in human isolates shifting from K (lysine) to N to D by the early 2010s, with G becoming prevalent as of 2023 (Supplementary Fig. 6c and d). Swine H3N2 viruses initially carried K93, gradually replaced by N93 (Supplementary Fig. 6c, bottom panel), which became fixed by the 2010s and has not changed ever since. This change at position 93 appears to affect subsequent residues, potentially explaining the altered ϕ angle between G111 and G112 (Supplementary Fig. 6e). This is further supported by comparing NA activity of swine- and human-origin H3N2 FLUAVs at different Ca²⁺ concentrations. Human H3N2 FLUAVs have exhibited reduced tolerance to Ca²⁺ depletion since the early 2000s (Supplementary Fig. 7a–i), contrasting with swine H3N2 strains, which largely retain high sialidase activity in the absence of Ca²⁺ (Supplementary Fig. 7j–q). This effect on NA activity seems to be associated with the residue at position 93 (Supplementary Table S1).

**Fig. 4: Amino acid 93 modulates Ca²⁺ binding in the low-affinity Ca²⁺ -binding pocket and stabilizes the NA tetramer under Ca²⁺-depleted conditions.**

To investigate the influence of residue 93 on NA physiochemical properties, the N93 substitution was introduced into the hVIC/11 NA, generating both hVIC/11-G93N and hVIC/11-A138S/G93N mutants. While the presence of Ca²⁺ led to a slight decrease in T50 compared to the unmodified hVIC/11, the absence of Ca²⁺ revealed a distinct pattern. In this Ca²⁺-depleted environment, the hVIC/11-G93N mutant exhibited a significant increase in T50 (47.22 °C) compared to both hVIC/11 and hVIC/11-A138S/G93N (Fig. 4d, e). The impact of residue 93 was not limited to the hVIC/11 NA. The N93G mutation in sOH/04-N93G NA also affected stability, resulting in reduced T50 values under both Ca²⁺-replete and Ca²⁺-depleted conditions compared to the sOH/04 control virus. These observations underscore the consistent influence of residue 93 on NA stability, regardless of the specific NA mutant or Ca²⁺ availability. Interestingly, this influence on stability extended to NA activity. The hVIC/11 mutants containing N93 demonstrated a remarkable 6-fold increase in sialidase activity compared to hVIC/11 in the absence of Ca²⁺ (hVIC/11-G93N, 22.14%; hVIC/11-A138S/G93N, 31.02%; hVIC/11, 3.8%; Fig. 4f, g). Conversely, the G93 mutation in sOH/04-N93G led to a reduction in activity compared to the sOH/04 control. These contrasting effects suggest that residue 93 not only modulates stability but also plays a crucial role in fine-tuning NA activity in response to Ca²⁺ availability.

Amino acids 93 and 113 differentially modulate neuraminidase enzymatic parameters

The impact of amino acids 93 and 113 on NA enzymatic activity was investigated by analyzing substrate affinity and sialic acid hydrolysis velocity. Recombinant NA constructs (rNA) consisting of the NA ectodomain fused to various tags (Fig. 5a) were expressed, purified, and evaluated for purity (Supplementary Fig. 8a). Upon cleavage of the introduced domains (Fig. 5b, Supplementary Fig. 8b and c), the released NA ectodomain was used to assess reaction velocity using MUNANA. While D113A did not affect the velocity of hVIC/11-D113A rNA, it reduced the reaction velocity in sOH/04-D113A rNA (Fig. 5c, d). Conversely, introduction of N93 increased the reaction velocity five-fold in hVIC/11-G93N rNA compared to hVIC/11 rNA (0.017 μM/min and 0.0033 μM/min, respectively), while G93 decreased the velocity of sOH/04-N93G rNA compared to sOH/04 rNA (0.011 μM/min versus 0.036 μM/min, Fig. 5d). These findings suggest that residues 93 and 113, although not part of the active site, influence NA catalytic properties.

**Fig. 5: Amino acids 93 and 113 distinctly affect NA kinetic parameters and promote NA activity under Ca²⁺-depleted conditions by stabilizing the NA tetramer.**

Further analysis of substrate affinity using the Michaelis constant (K_M) revealed that neither G93N nor D113A affected the K_M of hVIC/11-G93N rNA or hVIC/11-D113A rNA compared to hVIC/11 rNA (Fig. 5e, f). Conversely, both sOH/04-N93G rNA and sOH/04-D113A rNA exhibited increased K_M, indicating reduced substrate affinity compared to sOH/04 rNA. Assessment of maximum velocity (V_max) showed that hVIC/11-G93N rNA displayed the highest V_max among hVIC/11-derived NAs, while hVIC/11-D113A rNA had a reduced V_max compared to hVIC/11 rNA (Fig. 5g). Similarly, G93 and A113 reduced V_max in sOH/04 NA. These results suggest that residue N93 enhances the reaction V_max, whereas A113 decreases it.

Calculation of the turnover number (k_cat) further supported these observations (Supplementary Fig. 8d, e, Supplementary Table 2), demonstrating increased k_cat in hVIC/11-G93N rNA and decreased k_cat in hVIC/11-D113A rNA compared to hVIC/11 rNA. The opposite effect was observed in sOH/04 NA mutants. Additionally, the specificity constant (k_cat/K_M) was increased in hVIC/11-G93N rNA and reduced in both hVIC/11-D113A rNA and sOH/04 NA mutants (Supplementary Fig. 8f, Supplementary Table 2). This analysis indicates that N93 modulates catalytic power by affecting interactions after enzyme-substrate complex formation, while A113 decreases the catalytic efficiency noted as a reduced k_cat/K_M.

