MHC class II is a functional receptor for H3N2 Influenza A viruses and mediates host-specificity

MHC class II is a functional receptor for H3N2 Influenza A viruses and mediates host-specificity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MHC class II is a functional receptor for H3N2 Influenza A viruses and mediates host-specificity Daniela Rajao, Matias Cardenas, Sasha Compton, C. Joaquin Caceres, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6278351/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Influenza A viruses (FLUAV) utilize sialic acid to enter host cells via the envelope’s hemagglutinin (HA), and its affinity for host-specific sialic acid conformations is a major host range determinant. However, some FLUAV subtypes (H17, H18, H19) were recently shown to use the major histocompatibility complex class II (MHCII) as an entry receptor instead of sialic acid (SA), challenging our knowledge about FLUAV tropism and interspecies transmission potential. Here we show that H3N2 viruses can use MHCII as an alternative entry receptor in a host-specific manner, and adaptation of human viruses to pigs increases affinity for the MHCII swine leukocyte antigen (SLA). By using two prototypic human-seasonal (hVIC/11) and swine-adapted (sOH/04) H3N2 viruses we found that expression of the human (HLA) but not the swine MHCII conferred replication of hVIC/11 in deacetylated, non-susceptible cells which ultimately led to cell death. Further, expression of SLA in deacetylated, non-susceptible cells conferred susceptibility to infection by sOH/04. Introduction of point mutations near the hVIC/11 HA receptor-binding site (RBS) allowed the use of both human and swine MHCII. Our findings revealed that MHCII can serve as a sialic acid-independent entry receptor to H3N2 FLUAV in a host-specific manner, expanding the cell tropism and host range of the virus, with potential implications for the viral pathogenesis and adaptation to a new species. Biological sciences/Microbiology/Virology/Virus–host interactions Biological sciences/Microbiology/Virology/Influenza virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sialic acid (SA) is the canonical receptor used by influenza A viruses (FLUAV). These SAs on the cell surface are recognized by the viral hemagglutinin (HA) protein to promote entry and begin the replication cycle 1 , 2 . The SA structure is linked to FLUAV host range since avian-origin FLUAVs mainly recognize sialic acids linked to galactose by an α2,3 linkage while mammalian-origin FLUAVs recognize α2,6-linked SA 3 . Therefore, mutations in the HA protein in or near the receptor-binding site (RBS) can modify the binding preference of the virus 4 – 6 and may lead to a switch in the host specificity 3 . By contrast, recently discovered bat-origin H17 and H18 HA subtypes, as well as duck-origin H19, were shown to exclusively use the Major Histocompatibility Complex II (MHCII) as an entry receptor 7 – 9 . This interaction is completely independent of sialic acid as multiple glycan array studies have shown H17-H19 fail to bind to all glycans tested 9 , 10 . Interestingly, H17 viruses displayed a species-specific affinity for MHCII, in which only the human leukocyte antigen isotype DR (HLA-DR) but not the swine homolog (SLA-DR) MHCII allowed infection of non-permissive cells 8 . Conserved residues in the α1, α2, and β1 domains of the HLA-DR molecule were shown to be critical for H18N11 infection of human cells 11 . More recently, human H2N2 and related avian H2N2 FLUAV were shown to possess dual receptor specificity for sialic acid and MHC class II, with entry via MHC class II being independent of sialic acid 12 . This suggests that MHCII could play a role in the host range of FLUAV although the ability of other sialic acid-binding HA subtypes to use MHCII as an entry receptor remains unknown. Pigs are a natural host of FLUAV and have historically been pointed as important intermediary hosts for the generation of viruses with pandemic potential 13 . Frequent spillover of FLUAV between humans and swine, followed by subsequent evolution of few of these strains in the new host, has impacted the epidemiology of circulating strains in both species 14 , 15 . We previously showed that adaptation of a human-origin H3N2 virus to pigs selected for a single HA mutation (A138S, H3 numbering) that increased affinity for porcine alveolar macrophages (PAMs) in vivo compared to the original HA 16 . This ultimately led to apoptosis and depletion of this cell population in the lungs of infected pigs. Given that alveolar macrophages express high quantities of MHCII on the cell surface 17 , we investigated the ability of two prototypic human- (hVIC/11) and swine- (sOH/04) adapted H3N2 viruses 18 to use HLA-DR or SLA-DR as receptors to initiate infection. We found that both viruses can use MHCII as an entry receptor in addition to SA in a host-specific manner. Moreover, introduction of point mutations in the hVIC/11 HA RBS changed the virus host range and allowed utilization of the SLA-DR. These mutations also affected sialic acid binding, suggesting that the MHCII-HA interaction is mediated by the RBS or its surroundings and that mutations in or near this pocket have a dual impact on the specificity of the virus for SA and MHCII. Results MHCII is needed for the infection of porcine alveolar macrophages (PAMs) by swine-adapted H3N2 FLUAV in the absence of SAs. To investigate whether SLA-DR-mediated entry plays a role in FLUAV infection in vivo , we leveraged samples from a previous study 16 that showed significant tropism and replication differences between human-origin (hVIC/11) and swine-adapted (sOH/04) FLUAVs in the lungs of pigs. Analysis of lung samples from sOH/04-infected pigs revealed a strong colocalization between SLA-DR and FLUAV HA antigens, suggesting that SLA-DR + cells are susceptible to infection with the swine-adapted FLUAV in vivo ( Fig. 1 a ) . Following our previous findings that PAMs can be infected with swine-adapted FLUAV 16 , we investigated the role of MHCII during FLUAV infection in alveolar macrophages collected from naïve pigs. Since SA is the primary receptor used by H3N2 FLUAV, depletion of these receptors from PAMs was needed to evaluate the role of MHCII during FLUAV entry. Two methods were evaluated to deplete SA on the cell surface: chemical oxidation and enzymatic SA removal with the neuraminidase from Clostridium perfringens . SA oxidation with sodium periodate (NaIO 4 ) was highly cytotoxic (Supplementary Fig. 1a) while SA removal using neuraminidase treatment showed little cytotoxicity (Supplementary Fig. 1b). After a 24-hour incubation with 100 mU/mL of neuraminidase (100% cell viability), most of α2,3 and α2,6 SA was successfully removed from PAMs ( Fig. 1 b, Supplementary Fig. 1c) . Further, the neuraminidase treatment did not affect SLA-DR expression in these cells ( Fig. 1 c ) . Inoculation of untreated PAMs with sOH/04 and hVIC/11 resulted in replication of both viruses, although hVIC/11 displayed lower titers overall compared to sOH/04 ( Fig. 1 d and e) , particularly at 48 hours post infection (hpi). Neuraminidase treatment partially reduced replication of sOH/04 and hVIC/11. Inhibition seemed proportional to previously observed replication, with sOH/04 reaching significantly higher titers than hVIC/11 at 48 and 72 hpi. Infection of PAMs was significantly reduced only when both sialic acid and SLA-DR were depleted by a neuraminidase treatment coupled with an anti-SLA-DR antibody incubation, in a dose-dependent manner ( Fig. 1 f ) . Incubation with a control antibody did not affect