Examination of T50 values linked the increased activity of hVIC/11-G93N rNA to enhanced thermostability, whereas hVIC/11-D113A rNA exhibited reduced T50 under 2 mM Ca²⁺ (Fig. 5h, i). Similar trends were observed in sOH/04 NA mutants. Notably, both hVIC/11-G93N rNA and hVIC/11-D113A rNA displayed increased stability and activity at 0 mM Ca²⁺ compared to hVIC/11 rNA (Fig. 5j, Supplementary Fig. 9), whereas sOH/04r-N93G rNA showed reduced activity. The correlation between NA thermostability and activity under Ca²⁺-depleted conditions was confirmed by a Pearson test (Fig. 5k). Thus, residues 93 and 113 differentially modulate NA catalytic power by influencing the tetramer stability. Notably, amino acids destabilizing Ca²⁺ binding in the low-affinity Ca²⁺-binding pocket, prevalent in recent swine H3N2 viruses, enhance NA stability and activity in Ca²⁺-depleted conditions.

Amino acids 93 and 113 impact the HA/NA balance

To assess the impact of NA mutations at positions 93 and 113 on the balance between HA and NA activities, a red blood cell elution assay was performed, measuring the time required for a 50% reduction in HA titers under NA activity (Fig. 6a–f). At 2 mM Ca²⁺, hVIC/11-A138S exhibited the fastest elution, while mutations at either residue 93 (G to N) or 113 (D to A) increased elution time. Similarly, sOH/04-N93G showed slower elution kinetics. In the absence of Ca²⁺, hVIC/11 and hVIC/11-A138S displayed a marked increase in elution time, which was reversed by introducing either N93 or A113. Conversely, sOH/04-N93G maintained a slower elution time even without Ca²⁺. No elution was observed in the presence of oseltamivir, confirming assay functionality. These differences were statistically significant, indicating that residues 93 and 113 modulate viral elution time, reflecting alterations in the HA/NA balance. The observed changes in elution kinetics suggest that these residues influence the functional interplay between HA and NA, highlighting their role in optimizing viral fitness and adaptation.

**Fig. 6: Amino acids 93 and 113 affect the HA/NA balance.**

Disruption of Ca²⁺ binding in the low-affinity site reduces Ca²⁺ dependency for viral replication and aerosol infection

Given the importance of NA activity in the FLUAV replication cycle, the biological significance of the D113A mutation in hVIC/11-A138S/D113A was assessed through plaque assays under varying Ca²⁺ concentrations (Supplementary Fig. 10). At both 2 mM and 0 mM Ca²⁺, hVIC/11-D113A and hVIC/11-A138S/D113A produced the largest plaques (Fig. 7a). sOH/04 plaques were larger than those of hVIC/11. The hVIC/11 strain exhibited strong Ca²⁺ dependency, with plaque size reduced by nearly 60% in the absence of Ca²⁺ compared to the 2 mM condition (Fig. 7b). Conversely, Ca²⁺ concentration had less impact on hVIC/11-A138S, hVIC/11-D113A, and sOH/04, with plaque size reductions around ≤40%. The hVIC/11-A138S/D113A mutant showed minimal plaque size reduction under 0 mM Ca²⁺.

**Fig. 7: D113A decreases Ca²⁺ requirements for viral replication and enhances aerosol infection under Ca²⁺ -depleted conditions.**

Viral particle release, predominantly mediated by NA activity, was also evaluated. The hVIC/11 and hVIC/11-A138S viruses showed decreased release in the absence of Ca²⁺ (Fig. 7c). In contrast, sOH/04 and hVIC/11-A138S/D113A, particularly the latter, exhibited negligible differences among tested Ca²⁺ concentrations. These findings suggest that impairing Ca²⁺ binding in the symmetry axis reduces optimal Ca²⁺ requirements for growth, likely due to enhanced NA activity under low Ca²⁺ conditions, leading to improved viral release. This effect was particularly pronounced in aerosol infectivity assessments. The hVIC/11 and hVIC/11-A138S viruses showed delayed kinetics in MDCK cells when aerosolized in Ca²⁺-free media, reaching lower titers at 72 hpi compared to Ca²⁺-supplemented media (Fig. 7d). In contrast, hVIC/11-D113A, hVIC/11-A138S/D113A, and sOH/04 showed no differences under both conditions, indicating that viruses with Ca²⁺-destabilizing mutations are less sensitive to Ca²⁺ concentrations during aerosolization. This phenomenon was even more evident in swine-origin PK15 cells. While no significant differences were observed among viruses upon direct inoculation (Fig. 3a), aerosol delivery revealed a striking contrast (Fig. 7e). The hVIC/11 strain exhibited minimal replication with Ca²⁺-supplemented media and no detectable titers without Ca²⁺. However, hVIC/11-D113A and hVIC/11-A138S/D113A replicated similarly under both conditions, although hVIC/11-D113A titers were ultimately lower. These results demonstrate that viruses with reduced Ca²⁺ requirements for replication (hVIC/11-A138S/D113A, hVIC/11-D113A, and sOH/04) exhibit reduced dependency on this ion during aerosol infection, with minimal differences in growth kinetics between Ca²⁺ conditions.

Discussion

The NA protein plays a critical role in FLUAV infection, facilitating viral movement through respiratory mucus and promoting viral release by hydrolyzing sialic acid^32,33. NA activity is strongly dependent on Ca²⁺ ^34,35, with some subtypes binding up to two Ca²⁺ ions per monomer plus a central ion in the symmetry axis³⁶. However, most NA subtypes, including N2, primarily bind two Ca²⁺ ions in distinct high- and low-affinity binding pockets¹⁷. The high-affinity pocket is consistently occupied and essential for NA catalysis, as Ca²⁺ binding repositions the catalytic residues for proper sialic acid binding²⁶. Recent studies have revealed that the low-affinity pocket’s role in modulating N1 NA activity by stabilizing the tetramer is crucial for optimal function^28,37. Findings in this report extend this understanding to N2 FLUAV, demonstrating that the low-affinity pocket also modulates N2 NA catalytic properties, potentially serving as a mechanism for regulating the balance between HA and NA activities, and providing a mechanism to modulate NA enzymatic properties without mutating the catalytic residues.

Leveraging a swine sustained transmission model and a mutant virus with a mutation in HA (hVIC/11-A138S) previously detected after transmission of a human strain to pigs⁸, we identified a potentially compensatory mutation, D113A, in the N2 NA low-affinity Ca²⁺-binding pocket. N2 crystal structures of prototypical human strains revealed that residue 113 is the sole amino acid involved in Ca²⁺ binding at this site in this FLUAV subtype. This contrasts with N1 NAs, where residues K111, D113, and particularly Y170 play key roles in Ca²⁺ binding in the symmetry axis²⁸. Unlike the 2009 pandemic H1N1 virus, acquisition of D113A by the human-origin N2 NA did not affect viral replication in the cell lines tested. Furthermore, the failure of hVIC/11-D113A to replicate in swine respiratory cells suggests that this mutation alone does not enhance replication in pigs (Fig. 3a), underscoring the critical role of the HA mutation A138S in adaptation to pigs due to increased affinity for swine cells^38,39,40. These findings argue for a sequential adaptation model for human FLUAVs in pigs: initial acquisition of adaptive HA mutations to facilitate binding to swine cells, followed by NA mutations, such as D113A, to restore HA/NA balance disrupted by the prior HA changes. This model emphasizes the dynamic interplay between HA and NA in viral adaptation to new hosts, highlighting the importance of compensatory mechanisms in maintaining viral fitness during host transitions.

While the D113A mutation did not affect viral fitness in vitro in this study, it did impact NA thermostability, which is closely linked to Ca²⁺ binding⁴¹. The importance of Ca²⁺ in enhancing N2 activity has been previously established⁴². Notably, the D113A mutation increased N2 thermostability under Ca²⁺-depleted conditions, accompanied by an increase in its activity. Interestingly, swine-adapted N2 NAs inherently exhibit higher sialidase activity in the absence of Ca²⁺, contrasting with human viruses. The observation of stronger Ca²⁺ dependency in human-origin N2 aligns with previous reports^28,42. The high activity of swine FLUAVs under Ca²⁺-depleted conditions, despite carrying D113, suggests a lack of Ca²⁺ binding in the low-affinity Ca²⁺-binding pocket. Modeling of sOH/04 NA confirmed this hypothesis, revealing that Ca²⁺ binding is prevented by repositioning of water molecules in the symmetry axis due to a change in D113 side chain location (Fig. 4). Further support for this mechanism was provided by the introduction of D113A in sOH/04 NA, which resulted in no changes in thermostability compared to sOH/04 (Figs. 4 and 5), confirming that the virus does not bind Ca²⁺ when either amino acid is present. This suggests that the presence of D113, even without direct Ca²⁺ interaction, contributes to the overall stability of the NA structure, highlighting the complex interplay between amino acid residues and Ca²⁺ binding in modulating NA function.

Residue 93 appears to be under different selective pressures in humans and swine. While K93 and N93 were present in both human and swine FLUAV in the early 1990s, human isolates rapidly transitioned to D93 and G93, while N93 was retained in swine H3N2 FLUAVs. Protein modeling suggests that the N93 residue destabilizes the interaction of D113 with Ca²⁺, thereby preventing Ca²⁺ binding. Interestingly, a similar mechanism has been reported for residue 106 in N1 NAs, which also modulates Ca²⁺ binding in the low-affinity Ca²⁺-binding pocket²⁸. This suggests a conserved mechanism between N1 and N2 NAs for modulating Ca²⁺ binding at this site, involving specific residues distant from the pocket itself. The presence of this mechanism across subtypes may be due to the high conservation of the low-affinity Ca²⁺-binding pocket. Such a mechanism could prevent modifications near the Ca²⁺-binding pocket that might interfere with tetramer formation, as NA oligomerization is driven by both the head and transmembrane domains⁴³. However, further investigation is needed to confirm this hypothesis.

Residues 93 and 113 exert similar effects on Ca²⁺ binding in both human and swine N2 FLUAVs, yet their impact on NA catalytic properties differs. Notably, while D113A minimally affects sOH/04 rNA V_max, it reduces hVIC/11 rNA velocity. Conversely, N93 drastically increases reaction velocity within the hVIC/11 rNA. Additionally, sOH/04 rNA displays higher substrate affinity than hVIC/11 rNA, with no difference observed in hVIC/11-G93N rNA, suggesting that N93 seems to influence steps occurring after the enzyme-substrate complex formation, while A113 decreases the catalytic efficiency of the enzyme. This aligns with previous reports of Ca²⁺ modulating reaction velocity by favoring enzyme-substrate interactions^44,45, although the mechanism remains unclear. The D113A mutation impacts substrate binding, decreasing affinity regardless of NA backbone, potentially explaining its rarity in natural isolates due to disruption of HA/NA balance^46,47. Previous work has demonstrated that hVIC/11-A138S exhibits higher affinity for α2,6-SA, indirectly enhancing NA activity⁹. Based on this, it was hypothesized that the D113A mutation emerged in vivo to compensate for the A138S change in HA and regulate NA activity. Indeed, data revealed a higher elution rate for hVIC/11-A138S compared to hVIC/11 and sOH/04, which was restored to sOH/04-like levels upon introduction of D113A (Fig. 6). These findings underscore the intricate interplay between HA and NA, whereby mutations in one protein can necessitate compensatory changes in the other to maintain optimal viral fitness. Notably, A113 negatively impacted tetramer stability in the hVIC/11 NA backbone, suggesting that other residues might prevent this effect in sOH/04 NA. These two amino acids thus represent distinct mechanisms for N2 NA modulation of activity during host jumps, explaining the tendency of human viruses to bind Ca²⁺ in the low-affinity Ca²⁺-binding pocket, while swine H3N2 viruses do not. This suggests a novel role for Ca²⁺ in FLUAV host range, where viral changes may modulate sialidase activity without modifying the conserved catalytic pocket, by influencing Ca²⁺ binding in the low-affinity site. This is supported by previous studies on the 2009 pandemic H1N1 NA crystal structures²⁸. Early isolates, upon jumping from pigs to humans carried V106 that destabilized the tetramer at 2 mM Ca²⁺, but it was quickly replaced by I106 that enhanced N1 NA thermostability and activity under this condition. While that study did not directly link FLUAV host range and Ca²⁺ binding, the order of events aligns with the hypothesis that HA changes are required early in host adaptation, followed by NA activity modification through altered substrate affinity or reaction velocity, depending on specific residues involved in Ca²⁺ binding. However, this process seems to cycle between binding and not binding calcium in the symmetry axis²⁸, which could represent a mechanism to compensate for changes in the HA, as the D113A mutation alone cannot grant replication in swine airway cells (Fig. 3).