replication in deacetylated PAMs ( Fig. 1 g ). These results suggest that FLUAV can infect porcine alveolar macrophages in absence of sialic acid, and these infections might be dependent on MHCII availability. Transient expression of MHCII in deacetylated cells enables FLUAV entry and replication. To confirm that FLUAV uses MHCII as an entry receptor, we transfected HEK-293T cells with plasmids encoding the alpha and beta chain of either HLA-DR or SLA-DR. HEK-293T cells were selected due to their high transfection efficiency 19 . Transient expression of MHCII was demonstrated by immunofluorescence analysis (Supplementary Fig. 2a) . Subsequently, transfected cells were deacetylated using exogenous neuraminidase (Supplementary Fig. 2b). MHCII + HEK-293T cells depleted or not of SAs were inoculated with sOH/04 and hVIC/11. Under sialic acid-depleted conditions, FLUAV only infected MHCII-expressing cells ( Fig. 2 a ) . This was further confirmed by flow cytometry (Supplementary Fig. 3a). Moreover, removal of SAs completely prevented FLUAV infection when cells were transfected with an empty plasmid (Supplementary Fig. 3a-b) . Levels of FLUAV-positive cells among those bearing sialic acid were similar regardless of MHCII expression (Supplementary Fig. 3c) . When only MHCII-expressing cells were considered ( Fig. 2 b and c) , we found a clear trend in which hVIC/11 preferably infected HLA-DR-expressing cells. Similarly, transient expression of SLA-DR in deacetylated cells significantly increased the affinity of sOH/04 to higher levels than when HLA-DR was present. Quantitatively, hVIC/11 infected 17.9% of HLA-DR + cells vs 5.1%of SLA-DR-expressing cells ( Fig. 2 d ) . On this line, sOH/04 preferentially infected SLA-DR + cells in which 22.7% of cells were infected, contrasting with the 6.7% of infected cells when HLA-DR was present ( Fig. 2 d ) . Control infections performed in untreated cells or deacetylated cells transfected with an empty plasmid supported that the transfection process alone did not allow FLUAV infection of non-sialilated cells ( Fig. 2 e ) . Alignment of the HLA-DR and SLA-DR alpha and beta chains showed multiple amino acid differences between the two homologue genes that could account for this effect (Supplementary Fig. 4) . As a negative control we used the 2009 pandemic H1N1 A/California/04/2009 virus (pH1N1), a subtype previously shown to not use MHCII as an entry receptor 12 . pH1N1 successfully infected untreated HEK-293T cells but failed to infect deacetylated HEK-293T (Supplementary Fig. 5a) , even when HLA-DR or SLA-DR were expressed (Supplementary Fig. 5b and c) . After recognition of sialic acid, FLUAV is internalized in endosomes that are further acidified, allowing the virus particle disassembly and genome release into the cytoplasm 20 . Therefore, we evaluated if FLUAV follows a similar route after interacting with MHCII. Prevention of endosomal acidification by ammonium chloride (NH 4 Cl) 21 ( Fig. 2 f ) and bafilomycin A1 22 ( Fig. 2 g ) strongly reduced FLUAV infection of deacetylated MHCII + HEK-293T cells suggesting that upon interaction with MHCII, FLUAV follows a similar endocytic pathway as when SA is used as a receptor. HEK-293T cells allow entry of FLUAV but they do not support efficient FLUAV replication. Therefore, to assess whether MHCII allows replication of FLUAV in deacetylated airway epithelial cells, we used A549 cells due to their high transfection efficiency, high permissibility to FLUAV replication, and as a representative of the primary target cells of FLUAV in mammalian hosts. Treatment with neuraminidase efficiently removed sialic acid from the cell surface ( Fig. 3 a, Supplementary Fig. 6a) but the neuraminidase treatment alone did not fully prevent FLUAV replication (Supplementary Fig. 6b and c). Replication was only abolished when cells were also incubated with SNA and MALII lectins in addition to the peanut agglutinin (PNA, Supplementary Fig. 6b and c) to block any remaining SAs. A549 cells expressing either HLA-DR or SLA-DR ( Fig. 3 b ) were inoculated with hVIC/11 and sOH/04. Deacetylation by neuraminidase treatment followed by lectins and PNA treatment prevented H3N2 FLUAV replication. However, MHCII expression granted replication in a species-specific manner: HLA-DR expression allowed replication of hVIC/11 in non-sialylated cells ( Fig. 3 c ) while SLA-DR expression enabled replication of sOH/04 to similar levels as the untreated control group ( Fig. 3 d ) . Interestingly, sOH/04 also replicated in HLA-DR + cells, although titers at all timepoint were lower compared to infection in SLA-DR + cells. MHCII expression in presence of sialic acid did not boost replication of the viruses ( Fig. 3 c and d) . We then tested whether anti-MHCII antibodies inhibit FLUAV replication in deacetylated MHCII + A549 cells. The anti-HLA-DR antibody strongly inhibited hVIC/11 infection of deacetylated HLA-DR + cells ( Fig. 3 e ) , while the anti-SLA-DR antibody significantly reduced sOH/04 titers in SLA-DR + cells ( Fig. 3 f ) . Finally, we evaluated if infection of MHCII + cells can induce cell death. hVIC/11 replication led to a significant increase in HLA-DR + cells mortality at 48 hpi ( Fig. 3 g ) while sOH/04 replication resulted in significant increase in cell death in SLA-DR + cells, although it also affected HLA-DR + cells’ viability ( Fig. 3 h ). Altogether, these results show that MHCII can be used as an entry receptor, and MHCII-mediated entry of FLUAV in cells lacking sialic acid leads to productive infection and cell death. Mutations near the sialic acid receptor binding site of H3N2 FLUAV HA alter species-specificity of MHCII-mediated entry. FLUAV interacts with terminal sialic acids on the susceptible cell surface via the RBS on the HA head domain 23 . Previously, we showed that adaptation of hVIC/11 to pigs selected for three mutations within the HA RBS: A138S, V186G, and F193Y (H3 numbering), which resulted in increased replication in SA-bearing swine cells 18 . All these residues are located in the surrounding of the sialic acid-binding pocket, although they do not directly interact with sialic acid ( Fig. 4 a ) . Furthermore, the A138S amino acid change significantly increases affinity for PAMs in vivo 16 . Therefore, we evaluated the ability of mutants hVIC/11-A138S, hVIC/11-V186G, and hVIC/11-F193Y to utilize either HLA-DR or SLA-DR as a receptor in vitro . Infection of deacetylated MHCII + HEK-293T cells revealed a stronger preference for SLA-DR over HLA-DR ( Fig. 4 b and c). When only MHCII + cells were considered, a similar trend was observed, with all three mutant viruses using both HLA-DR and SLA-DR as entry receptors, but with a statistically significant higher preference for the swine homologue ( Fig. 4 d ) . Quantification of FLUAV-positive cells among total cells (regardless of MHCII expression) supported that infection of deacetylated cells is completely abolished even when cells are transfected with an empty plasmid ( Fig. 4 e ). Evaluation of the replication capacity of the viruses in MHCII-transfected A549 cells, revealed an increase of the viral titers overtime in both HLA-DR- and SLA-DR-expressing deacetylated cells, with no differences observed between the MHCII from both species ( Fig. 4 f-h ) . This contrasts with hVIC/11 ( Fig. 3 c ) which failed to replicate in deacetylated, SLA-DR-expressing cells. Overall, these data demonstrate that the introduction of swine-adapting point mutations near the receptor binding site of hVIC/11 increase the affinity of the virus for SLA-DR. This is consistent with previous reports suggesting that these mutations