Increased NA thermostability conferred by the D113A substitution reduced the Ca²⁺ requirement for optimal virus replication to levels comparable to sOH/04. This is likely due to enhanced NA activity under low Ca²⁺ conditions, leading to more efficient virus release from infected cells²⁸. The increased stability of A113-containing mutants also resulted in more efficient aerosol infection of MDCK cells when the inoculum was aerosolized in the absence of Ca²⁺, similar to sOH/04. This effect was even more pronounced in PK15 cells, with limited to no replication observed for hVIC/11 and hVIC/11-D113A. Notably, hVIC/11-A138S/D113A demonstrated similar replication kinetics to sOH/04 across all tested conditions. These findings suggest that the absence of Ca²⁺ in the symmetry axis enhances aerosol infection under Ca²⁺-depleted conditions, potentially reflecting differences in mucus composition between human and swine, which could select for viruses with distinct aerosol stability. This could explain why hVIC/11 fails to transmit in pigs⁹ and only replicates in the upper respiratory tract of swine (~37 °C) but not in the lungs (~39 °C)^8,9, given that the NA of hVIC/11 is almost inactive at 39 °C in the absence of calcium (Figs. 3, 4, and 5). However, further investigation is needed to confirm this hypothesis.

Overall, this study demonstrates distinct Ca²⁺ binding profiles in human and swine H3N2 FLUAVs, with swine strains lacking Ca²⁺ binding in the low-affinity pocket. This difference leads to enhanced stability and activity of swine-origin FLUAVs under Ca²⁺-depleted conditions compared to human-origin FLUAVs, which are almost inactivated under these conditions. Residue 93 modulates Ca²⁺ binding and significantly impacts reaction velocity by affecting steps after SA binding in the NA catalytic pocket. This difference in reaction velocity and Ca²⁺ binding suggests a role of Ca²⁺ in the host range of FLUAV, with those from humans requiring higher Ca²⁺ concentrations for replication while their swine counterparts have reduced Ca²⁺ requirements. A better understanding of the biochemical differences of the upper and lower respiratory tract of humans and pigs is needed to understand why swine N2s need to retain high activity under calcium-depleted conditions. Nonetheless, the identification of residues that lead to enhanced fitness in swine cells (such as N93 and A113) can help recognize new spillover events that are more likely to be sustained in pigs and help improve biosecurity measures to decrease viral spread and prevent establishment of novel lineages. These results provide new insights into the mechanisms utilized by FLUAV to regulate NA activity during host adaptation and the potential effects on the HA/NA balance.

Materials and methods

Ethics statement

Animal studies were approved by the Institutional Care and Use Committee (IACUC) at the University of Georgia (protocol A2019 03-031-Y3-A9). Studies were performed under animal biosafety level 2+ (ABSL2+) containment and animals were cared for in accordance with the guidelines set forth in the “Guide for the Care and Use of Agricultural Animals in Research and Teaching (Ag Guide)” (American Dairy Science Association, American Society of Animal Science, and Poultry Science Association, 2020). At the end of the study, animals were euthanized following the American Veterinary Medical Association (AVMA) guidelines. All experiments with mutant viruses were performed under BSL 2+ conditions at University of Georgia (Institutional Biosafety Committee approval number 2020-0035).

Cells and viruses

Madin-Darby canine kidney (MDCK), human lung carcinoma (A549), and porcine kidney (PK15) cells were grown in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St Louis, MO) supplemented with 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO), 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO), and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂. MDCK cells were a kind gift from Dr. Robert Webster (St Jude Children’s Research Hospital, SJCRH). A549 and PK15 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Human airway epithelial cells (BCi.NS1.1)⁴⁸ were kindly provided by Dr. Ronald Crystal (Weill Cornell Medicine, New York) and maintained in PneumaCult-Ex Plus Basal Media (STEMCELL Technologies, Vancouver, Canada) supplemented with PneumaCult-Ex Plus Supplement (STEMCELL Technologies, Vancouver, Canada), 0.1% hydrocortisone (STEMCELL Technologies, Vancouver, Canada), 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO), and 0.5% gentamycin (Sigma-Aldrich, St Louis, MO). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂ and media was replaced every two days. Differentiation of BCi.NS1.1 cells was performed in type IV collagen-coated 12 mm trans well plates (0.4 μm pore size, Corning Inc., NY, USA). Cells were plated at 3 × 10⁵ cells/well and cultures were maintained at 37 °C in a humidified incubator with 5% CO₂ until a 100% confluency was reached. After reaching confluency, cells were differentiated by changing to air-liquid interface (ALI) conditions by removing the apical media and replacing the basal media with PneumaCult ALI Base Media (STEMCELL Technologies, Vancouver, Canada), supplemented with PneumaCult ALI Supplement (STEMCELL Technologies, Vancouver, Canada), 1% PneumaCult ALI Maintenance Supplement (STEMCELL Technologies, Vancouver, Canada), 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO), and 0.5% gentamycin (Sigma-Aldrich, St Louis, MO), 0.2% heparin (STEMCELL Technologies, Vancouver, Canada), and 0.5% hydrocortisone (STEMCELL Technologies, Vancouver, Canada). Cells were maintained at 37 °C in a humidified incubator with 8% CO₂ for 5 days and then were cultured at 37 °C in a humidified incubator with 5% CO₂ for 16 more days.

Spodoptera frugiperda pupal ovarian tissue (Sf9) cells were purchased from ThermoFisher Scientific (Waltham, MA) and maintained in SF-900 II SFM media (ThermoFisher Scientific, Waltham, MA) supplemented with 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). High Five™ Insect cells (BTI-TN-5B1-4) were obtained from ThermoFisher Scientific (Waltham, MA) and cultured in Express Five SFM media (ThermoFisher Scientific, Waltham, MA) supplemented with 18 mM L-glutamine (ThermoFisher Scientific, Waltham, MA) and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). Both insect cell lines were maintained in suspension at 28 °C.

FLUAV viruses used in this study (Table 1) were generated using an eight-plasmid reverse genetics system as previously described⁴⁹. Parental viruses (hVIC/11, sOH/04, and hVIC/11-A138S) were described before^8,9. D113A and G93N/N93G mutations were introduced into hVIC/11 or sOH/04 NA by site-directed mutagenesis using the Phusion site-directed mutagenesis kit (ThermoFisher Scientific, Waltham, MA), and the primers listed in Supplementary Table 3 according to the manufacturer’s instructions and plasmids’ sequences were confirmed by whole plasmid sequencing. Viruses were propagated in MDCK cells using Opti-MEM (ThermoFisher Scientific, Waltham, MA) containing 1 μg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemicals, Lakewood, NJ) at 37 °C. Viral titers were determined by TCID₅₀ using the Reed and Muench method⁵⁰.

Table 1 Swine and human H3N2 viruses used in this study^a

Full size table

In vivo studies

3-weeks-old cross-bred pigs obtained from Midwest Research Swine Inc (Glencoe, MN, USA) and housed in animal biosafety level 2 (ABSL2) facilities at the University of Georgia. Animals were negative for anti-FLUAV NP antibodies by competitive ELISA (IDEXX, Westbrook, ME) and randomly distributed into two groups. Pigs (n = 3, seeders) were inoculated intratracheally and intranasally under anesthesia using a cocktail of ketamine (6 mg/kg), xylazine (3 mg/kg), and Telazol (6 mg/kg) with 3 × 10⁶ TCID₅₀ per pig of hVIC/11-A138S or sOH/04. Animals were checked daily for clinical signs and nasal swabs were collected at 0, 1, 2, 3, and 5 days-post infection (dpi) in 2 mL of brain hart infusion broth (Sigma-Aldrich, St Louis, MO). At 2 dpi, naïve pigs (n = 3) were placed as contacts (contact 1, C1) with the directly inoculated animals and were kept together until 5 dpi. At 5 dpi, seeders pigs were sedated using a cocktail of ketamine (3 mg/kg), xylazine (1.5 mg/kg), and Telazol (3 mg/kg) and then humanely euthanized by an intravenous pentobarbital overdose (Euthasol, 390 mg/10 lb). Contact 1 pigs were moved into a clean enclosure after removal of seeder pigs, and additional naïve pigs (n = 3) were placed as new contacts (contact 2, C2). This process was repeated for a total of 4 contacts. Nasal swabs from contact pigs were collected at 0, 1, 3, 5, and 6 days-post contact (dpc). At 6 dpc, contact pigs were sedated and humanely euthanized as described above. During necropsies, bronchoalveolar lavage fluid (BALF) and lung tissue were collected from all pigs.

Virus titration from lung tissue, nasal swabs, and BALF samples

Lung samples were homogenized using the Tissue Lizer II (Qiagen, Gaithersburg, MD) and a Tungsten carbide 3 mm bead (Qiagen, Gaithersburg, MD) for 10 min at 30 Hz. Subsequently, RNA from lung tissues, nasal swabs, and BALF samples was extracted using the MagMax-96 AI/ND viral RNA (ThermoFisher Scientific, Waltham, MA) isolation kit according to manufacturer’s instructions. RNA from lung tissue was normalized to 1 μg in 20 μL of nuclease-free water while RNA from nasal swabs and BALF samples were used directly for titration. Samples were titrated using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly MA) targeting the M segment (Supplementary Table 3) according to manufacturer’s instructions.

Primary swine tracheal epithelial cells isolation

Trachea samples were obtained from three 7-weeks old cross-bred pigs obtained from Midwest Research Swine Inc (Glencoe, MN) and airway epithelial cells were isolated as previously described⁵¹ with minor modifications. Briefly, trachea sections were digested for 12 h at 4 °C in DMEM/F12 (Sigma-Aldrich, St Louis, MO) supplemented with 1.5 mg/mL pronase (Sigma-Aldrich, St Louis, MO), and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). After overnight incubation, trachea sections were scraped to release individual cells, and fibroblasts were discarded by plastic adherence for 4 h⁵². Non-adherent cells were then plated in type I collagen-coated flasks and cultured with BEGM^TM Bronchial Epithelial Cell Growth Medium BulletKit^TM (Lonza, Bend, OR, USA) at 37 °C in a humidified incubator with 5% CO₂. Differentiation was performed by seeding 3 × 10⁵ cells/well in type I collagen-coated 12 mm transwell plates (0.4 μm pore size, Corning Inc., NY, USA) in BEGM^TM media at 37 °C in a humidified incubator with 5% CO₂ until a 100% confluency was reached. After reaching confluency, BEGM^TM media was removed from the apical and basal compartments and ALI media composed of DMEM/F12 supplemented with 2% NuSerum (Corning Inc., NY, USA), 2mM L-glutamine (Sigma-Aldrich, St Louis, MO), 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO), and 15 ng/mL retinoic acid (Sigma-Aldrich, St Louis, MO) was added to the basal compartment. ALI media was changed every two days and ALI conditions were maintained for 3 weeks prior to use.