represent an advantage for the virus in the swine host 18 . Additionally, we tested if these mutations could also affect the SA-binding preference of the virus. By performing a solid-phase SA receptor-binding assay, we observed that hVIC/11-A138S and hVIC/11-F193Y exhibited higher affinity for α2,6 SA compared to hVIC/11 ( Fig. 5 a-b,d ) although no changes were seen for α2,3 binding. Contrary, hVIC/11-V186G ( Fig. 5 c ) had a reduced binding to both α2,3 and α2,6 compared to all the mutants, including the parental hVIC/11. Taken together, these results demonstrate that mutations in the proximity of the sialic acid RBS can have a dual effect and impact MHCII recognition and SA binding at the same time. Discussion Recent methodological advances have increased our understanding on the tropism and receptor-specificity of FLUAV beyond the sialic acid linkage to galactose, including the description of numerous non-canonical entry receptors 24 . However, these newly identified molecules often rely on sialic acid to act as a functional receptor 25 , 26 , with the exception of a subset of phosphoglycans 27 . Bat-derived H17 and H18 and duck-derived H19 HAs were recently shown to be unable to bind sialic acid and instead use MHCII as a sialic acid-free entry mechanism 9 , 10 , 28 . Interestingly, MHCII was suggested to promote entry of H2N2 FLUAV into dendritic cells 29 , which was further recently demonstrated 12 . In this report we found that MHCII acts as a functional SA-independent entry receptor for two prototypic H3N2 viruses from human- and swine-origin and that binding is likely mediated by the vicinity of the HA RBS. To our knowledge, this is the first report for MHCII-mediated infection of group 2 HAs. Moreover, affinity for different MHCII homologues seems to correlate with the host from which the virus originated, implying a role of the MHCII complex in the adaptation process of FLUAV to a new host. Infection of deacetylated PAMs revealed that the swine-adapted sOH/04 was still able to replicate but replication was inhibited by blocking SLA-DR. Findings were similar for the human-seasonal hVIC/11, although replication was lower. Previous literature has shown that human and swine alveolar macrophages exhibit similar levels of α2,3 and α2,6 SA 30 , 31 . Hence, it is likely that the observed differences in affinity for these swine cells correlate with the ability of the viruses to recognize the SLA-DR more than the affinity for host-specific SA conformations. Moreover, our findings suggest that binding to MHCII in addition to SAs increases the affinity of FLUAV to PAMs but not in an additive effect. A recent report showed that replication of a seasonal H3N2 was not boosted by HLA-DR expression in the presence of sialic acid 12 . This is in line with our results in which neither HLA-DR nor SLA-DR enhanced viral replication when SA was present. We hypothesize that MHCII acts as an alternative host-specific receptor, increasing the availability of receptors when there is a potential sialic acid mismatch. Further, since our previous findings revealed that MHCII-mediated FLUAV infection of macrophages can induce cell death 16 , this could be a mechanism used by FLUAV to deplete AMs in the lungs 16 , 32 and reduce their antiviral activity 33 – 36 ; hence, facilitating virus spread in the respiratory tract. Future studies beyond the scope of this research will be needed to confirm this hypothesis. Further, the mechanism in which MHCII-mediated entry occurs and how it differs from the canonical sialic acid-triggered entry remains unknown. However, our data showed that MHCII-mediated entry and infection was disrupted by NH 4 Cl and bafilomycin A1. These compounds prevent endosomal acidification 37 and acid lysosome formation 38 , respectively, and inhibit FLUAV infection by preventing fusion with the endosome membrane and viral particle disassembly 39 . Hence, our data suggest that upon MHCII-mediated internalization, FLUAV follows a similar trafficking pathway as occurs with sialic acid-triggered entry. Similar to H17, H18, and H19 viruses that can use a wide range of MHCII homologues from different bat species, humans, swine, and chickens 8 , the H3N2 strains used here were shown to utilize both HLA-DR or SLA-DR as entry receptors in HEK-293T cells. As observed in PAMs, this promiscuity may have implications for the amplified tropism of H3N2 viruses that would facilitate host jumps. However, the human-adapted hVIC/11 virus exhibited preference for HLA-DR, and similarly, the swine-adapted sOH/04 preferentially used SLA-DR. The same pattern was observed in deacetylated A549 cells. Such host-specificity could have implications on the host range of influenza viruses but the ability to still bind to MHCII from other species, even if less efficiently, could play a role in facilitating the adaptation to a new host. In a previous report we found that the A138S mutation in a hVIC/11 HA increased affinity for PAMs in vivo but did not reach the same level of affinity as the swine-adapted sOH/04 16 . Other mutations near the RBS (V186G and F193Y) were also found after transmission in pigs and resulted in a fitness advantage for the viruses in swine respiratory cells 18 . Here, introduction of these single point mutations in the HA, near the RBS of hVIC/11, modified the MHCII recognition pattern of the virus and allowed utilization of both HLA-DR and SLA-DR. Although computational analysis suggests that the H18 binds to MHCII via the head domain 11 , the specific site on the HA protein responsible for MHCII recognition by H17-H19 remains unknown. Our data are in agreement with these observations and suggest that the interaction between MHCII and H3 is mediated by the HA sialic acid RBS or its surroundings, as the residues evaluated here do not directly interact with sialic acid but may affect conformation of the pocket 40 . Interestingly, a recent report found that antibodies targeting the H2 RBS can also inhibit MHCII binding 12 , which support that residues near the HA RBS play a role in MHCII binding. Furthermore, we found that all three mutations also affected SA binding, demonstrating that mutations in the HA RBS can have a dual impact on MHCII and sialic acid recognition. However, it is possible that residues 138, 186, and 193 do not directly interact with MHCII but simply induce conformational changes in the RBS that could indirectly favor or prevent recognition of MHCII. Crystallization of the HA-MHCII complex in future studies will be critical to understand the molecular basis of this interaction. Overall, our results demonstrate that H3N2 viruses can use the MHCII complex as an alternative entry receptor, resulting in a dual receptor preference (sialic acid and MHCII), and binding to MHCII is likely mediated by the sialic acid RBS of the HA. Similar to SA, affinity of the viruses for MHCII seems to be host-specific and adaptation of human viruses to pigs selects for mutations in the HA that enhance affinity for SLA-DR. Interestingly, during early stages of adaptation, the virus is still able to utilize both HLA-DR and SLA-DR, enabling the virus to expand its host range. Further, mutations occurring in the vicinity of the H3 RBS can affect the affinity of the HA for both SAs and MHCII receptors simultaneously. This is particularly important as MHCII might act as a tropism determinant that selectively promotes infection of antigen-presenting cells and ultimately favors virus replication in the lungs. Materials and Methods Ethics statement Animal studies described in this report were approved by Institutional Animal Care and