Next-generation sequencing

Viral genomic RNA was extracted from nasal swabs collected at 1, 2, and 5 dpi or 1, 3, and 6 dpc and BALF samples using the MagMax-96 AI/ND viral RNA isolation kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer’s instructions. After extraction, HA and NA segments were amplified using specific primers (Supplementary Table 3) and the SuperScript III One-Step PCR System (ThermoFisher Scientific, Waltham, MA) according to manufacturer’s instructions in duplicates. Duplicate PCR products from both HA and NA PCRs from the same sample were pooled in equal volumes and cleaned with 0.45X Agencourt AMPure XP Magnetic Beads (Beckman Colter, Brea, CA, USA) according to manufacturer’s instructions. Clean PCR product concentration was measured using the Qubit dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA) on a Qubit 3.0 fluorometer (ThermoFisher Scientific, Waltham, MA) and normalized to 0.2 ng/μL. Sequencing libraries were prepared with the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). Indexed libraries were cleaned with 0.7X Agencourt AMPure XP Magnetic Beads and samples size distribution was analyzed using the Agilent Bioanalyzer 2100 DNA-HS assay (Agilent, Santa Clara, CA, USA). Libraries were then pooled, denatured, and further diluted to 10 pM. Finally, samples were sequenced using a MiSeq Reagent Kit V2, 300 cycles (Illumina, San Diego, CA, USA).

Variant analysis

Analyses of FLUAV variants were performed as previously described^53,54. Briefly, sequencing adapters were removed using Cutadapt (version 3.4) and then mapped back to their respective reference sequence using option mem from BWA (version 0.7.17)⁵⁵. Read-pairing errors that may have been introduced by the alignment were corrected using option fixmate, and secondary and unmapped reads were removed. The resulting bam file was used by LoFreq⁵⁶ following practices outlined in the Genome Analysis Toolkit⁵⁷ to identify non-consensus variants. Only variants with a frequency of 0.01 (1%) and coverage equal to or above 100 were used. Synonymous and nonsynonymous mutations were identified using SNPdat (version v1.0.5)⁵⁸. The output from SNPdat was inputted into RStudio (R version 4.1.2) for visualization. Variants with a frequency equal to or above 0.5 (50%) were considered major variants.

Diversity analysis

Nucleotide diversity π was calculated using the πN (nonsynonymous) to πS (synonymous) ratio statistics determined using SNPGenie⁵⁹ for estimation of nucleotide diversity from next-generation sequencing. An arbitrary minimal variant frequency of 0.01 was set for π determination.

Amino acid temporal frequency analysis

55,590 human origin and 15,514 swine origin N2 sequences from H3N2 FLUAVS from 1992 to 2022 were obtained from GISAID⁶⁰. Duplicated sequences were discarded, and samples were aligned using Clustal Omega version 1.2.2⁶¹. Alignments were exported and amino acid frequency at position 93 and 113 was assessed using Geneious version 2024.0.2.

NA homology model

FLUAV NA sialidase domains were modeled using the SWISS-MODEL server (https://swissmodel.expasy.org/)⁶². hVIC/11 NA model was made using A/Tanzania/205/2010 as template (PDB accession code: 4GZO⁶³) while A/Moscow/10/1999 (PDB accession code: 8DWB⁶⁴) was used to generate the sOH/04 NA 3D structure. NA structures were visually inspected using PyMOL 3.0 and model quality was assessed using PROCHECK⁶⁵.

Growth kinetics

MDCK, A549, and PK15 cells were seeded in 6 well-plates and incubated at 37 °C in a humidified incubator with 5% CO₂ until an 80% confluency before use. Cells were infected at a MOI of 0.01 for 1 h at 37 °C. After incubation, cells were washed three times with PBS and supplemented with fresh Opti-MEM containing 1 μg/ml TPCK-treated trypsin. Timepoints were collected at 0, 12, 24, 48, and 72 hpi, and viral titers were determined by RT-qPCR. HAE and differentiated swine tracheal cells were infected by adding 200 μL of virus in the respective ALI media into the apical compartment and incubated for 1 h at 37 °C. After incubation, cells were washed 5 times with PBS. Timepoints were collected at 0, 12, 24, 48, and 72 hpi by adding 200 μL of PBS onto the cells and incubating them at 37 °C for 15 min. Viral titers were determined as described above.

Neuraminidase activity and thermostability

Neuraminidase sialidase activity was evaluated using the whole virus as previously described⁶⁶ or purified recombinant NA (rNA). Briefly, viruses were diluted in reaction buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid -MES-, 2 mM CaCl₂ -unless stated otherwise-, pH 6.5) and incubated at 37 °C for 30 min. To evaluate purified recombinant proteins, samples were normalized to 50 ng for all of the assays described below. After incubation, 100 μM 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA, Sigma-Aldrich, St Louis, MO) was added and reactions were incubated for 60 min at 37 °C. Fluorescence was measured every 60 s at excitation and emission wavelengths of 360 nm and 460 nm, respectively, using a Synergy HTX Multi-Mode Microplate Reader (Agilent BioTek, Santa Clara, CA).

NA thermostability was assessed by incubating equal amounts of each virus at 33, 37, 40, 43, 46, 49, 52, 55, 58, 61, and 64 °C for 10 min. After incubation, viruses’ NA sialidase activity was measured as mentioned above. NA relative activity was calculated as percentage of the activity at 33 °C. T₅₀ values were obtained by adjusting the curves to a non-linear dose-response logarithmic four parameters equation of the form:

$$Y=\frac{a+(a-b)}{1+{10}^{\left(\left(\log \left({T}_{50}-X\right)\, * \, H\right)\right.}}$$

Where Y is NA activity in percentage, X is the log(temperature), a is the theoretical maximum activity, b is the theoretical minimum activity, and H is the Hill slope.