Use Committee (IACUC) at the University of Georgia (protocol A2019 03-031-Y3-A9). Animals were maintained under biosafety level 2 conditions according to the Guide for the Care and Use of Agricultural Animals in Research and Teaching. At the end of the study, animals were euthanized following the American Veterinary Medical Association (AVMA) guidelines. Cells and viruses Madin-Darby canine kidney (MDCK), human lung carcinoma (A549), and human embryonic 293T (HEK-293T) cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St Louis, MO) supplemented with 2mM 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 incubated at 37°C under 5% CO 2 . FLUAV viruses in this study were generated using an 8-plasmid reverse genetic system as previously described 41 , 42 using an isogenic backbone. HA sequences were confirmed by Sanger sequencing and viral stocks were made in MDCK cells in opti-MEM containing 1 µg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemicals, Lakewood, NJ) at 37°C. Viral titers were determined by TCID 50 using the Reed and Muench method 43 . Porcine alveolar macrophages isolation Swine alveolar macrophages were collected and isolated as previously described 44 , 45 with minor modifications. Three-weeks-old cross-bred pigs were obtained from Midwest Research Swine Inc (Glencoe, MN). Animals were housed under biosafety level 2 conditions at the University of Georgia. After a 7-days acclimatation period, animals were confirmed negative for anti-FLUAV antibodies by competitive ELISA. For euthanasia, pigs were anesthetized with a cocktail of ketamine (6 mg/kg), xylazine (3 mg/kg), and telazol (6 mg/kg), and an intravenous pentobarbital overdose (Euthasol, 87 mg/kg). Lungs were aseptically collected, and alveolar macrophages were harvested by rinsing the lungs twice with calcium-free phosphate buffered saline (PBS). Samples were pelleted at 1,500 rpm for 10 minutes at 4°C and washed twice with calcium-free PBS. Subsequently, the pellet was resuspended in RPMI-1640 media (Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS, 1% antibiotics/antimycotic, 2 mM L-glutamine, and 1% non-essential amino acids. Cells were passed through a 40 µm cell strainer and then were incubated for 2 hours at 37°C in 10-cm petri dishes. After incubation, non-adherent cells were discarded, and PAMs were transferred to a new petri dish and incubated at 37°C under 5% CO 2 using the media described above. Tissue immunofluorescence : Right cranial lobe samples were collected from infected (sOH/04) and non-infected pigs in neutral-buffered formalin and paraffin embedded in a previous study 16 . For SLA-DR and HA detection, samples were deparaffinized and stained as previously described 46 with minor modification. Briefly, lung sections were deparaffinized and rehydrated followed by an antigen retrieval step in citrate buffer (10 mM sodium citrate, pH 6.0) for 40 minutes. Samples were permeabilized with 0.3% Triton-X100 (Sigma-Aldrich, St Louis, MO) and blocked with 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, St Louis, MO) for 1 hour. FLUAV was detected using an anti-Multi-Hemagglutinin (H3N2) polyclonal antibody (eEnzyme, Gaithersburg, MD) while an anti-pig SLA class II DR clone 2E9/13 (Bio-Rad, Hercules, CA) for MHCII detection was used. Samples were then incubated with a secondary Alexa 594-conjugated anti-rabbit antibody in a 1:1,000 dilution for HA detection and an Alexa 488-conjugated anti-mice antibody in a 1:1000 dilution for SLA-DR. After a 1-hour incubation, tissue samples were stained with 0.5 µg/mL 4’,6-diamine-2- phenylindole (DAPI, Sigma-Aldrich, St Louis, MO) for 15 minutes. Finally, samples were mounted on glass slides with mounting media (Vector Laboratories, Newark, CA) and imaged using a ZEISS LSM 900 confocal microscope. Sialic acid depletion : α2,3 and α2,6-SA were removed from the cell surface by incubating cells with 100 mU/mL of neuraminidase from Clostridium perfringens (Sigma-Aldrich, St Louis, MO) for 24 hours prior to infection. After infection, fresh neuraminidase was added, and new neuraminidase was added every 24 hours post infection. To prevent binding to uncleaved SA, cells were also supplemented with 15 µg/mL of Sambucus Nigra Lectin (SNA, Vector Laboratories, Newark, CA) and Maackia Amurensis Lectin II (MAL II, Vector Laboratories, Newark, CA). Finally, cells were supplemented with 20 µg/mL of Peanut Agglutinin (PNA, Sigma-Aldrich, St Louis, MO) to prevent any unspecific binding to exposed β-1,3- N -acetylgalactosamine. Sialic acid depletion was confirmed by immunofluorescence using fluorescein-conjugated SNA (Vector Laboratories, Newark, CA) and biotinylated MALII (Vector Laboratories, Newark, CA). PAMs viral growth kinetics Isolated PAMs were seeded in a 12-well plate at a density of 1.5x10 5 cells/well. Similarly, A549 cells were seeded in a 6-well plate at a density of 5x10 5 cells/well. All cells were incubated at 37°C overnight and then infected at an MOI of 0.01 for 1 hour at 37°C. After infection, plates were washed three times with PBS and cells were supplemented with Opti-MEM (ThermoFisher Scientific, Waltham, MA) containing 0.1 µg/mL of TPCK-treated trypsin. Supernatant aliquots were collected at 0, 12, 24, 48, and 72 h post-infection and kept at -80°C until processing. Viral RNA was extracted using the MagMax-96 AI/ND viral RNA (ThermoFisher Scientific, Waltham, MA) isolation kit according to manufacturer’s instructions. Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly MA) and specific primers targeting the M segment of the virus (Supplementary Table 1) . FLUAV titers were determined by comparison to a TCID 50 eq standard curve of an exact virus match of known titer previously determined using the Reed and Muench method. HLA-DR and SLA-DR antibody blocking PAMs were seeded in 12-well plates at a density of 1.5x10 5 cells/well while A549 cells were plated in a 6-well plate at a density of 5x10 5 cells/well. Plates were incubated overnight at 37°C. The next day, cells were incubated for 1 hour at 4°C with an anti-SLA-DR (Bio-Rad, Hercules, CA) or an anti-HLA-DR (ThermoFisher Scientific, Waltham, MA) antibody at concentrations ranging from 1 to 10 µg/mL or an anti-GFP antibody as a control (Sigma-Aldrich, St Louis, MO). After incubation cells were infected at an MOI of 0.1 for 1 hour at 37°C in the presence of the indicated antibody concentrations. Finally, cells were washed three times with PBS and supplemented with Opti-MEM containing 0.1 µg/mL of TPCK-treated trypsin and the desired antibody concentrations. Samples were collected at 48 hpi for further virus titration. Plasmids : HLA-DRA (GenBank accession number: NM_019111.5), HLA-DRB1 (GenBank accession number: NM_002124.4), SLA-DRA (GenBank accession number: MF498819.1), and SLA-DRB1 (GenBank accession number: MF498811.1) gene fragments were synthetized (Twist Bioscience, San Francisco, CA) and amplified by PCR to introduce EcoRI and BglII restriction sites using the primers listed in Supplementary Table 1. pCAGGS plasmid and MHCII fragments were digested using EcoRI and BglII restriction enzymes (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions for 1 hour at 37°C for further ligation and transformation. Plasmids sequences were confirmed by whole plasmid sequencing. MHCII expression in A549 and HEK-293T cells : Cells were seeded at a density of 8x10 5 cells/well in 6-well plates and were incubated at 37°C until a 70% confluence was reached. MHCII subunits were transfected using equimolar quantities of either HLA-DRA and HLA-DRB1 or SLA-DRA and SLA-DRB1 and TransiT LT1 (Mirus Bio, Madison, WI) in a 1:2 ratio DNA:TransiT. HLA-DR and SLA-DR complexes expression was assessed by flow cytometry and immunofluorescence as described below. At 24 hours post transfection, cells were deacetylated as described above and non-sialylated cells were infected at 48 hours post transfection. MHCII-mediated entry assessment : HEK-293T and A549 cells were seeded and transfected as described above. At 24 hours post transfection, cells were deacetylated with 100 mU/mL of neuraminidase from Clostridium perfringens for 24 hours at 37°C. Non-deacetylated cells were left untouched until 48 hours post transfection. 1 hour before infection, deacetylated cells were incubated with 15 µg/mL of SNA, 15 µg/mL of MALII, and 20 µg/mL of PNA. At 48 hours post transfection, cells were infected at an MOI of 0.1 for 1 hour at 37°C. After incubation, virus-containing supernatant was discarded, and fresh media supplemented with 0.1 µg/mL TPCK-treated trypsin and 100 mU/mL of neuraminidase was added. Neuraminidase was not included in the non-deacetylated control groups. Finally, infections were incubated for 24 hours at 37°C and cells were then processed for either flow cytometry or immunofluorescence. Flow cytometry analysis : 24 hours after infection as described above, supernatants from A549- and HEK-293T-infected cells were collected, and cells were trypsinized and centrifuged at 1,500 rpm for 5 minutes. Cells were resuspended in PBS supplemented with 5% FBS and stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by a 15-minute permeabilization with 0.3% Triton-X (Sigma-Aldrich, St Louis, MO). Afterwards, cells were washed three times and incubated for 1 hour with the following primary antibodies: Alexa 647-conjugated anti-SLA-DR clone 2E9/13 (Bio-Rad, Hercules, CA), anti-HLA-DR clone LN3 (ThermoFisher Scientific, Waltham, MA) at a 1:200 dilution in PBS and an anti-H3 HA antibody (stem specific antibody) clone CR8020 at a 1:500 dilution. Following incubation, cells were washed three times with PBS. For simultaneous detection of FLUAV and MHCII, samples were incubated for 1 hour with an Alexa 488-conjugated anti-human secondary antibody and an APC-conjugated anti-mice secondary antibody respectively (ThermoFisher Scientific, Waltham, MA), both in a 1:1,000 dilution. Finally, cells were washed three times with PBS and analyzed using a NovoCyte Quanteon flow cytometer system (Agilent, Santa Clara, CA) using the gating strategy exemplified in Supplementary Fig. 7 . Data was analyzed using FlowJo version 10.8.2 (FlowJo, Ashland, OR). Immunofluorescence staining : HEK-293T and A549 cells were plated in poly-D-Lysine-coated glass slides and incubated at 37°C until an 80% confluence was reached. Cells were transfected and deacetylated as described above. Cells were infected at an MOI of 0.01 and incubated for 24 hours at 37°C as described above. After infection, cells were fixed with 4% formaldehyde for 30 minutes at room temperature, followed by a 10-minute permeabilization with 0.3% Triton-X100 and a 1-hour blocking step using 3% bovine serum albumin (BSA, ThermoFisher Scientific, Waltham, MA) and 0.1% Triton-X100 in PBS. HLA-DR, SLA-DR, and FLUAV were stained using the specific antibodies described above in a 1:200 dilution for 1 hour at room temperature in 3% BSA and 0.1% Triton-X100 in PBS. After incubation, samples were washed three times with PBS and incubated with the secondary antibodies described above in a 1:1,000 dilution with 0.5 µg/mL 4’,6-diamine-2- phenylindole (DAPI, Sigma-Aldrich, St Louis, MO), 3% BSA, and 0.1% Triton-X100 in PBS for 1 hour. Finally, cells were washed 5 times with PBS and mounted on glass slides using mounting media. For sialic acid staining, samples were incubated with either biotin-conjugated or fluorescein-labelled SNA and biotin-conjugated MAL II lectins in a 1:250 dilution in PBS for 30 minutes prior to permeabilization. After incubation with lectins, cells were incubated for 30 minutes with a secondary Alexa 594-conjugated streptavidin in a 1:1,000 dilution. Samples were imaged using a ZEISS LSM 900 confocal microscope. FLUAV entry blockage and cell viability assay HEK-293T cells were plated and transfected as described above. 1 hour before infection, cells were incubated with 20 mM of NH 4 Cl (Sigma-Aldrich, St Louis, MO) or 10 nM Bafilomycin A1 (Cells Signaling Technologies, Danvers, MA). After incubation, cells were infected at an MOI of 0.1 as described above and kept at 37°C for 24 hours in presence of the desired inhibitor. At 24 hpi, cells were washed three times with PBS and total cellular RNA was extracted using the MagMax-96 AI/ND RNA (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. RNA samples were normalized to 1 µg and FLUAV M segment copy number was determined by RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly MA) and a standard curve of a M-cloned segment plasmid of known concentration. Cellular viability was assessed using the CellTiter Glo Cell Viability kit (Promega, Madison, WI) according to the manufacturer’s instruction. MHCII-mediated replication in A549 cells A549 cells were seeded and transfected as described above. At 24 hours post transfection, sialic acid was removed with 100 mU/mL of neuraminidase for 24 hours. Following incubation, deacetylated cells were further incubated with 15 µg/mL of SNA, 15 µg/mL of MALII, and 20 µg/mL of PNA for 1 hour before infection. Non-deacetylated control cells remained untouched until infection. At 48 hours post transfection, cells were infected at an MOI of 0.01 for 1 hour at 37°C. After incubation, supernatant was discarded and replaced with media containing 0.1 µg/mL TPCK-treated trypsin and 100 mU/mL of neuraminidase. Fresh neuraminidase was added every 24 hours. No neuraminidase was used in non-deacetylated cells. Time-points were collected at 0, 12, 24, 48, and 72 hours post infection and viral titers were determined by RT-qPCR as described above. Solid-phase binding assay : Virus affinity for α2,3 and α2,6 SA was assessed as previously described 47 . Briefly, viruses were purified by ultracentrifugation at 28,000 rpm for 3 hours using a 20% sucrose cushion. Pelleted viruses were resuspended in TNE buffer (0.01M Tris, 0.001M EDTA, 0.1 M NaCl, pH 7.2). Samples were normalized to 128 HAU and adsorbed in fetuin-coated plates at 4°C overnight. Unbound virus was washed using 0.02% Tween-80 in PBS (washing buffer) and plates were blocked for 2 hours with deacetylated BSA (BSA-NA). After blocking, plates were washed three times with washing buffer and incubated with different concentrations of Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (α2,3 SA, Glycotech, Gaithersburg, MD) or Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (α2,6 SA, Glycotech, Gaithersburg, MD) in 0.02% Tween-80, 0.1% BSA-NA, and 2 µM oseltamivir in PBS (reaction buffer). After a 1-hour incubation, plates were washed five times with washing buffer and incubated with HRP-conjugated streptavidin (ThermoFisher Scientific, Waltham, MA) in a 1:1,000 dilution for 1 hour at 4°C. Finally, plates were washed 5 times with washing buffer and incubated with TMB (ThermoFisher Scientific, Waltham, MA) for 10 minutes. Reaction was stopped using a stop solution (ThermoFisher Scientific, Waltham, MA), and absorbance was measured at 450 nm using a Synergy HTX Multi-Mode Microplate Reader Agilent BioTek, Santa Clara, CA). Statistical analysis Statistical analyses were performed using GraphPad Prism 10 software. Data is presented as the mean ± standard error of the mean of at least three independent experiments unless stated otherwise in the figure legend. Each data point represents