NA sialidase activity under different concentrations of Ca²⁺ was performed by adding 2, 1.5, 1, 0.5, or 0 mM CaCl₂ to the reaction buffer (32.5 mM MES, pH 6.5). Reaction velocity was measured as described above, and relative NA activity was calculated by comparing the reaction velocity under each condition to the 37 °C and 2 mM Ca²⁺ condition.

Enzyme kinetics

rNA samples were diluted in NA reaction buffer and incubated at 37 °C for 30 min. Then 4-MU production over time was evaluated under different MUNANA concentrations (1.17, 2.34, 4.68, 9.37, 18.75, 37.5, 75, 150, 300, and 600 μM). Fluorescence was measured every 60 s at excitation and emission wavelengths of 360 nm and 460 nm and data was fitted to a simple saturation-kinetics Eq. (1) and k_cat was obtained by rearranging the Michaelis–Menten Eq. (2) assuming saturated substrate concentrations and a steady-state model:

$$v=\frac{{v}_{\max }* [{MUNANA}]}{{K}_{M}+[{MUNANA}]}$$

(1)

$${k}_{{cat}}=\frac{{v}_{\max }}{[{E}_{t}]}$$

(2)

Red blood cells elusion assay

Viruses were normalized at 32 HA units (HAU) in Ca²⁺ -free PBS or PBS supplemented with 2 mM Ca²⁺. Normalized samples were then mixed with 0.5% turkey red blood cells and incubated at 4 °C for 1 h. Then, samples were incubated at 37 °C, and aliquots were collected every 30 min until all HAU were lost. As a control, a duplicate sample supplemented with 1 μM oseltamivir (Sigma-Aldrich, St Louis, MO) was included. Elution due to NA activity was confirmed by incubation at 4 °C for 1 h to allow re-agglutination.

NA protein expression and purification

NA ectodomain from hVIC/11 and sOH/04 was cloned into the pFastBac1 transfer vector. The stalk domain was replaced and fused in frame to a GG linker, a thrombin cleavage site, a tetramerization domain from the human vasodilator-stimulated phosphoprotein (VASP), 6x His tag, and a secretion signal as previously described⁶⁷. D113A and G93N/N93G mutations were introduced as described above. Cloned pFastBac1 plasmids were then used to generate the recombinant bacmids by transforming DH10Bac competent cells (ThermoFisher Scientific, Waltham, MA), and insert transposition was confirmed by colony PCR using M13 primers (Supplementary Table 3). Bacmids were purified by alkaline lysis and used to rescue recombinant baculoviruses in Sf9 cells according to the Bac-to-Bac system manual (ThermoFisher Scientific, Waltham, MA).

NA proteins were produced by infecting High Five cells at an MOI of 10. When cells reached an 80% mortality (∼72 hpi), the supernatant was cleared by centrifugation at 3000 rpm. NA constructs were purified from clarified supernatant by immobilized metal affinity chromatography (IMAC) using the HisPur Ni-NTA Spin purification kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions and eluted in elution buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, and 300 mM imidazole. Subsequently, eluted NAs were further purified by size exclusion chromatography using a Superdex-200 (Sigma-Aldrich, St Louis, MO) column with a running buffer composed of 20 mM Tris-HCl, 50 mM NaCl, pH 8.00. NA activity was monitored for each fraction. Finally, purified samples were digested using the Thrombin CleanCleave Kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s instructions, and digestion products were passed through a Superdex-200 column.

SDS-PAGE and western blot

Protein concentration was determined using the Pierce BCA Protein Assay kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. 100 ng of either cleaved or non-cleaved NA constructs were boiled for 5 min and resolved by SDS-PAGE using a Mini-Protean TGX Stain-Free Precast Gel (Bio-Rad, Hercules, CA). For total protein imagining, gels were then fixed with 40% ethanol and 10% acetic acid in water for 1 h. Then samples were sensitized for 1 min with 0.02% Na₂S₂O₃ and incubated for 20 min with staining solution (0.1% AgNO₃ and 0.02% formaldehyde in water). Finally, gels were developed by incubating them in 3% NaCO₃ and 0.05% formaldehyde in water. Gels used for western blot were transferred to a TransBlot Turbo Mini Nitrocellulose membrane and blocked for 2 h in blocking buffer (5% BSA in PBS) at room temperature. After incubation, membranes were washed two times with T-PBS (0.05% Tween-20 in PBS, washing buffer) and incubated for 1 h with an anti-His tag primary antibody (Sigma-Aldrich, St Louis, MO). Membranes were then washed three times (5 min each) and incubated for 1 h with a secondary HRP-conjugated anti-mouse antibody in a 1:1,000 dilution in T-PBS. Finally, membranes were washed 5 times and then developed with the SuperSignal West Femto Maximun Sensibility Substrate kit (ThermoFisher Scientific, Waltham, MA). Membranes were imaged using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).

Plaque assay

MDCK cells were seeded at a density of 10⁵ cells/cm² and incubated at 37 °C in a humidified incubator with 5% CO₂ in Opti-MEM (Life Technologies, Carlsbad, CA, USA) until a 100% confluency before use. The day of infection, culture media was discarded, cells were washed three times with Ca²⁺ -free DMEM (ThermoFisher Scientific, Waltham, MA), and were infected using 10-fold serial dilution of each virus in Ca²⁺ -free DMEM. After 1 h incubation at 37 °C, unbound virus was removed by washing cells three times with Ca²⁺ -free DMEM and then cells were supplemented with DMEM containing 2 mM L-glutamine, 0.3% bovine serum albumin (BSA, ThermoFisher Scientific, Waltham, MA), 1% antibiotic/antimycotic, 1 mM sodium pyruvate (ThermoFisher Scientific, Waltham, MA), 0.8% avicel, and variable concentrations of Ca²⁺ chloride (2, 0.5 or 0 mM, Sigma-Aldrich, St Louis, MO). Infections were incubated for 72 h at 37 °C. Finally, cells were fixed with 37% formaldehyde (Sigma-Aldrich, St Louis, MO) and stained for 15 min with 0.5% crystal violet in 20% methanol.

Viral particle release

Viral particles release was evaluated as previously described²⁸. Briefly, MDCK cells were infected at a multiplicity of infection (MOI) of 0.1 and were incubated at 4 °C for 30 min. Unbound virus was removed by washing cells three times with PBS and cells were supplemented with fresh Opti-MEM containing 1 μg/ml TPCK-treated trypsin (Worthington Biochemicals, Lakewood, NJ). At 5 hpi, media was discarded and 3 mL of DMEM containing 2 mM L-glutamine, 0.3% BSA, 1% antibiotic/antimycotic, 1 mM sodium pyruvate, 1 μg/ml TPCK-treated trypsin, and variable concentrations of CaCl₂ was added. Starting at 6 hpi, 100 μL was collected from each condition every 1 h until 12 hpi, and HA titers were measured using 50 μL of sample and 50 μL 0.5% turkey red blood cells.

Aerosol infection

MDCK and PK15 cells were seeded and incubated as described above until 80% confluence was reached. Viruses were diluted in Ca²⁺ -free DMEM supplemented with 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO), 0.3% BSA (Sigma-Aldrich, St Louis, MO), and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO) and used at an MOI of 0.01. For infections performed with Ca²⁺, 2 mM CaCl₂ was added to the infection media. A total of 6 mL of inoculum was aerosolized using the Aeroneb Lab Neubilizer (Small VMD; Kent Scientific, CT, USA) with an expected particle size of 2.5–4 μm. Aerosol was passed through an exposure chamber at a flow rate of 0.1 mL/min. Cells were exposed for 15 min at room temperature, followed by a 5 min purge. Infections were incubated for 1 h at 37 °C and then washed three times with PBS. Finally, Opti-MEM media supplemented with 1 μg/ml TPCK-treated trypsin was added, and time points were collected at 0, 12, 24, 48, and 72 hpi.

Statistics and reproducibility

All statistical analyses were performed using GraphPad Prism Version 10.3.1. P values were obtained by ordinary one-way ANOVA with Tukey’s multiple comparison test. No data was excluded from the analyses. All data is presented as the mean ± standard deviation. Animal sample size of three animals per group (seeders or contacts) was based on previously published work^68,69. Animals were randomly allocated to each group to ensure reproducibility. In vitro experiments were performed at least three times independently in triplicates (n = 3 biological replicates) unless stated otherwise in the figure legend, and results were reproducible between experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data relevant for this manuscript is provided as a supplementary data file. Sequencing data is available through NCBI’s short read archive BioProject accession number PRJNA1117070.

Code availability

Custom computer code used in this study are available on GitHub (https://github.com/genferreri/Intra--and-inter-host-evolution-of-H9N2-influenza-A-virus-in-Japanese-quail).

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Acknowledgements

The authors thank the University Research Animal Resources personnel at the University of Georgia for assistance with animal care. We thank Dr. Lucas Ferreri for the development of the pipeline used for variant analysis. This research was supported by Agriculture and Food Research Initiative grant no. 2020-67015-31563/project accession no. 1022827 from the USDA National Institute of Food and Agriculture to DSR. Funding was also provided, in part, by The National Pork Board to DSR under Project #21-085. DSR is also funded by Agriculture and Food Research Initiative grant no. 2022-67015-37205/project accession no. 1028058 from the USDA National Institute of Food and Agriculture. DRP is funded by subcontract 75N93021C00014 Centers for Influenza Research and Response (CEIRR) from the National Institute of Allergy and Infectious Diseases (NIAID) and GRANT12901999, project accession no. 1022658 from the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture. DRP receives additional support from the Georgia Research Alliance and the Caswell S. Eidson endowment funds from The University of Georgia. This study was partly supported by resources and technical expertize from the Georgia Advanced Computing Resource Center, a partnership between the University of Georgia’s Office of the Vice President for Research and the Office of the Vice President for Information Technology. A.L.B. and T.K.A. are funded by the USDA-ARS project number 5030-32000-231-000D and the NIAID, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00015. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA or UGA. USDA is an equal opportunity provider and employer.

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Brittany Seibert
Present address: W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Authors and Affiliations

Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Matias Cardenas, Brittany Seibert, Brianna Cowan, C. Joaquin Caceres, L. Claire Gay, Flavio Cargnin Faccin, Daniel R. Perez & Daniela S. Rajao
National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA
Amy L. Baker & Tavis K. Anderson

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Matias Cardenas
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Brittany Seibert
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Brianna Cowan
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L. Claire Gay
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Daniel R. Perez
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Amy L. Baker
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Contributions

D.S.R. and M.C. designed the experiments. M.C., B.S., C.J.C., B.C., L.C.G., and F.C.F. performed the in vivo experiments. M.C. and B.C. processed the samples from the animal studies. M.C., B.C., and B.S. constructed the NGS libraries and sequenced the samples. B.S. performed the genetic diversity analysis and variant analysis. M.C. rescued and grew the viruses used in this study. M.C. cloned the pFastBac1 plasmids, expressed, and purified the recombinant NA proteins. M.C. performed the thermostability and NA activity assays. M.C. collected and isolated the swine tracheal cells. M.C. performed the viral growth kinetics. M.C. and B.S. performed the plaque assays and aerosol infections. M.C. performed the data analysis. M.C. and D.S.R. wrote the manuscript. D.R.P., T.K.A., and A.L.B. edited the manuscript.

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Correspondence to Daniela S. Rajao.

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Communications Biology thanks Wenjun Song and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editors: Christina Karlsson Rosenthal, Laura Rodríguez Pérez and Gavin Mason.

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Cardenas, M., Seibert, B., Cowan, B. et al. Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket. Commun Biol 7, 1230 (2024). https://doi.org/10.1038/s42003-024-06928-6

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Received: 06 June 2024
Accepted: 19 September 2024
Published: 01 October 2024
DOI: https://doi.org/10.1038/s42003-024-06928-6