the average of three technical replicates for a total of three biological replicates per experiment. P values were obtained by ordinary two-way ANOVA with Tukey’s multiple comparison test. Declarations Data availability: All source data relevant for this study is available within the manuscript and has been provided as a source data file and supplementary information. Acknowledgement We thank the University Research Animal Resources personnel at the University of Georgia for assistance with animal care. Authors contribution: MCP, DSR, and CJC designed the experiments. MCP performed the cloning, growth kinetics, immunofluorescence, and MHCII expression experiments. MCP performed the cell viability assays. MCP and SC performed the flow cytometry experiments. MCP analyzed the data. MCP and DSR wrote the manuscript. CJC, AGS, and DRP edited the manuscript. Funding: 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 to DRP and by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a National Institute of Allergy and Infectious Diseases (NIAID) funded Center for Influenza Research and Response (CEIRR, contract 75N93021C00014 to DRP and AG-S. 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 also funded by 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 expertise 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. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Conflict of interest The A.G.-S. laboratory has received research support from Avimex, Dynavax, Pharmamar, 7Hills Pharma, ImmunityBio and Accurius, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, Virofend and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The other authors declare no conflict of interest. References Zhao, C. & Pu, J. Influence of Host Sialic Acid Receptors Structure on the Host Specificity of Influenza Viruses. Viruses 14 (2022). https://doi.org:10.3390/v14102141 Samji, T. 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Porcine alveolar macrophages, isolation, morphological and functional characteristics. Zentralbl Veterinarmed A 31 , 59-71 (1984). https://doi.org:10.1111/j.1439-0442.1984.tb01262.x Goatley, L. C., Nash, R. & Netherton, C. L. Primary Macrophage Culture from Porcine Blood and Lungs. Methods Mol Biol 2503 , 63-72 (2022). https://doi.org:10.1007/978-1-0716-2333-6_4 Zaqout, S., Becker, L. L. & Kaindl, A. M. Immunofluorescence Staining of Paraffin Sections Step by Step. Front Neuroanat 14 , 582218 (2020). https://doi.org:10.3389/fnana.2020.582218 Matrosovich, M. N. & Gambaryan, A. S. Solid-phase assays of receptor-binding specificity. Methods Mol Biol 865 , 71-94 (2012). https://doi.org:10.1007/978-1-61779-621-0_5 Yang, H. et al. Structure and receptor binding preferences of recombinant human A(H3N2) virus hemagglutinins. Virology 477 , 18-31 (2015). https://doi.org:10.1016/j.virol.2014.12.024 Additional Declarations Yes there is potential Competing Interest. The A.G.-S. laboratory has received research support from Avimex, Dynavax, Pharmamar, 7Hills Pharma, ImmunityBio and Accurius, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, Virofend and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The other authors declare no conflict of interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6278351","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":450146950,"identity":"32f1b2aa-792e-4c9b-9f2a-5165ae42e500","order_by":0,"name":"Daniela Rajao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDCCAyBUAGYmMDBUQAQlCGsxgGk5Q6QWBqgWBgbGNiK08B0/e/DABwM7BvP2Aw8/fJx32K6/gfngbR48WiTP5CUcnGGQzCBzJiFZcua2w8kzDrAlW+PTYnAgx+AwjwEz0C0JCdK8QC0MB3jMpPFqOf/G4PAfg3oGCf4Hyb955xxOlj/A/w2/lhtAWxiASEIiIU2at+GwncEBHja8WiRvvDE42GNwnEdC4kGa5Yxj6QmGh9mMLefg0cJ3Psf4w4+KajkJ/pzkGx9qrO3ljjc/vPEGjxYYALqEJwHESGxgJkI5FLAfAJH2xGsYBaNgFIyCkQIA1G5Ppo6ifKYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0772-0065","institution":"University of Georgia","correspondingAuthor":true,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Rajao","suffix":""},{"id":450146951,"identity":"a07b78ec-566a-46c5-b8c2-0b10003300c3","order_by":1,"name":"Matias Cardenas","email":"","orcid":"https://orcid.org/0000-0001-8247-3669","institution":"University of Georgia","correspondingAuthor":false,"prefix":"","firstName":"Matias","middleName":"","lastName":"Cardenas","suffix":""},{"id":450146952,"identity":"8422ec3d-ea86-4849-a113-6925b1831a88","order_by":2,"name":"Sasha Compton","email":"","orcid":"","institution":"University of Georgia","correspondingAuthor":false,"prefix":"","firstName":"Sasha","middleName":"","lastName":"Compton","suffix":""},{"id":450146953,"identity":"8180d8be-1481-462b-bb7f-eb9457d0a78b","order_by":3,"name":"C. 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Nuclei were stained using DAPI (blue). Scale bar represents 30 mm.\u003cstrong\u003eb, \u003c/strong\u003eNeuraminidase treatment efficiently removed sialic acid from the surface of PAMs. Macrophages were incubated with 100 mU/mL of neuraminidase and sialic acid was stained at 24 hours post-treatment. Scale bar represents 30 mm. \u003cstrong\u003ec, \u003c/strong\u003eSLA-DR expression in deacetylated and non-deacetylated PAMs was evaluated at 24 hours post-neuraminidase treatment by immunofluorescence using an aSLA-DR specific antibody. Scale bar represents 30 mm. PAMs were left untreated or deacetylated and further infected \u003cem\u003eex vivo\u003c/em\u003e for 1 hour at an MOI of 0.01 of either sOH/04 (blue, \u003cstrong\u003ed\u003c/strong\u003e) or hVIC/11 (orange, \u003cstrong\u003ee\u003c/strong\u003e) and viral titers in the supernatant were determined at 0, 12, 24, 48, and 72 hpi. Data are presented as the mean ± SEM of three independent experiments. \u003cstrong\u003ef,\u003c/strong\u003e MHCII availability is critical for sOH/04 infection of deacetylated PAMs. 24 hours after deacetylation, PAMs were incubated with concentrations ranging 0-10 mg/mL of an aSLA-DR specific antibody or with 10 mg/mL of a control antibody \u003cstrong\u003e(g) \u003c/strong\u003efor 1 hour before infection. PAMs were infected at an MOI of 0.1 for 1 hour and then cells were supplemented with fresh media containing the desired antibody concentration and viral titers were measured at 48 hpi. \u0026nbsp;Data are presented as the mean ± SEM of three independent experiments. L.D= limit of detection.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/bfb5e65069198c6812056a29.png"},{"id":81814080,"identity":"4a4a63a6-de4b-4e73-b922-756d9df1725a","added_by":"auto","created_at":"2025-05-02 09:37:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8673439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHLA-DR and SLA-DR allow FLUAV entry into mammalian cells. a,\u003c/strong\u003e HEK-293T cells were transfected with equimolar quantities of either HLA-DRA+HLA-DRB1 or SLA-DRA+SLA-DRB1. MHCII-expressing cells and cells transfected with an empty plasmid were deacetylated at 24 hours post-transfection for 24 hours. Cells were infected with either sOH/04 or hVIC/11 at an MOI of 0.1 and infections were incubated for 24 hours before processing. Subsequently, cells were stained for MHCII (pink), sialic acid (red), and FLUAV (green) detection. Co-localization between MHCII and FLUAV can be seen as white. Scale bar represents 30 mm. Transient expression of MHCII allows FLUAV entry into deacetylated HEK-293T cells. Cells were transfected with either HLA-DR \u003cstrong\u003e(b)\u003c/strong\u003e or SLA-DR \u003cstrong\u003e(c) \u003c/strong\u003eand further deacetylated prior to infection with hVIC/11 or sOH/04 at an MOI of 0.01. At 24 hpi, cells were fixed and stained for MHCII (APC) and FLUAV (Alexa488). The percentage of FLUAV-positive cells was determined by flow cytometry (n=3). \u003cstrong\u003ed, \u003c/strong\u003ePercentage of FLUAV-positive cells among deacetylated MHCII-expressing cells determined by flow cytometry represented as the mean ± SEM of three independent experiments. sOH/04 is shown in blue while hVIC/11 can be seen in orange. \u003cstrong\u003ee, \u003c/strong\u003ePercentage of infected HEK-293T cells (regardless of MHCII expression) determined by flow cytometry. Data are presented as the mean ± SEM of three independent experiments. \u003cstrong\u003ef, \u003c/strong\u003eMHCII-expressing cells were incubated with 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl for 1 hour prior to infection with either sOH/04 (blue) or hVIC/11 (orange) at an MOI of 1. Infections were maintained at 37°C for 24 hours in presence of NH\u003csub\u003e4\u003c/sub\u003eCl and intracellular vRNA was determined at 24 hpi by RT-qPCR (n=3). \u003cstrong\u003eg,\u003c/strong\u003e Transfected HEK-293T cells were incubated with 10 nM Bafilomycin A1 before infection and were subsequently infected at an MOI of 1 with sOH/04 (blue) or hVIC/11 (orange). Intracellular FLUAV vRNA was quantified at 24 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D= limit of detection. Untreated: non-transfected, non-deacetylated cells. Empty vector: deacetylated cells transfected with an empty pCAGGS plasmid. HLA-DR: deacetylated cells expressing HLA-DR. SLA-DR: deacetylated cells expressing SLA-DR.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/5fbf7771de2e02e1fc34f0a2.png"},{"id":81814925,"identity":"ecec0744-91cd-4a82-8792-94aab8bec49e","added_by":"auto","created_at":"2025-05-02 09:45:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3966032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMHCII expression supports replication of FLUAV in deacetylated cells. a, \u003c/strong\u003eA549 cells were deacetylated using 100 mU/mL of neuraminidase for 24 hours. After incubation, a2,3 and a2,6 SA were stained and SA presence on the cells surface was analyzed by immunofluorescence. Scale bar represents 30 mm. \u003cstrong\u003eb, \u003c/strong\u003eMHCII expression in A549 cells was evaluated by immunofluorescence. Cells were transfected with equimolar quantities of either HLA-DRA+HLA-DRB1 or SLA-DRA+SLA-DRB1 and MHCII expression was assessed at 48 hours post-transfection. Scale bar represents 30 mm. Non-deacetylated (red), non-deacetylated cells expressing HLA-DR (SA+HLA-DR, green), non-deacetylated cells expressing SLA-DR (SA+SLA-DR, purple), deacetylated cells transfected with an empty plasmid (blue), deacetylated cells transfected with HLA-DR-expressing plasmids (black), and deacetylated A549 cells transfected with SLA-DR-expressing plasmids (yellow) were infected with either hVIC/11 \u003cstrong\u003e(c) \u003c/strong\u003eor sOH/04 \u003cstrong\u003e(d)\u003c/strong\u003e. Viral titers were measured in the supernatant at 0, 12, 24, 48, and 72 hpi by RT-qPCR. L.D= limit of detection. Data are presented as the mean ± SEM of three independent experiments. Similarly, cells left untreated or deacetylated and transfected with either HLA-DR or SLA-DR, were incubated with 10 mg/mL of an anti-HLA-DR, anti-SLA-DR, or a control antibody for 1 hour prior to infection with hVIC/11 \u003cstrong\u003e(e) \u003c/strong\u003eor sOH/04 \u003cstrong\u003e(f)\u003c/strong\u003e. At 48 hpi viral titers in the supernatant were quantified by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. The effect of FLUAV replication in deacetylated MHCII-expressing cells on the cell viability was evaluated for hVIC/11 \u003cstrong\u003e(g) \u003c/strong\u003eand sOH/04 \u003cstrong\u003e(h)\u003c/strong\u003e. A549 cells were transfected, deacetylated, and infected for further determination of cell viability at 48 hpi (n=3). Mock: non-infected cells, untreated: non-transfected, non-deacetylated cells. Empty vector: deacetylated cells transfected with an empty pCAGGS plasmid. HLA-DR: deacetylated cells expressing HLA-DR. SLA-DR: deacetylated cells expressing SLA-DR. For all experiments cells were also incubated with SNA and MALII lectins in addition to PNA to prevent binding to uncleaved SA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/2d2e0947c264a8d8d7a39748.png"},{"id":81814081,"identity":"072e174a-7a15-4f09-b7de-47c20838d5a1","added_by":"auto","created_at":"2025-05-02 09:37:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4220464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecognition of MHCII is mediated by the head domain of the hVIC/11 HA. a, \u003c/strong\u003ehVIC/11 HA trimer model made using pymol based on previously published crystal structure (PDB: 4WE8 \u003csup\u003e48\u003c/sup\u003e).\u003cstrong\u003e \u003c/strong\u003eSialic acid RBS is highlighted in pink while residues 138, 186, and 193 (H3 numbering) are shown in red.\u003cstrong\u003e \u003c/strong\u003eHEK-293T cells transfected with HLA-DR- \u003cstrong\u003e(b)\u003c/strong\u003e or SLA-DR-expressing plasmids \u003cstrong\u003e(c)\u003c/strong\u003e and then deacetylated with 100 mU/mL of neuraminidase. \u0026nbsp;Subsequently, cells were infected at an MOI of 0.01 with hVIC/11-A138S, hVIC/11-V186G, or hVIC/11-F193Y. At 24 hpi cells were stained for MHCII (APC) and FLUAV (Alexa488). \u003cstrong\u003ed\u003c/strong\u003e, FLUAV-positive cells among MHCII-expressing HEK-293T cells was determined by gating live, MHCII\u003csup\u003e+\u003c/sup\u003e cells only. Percentages were obtained by flow cytometry (n=3).\u003cstrong\u003e e,\u003c/strong\u003e Total amount of FLUAV-infected HEK-293T cells was determined by multi-color flow cytometry. \u0026nbsp;Data are presented as the mean ± SEM of three independent experiments. To evaluate virus replication in A549 cells, non-deacetylated (red), deacetylated cells transfected with an empty plasmid (blue), deacetylated cells transfected with HLA-DR-encoding plasmids (black), and deacetylated cells transfected with SLA-DR-expressing plasmids (yellow) were infected at an MOI of 0.01 with either hVIC/11-A138S \u003cstrong\u003e(f), \u003c/strong\u003ehVIC/11-V186G \u003cstrong\u003e(g)\u003c/strong\u003e, or hVIC/11-F193Y \u003cstrong\u003e(h)\u003c/strong\u003e. Viral titers in the supernatant were quantified at 0, 12, 24, 48, and 72 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D= limit of detection.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/8665663b3b9b09dafd1b4bb6.png"},{"id":81814086,"identity":"97fa5fbb-c01d-4a6a-87d5-db296fe84d60","added_by":"auto","created_at":"2025-05-02 09:37:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":770264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutations in the sialic acid HA RBS affecting MHCII recognition also influence SA binding. \u003c/strong\u003ePurified hVIC/11 \u003cstrong\u003e(a)\u003c/strong\u003e, hVIC/11-A138S \u003cstrong\u003e(b)\u003c/strong\u003e, hVIC/11-V186G \u003cstrong\u003e(c)\u003c/strong\u003e, and hVIC/11-F193Y \u003cstrong\u003e(d) \u003c/strong\u003ewere normalized to 100 HAU and adsorbed in fetuin-coated plates. Adsorbed viruses where then incubated with different concentrations Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (a2,3 SA) or Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (a2,6 SA). Data are presented as the mean ± SEM of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/f59fc70ceeeeb40b43b3398e.png"},{"id":104952649,"identity":"791d6181-2f3e-45ab-a70f-82d8657b1ee5","added_by":"auto","created_at":"2026-03-19 07:13:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22169102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/a7747ec6-da1b-4a67-898a-6874932bf175.pdf"},{"id":81814077,"identity":"0b414330-4536-4c20-a920-757b7347d254","added_by":"auto","created_at":"2025-05-02 09:37:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15697,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/569a08bbfda7d3360b961e0d.docx"},{"id":81814091,"identity":"220fe8b5-7487-4bbb-a42d-f3606ca35ff9","added_by":"auto","created_at":"2025-05-02 09:37:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14850841,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"MHCSupplementaryFiguresFV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6278351/v1/8055a0568ad999ec5207697b.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe A.G.-S. laboratory has received research support from Avimex, Dynavax, Pharmamar, 7Hills Pharma, ImmunityBio and Accurius, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, Virofend and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The other authors declare no conflict of interest.","formattedTitle":"MHC class II is a functional receptor for H3N2 Influenza A viruses and mediates host-specificity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSialic acid (SA) is the canonical receptor used by influenza A viruses (FLUAV). These SAs on the cell surface are recognized by the viral hemagglutinin (HA) protein to promote entry and begin the replication cycle \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The SA structure is linked to FLUAV host range since avian-origin FLUAVs mainly recognize sialic acids linked to galactose by an α2,3 linkage while mammalian-origin FLUAVs recognize α2,6-linked SA \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, mutations in the HA protein in or near the receptor-binding site (RBS) can modify the binding preference of the virus \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and may lead to a switch in the host specificity \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. By contrast, recently discovered bat-origin H17 and H18 HA subtypes, as well as duck-origin H19, were shown to exclusively use the Major Histocompatibility Complex II (MHCII) as an entry receptor \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This interaction is completely independent of sialic acid as multiple glycan array studies have shown H17-H19 fail to bind to all glycans tested \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Interestingly, H17 viruses displayed a species-specific affinity for MHCII, in which only the human leukocyte antigen isotype DR (HLA-DR) but not the swine homolog (SLA-DR) MHCII allowed infection of non-permissive cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Conserved residues in the α1, α2, and β1 domains of the HLA-DR molecule were shown to be critical for H18N11 infection of human cells \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. More recently, human H2N2 and related avian H2N2 FLUAV were shown to possess dual receptor specificity for sialic acid and\u003c/p\u003e \u003cp\u003eMHC class II, with entry via MHC class II being independent of sialic acid\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This suggests that MHCII could play a role in the host range of FLUAV although the ability of other sialic acid-binding HA subtypes to use MHCII as an entry receptor remains unknown.\u003c/p\u003e \u003cp\u003ePigs are a natural host of FLUAV and have historically been pointed as important intermediary hosts for the generation of viruses with pandemic potential \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Frequent spillover of FLUAV between humans and swine, followed by subsequent evolution of few of these strains in the new host, has impacted the epidemiology of circulating strains in both species\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We previously showed that adaptation of a human-origin H3N2 virus to pigs selected for a single HA mutation (A138S, H3 numbering) that increased affinity for porcine alveolar macrophages (PAMs) \u003cem\u003ein vivo\u003c/em\u003e compared to the original HA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This ultimately led to apoptosis and depletion of this cell population in the lungs of infected pigs. Given that alveolar macrophages express high quantities of MHCII on the cell surface \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, we investigated the ability of two prototypic human- (hVIC/11) and swine- (sOH/04) adapted H3N2 viruses \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e to use HLA-DR or SLA-DR as receptors to initiate infection. We found that both viruses can use MHCII as an entry receptor in addition to SA in a host-specific manner. Moreover, introduction of point mutations in the hVIC/11 HA RBS changed the virus host range and allowed utilization of the SLA-DR. These mutations also affected sialic acid binding, suggesting that the MHCII-HA interaction is mediated by the RBS or its surroundings and that mutations in or near this pocket have a dual impact on the specificity of the virus for SA and MHCII.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMHCII is needed for the infection of porcine alveolar macrophages (PAMs) by swine-adapted H3N2 FLUAV in the absence of SAs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether SLA-DR-mediated entry plays a role in FLUAV infection \u003cem\u003ein vivo\u003c/em\u003e, we leveraged samples from a previous study \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e that showed significant tropism and replication differences between human-origin (hVIC/11) and swine-adapted (sOH/04) FLUAVs in the lungs of pigs. Analysis of lung samples from sOH/04-infected pigs revealed a strong colocalization between SLA-DR and FLUAV HA antigens, suggesting that SLA-DR\u0026thinsp;+\u0026thinsp;cells are susceptible to infection with the swine-adapted FLUAV \u003cem\u003ein vivo\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Following our previous findings that PAMs can be infected with swine-adapted FLUAV \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, we investigated the role of MHCII during FLUAV infection in alveolar macrophages collected from na\u0026iuml;ve pigs. Since SA is the primary receptor used by H3N2 FLUAV, depletion of these receptors from PAMs was needed to evaluate the role of MHCII during FLUAV entry. Two methods were evaluated to deplete SA on the cell surface: chemical oxidation and enzymatic SA removal with the neuraminidase from \u003cem\u003eClostridium perfringens\u003c/em\u003e. SA oxidation with sodium periodate (NaIO\u003csub\u003e4\u003c/sub\u003e) was highly cytotoxic \u003cb\u003e(Supplementary Fig.\u0026nbsp;1a)\u003c/b\u003e while SA removal using neuraminidase treatment showed little cytotoxicity \u003cb\u003e(Supplementary Fig.\u0026nbsp;1b).\u003c/b\u003e After a 24-hour incubation with 100 mU/mL of neuraminidase (100% cell viability), most of α2,3 and α2,6 SA was successfully removed from PAMs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eSupplementary Fig.\u0026nbsp;1c)\u003c/b\u003e. Further, the neuraminidase treatment did not affect SLA-DR expression in these cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Inoculation of untreated PAMs with sOH/04 and hVIC/11 resulted in replication of both viruses, although hVIC/11 displayed lower titers overall compared to sOH/04 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed \u003cb\u003eand e)\u003c/b\u003e, particularly at 48 hours post infection (hpi). Neuraminidase treatment partially reduced replication of sOH/04 and hVIC/11. Inhibition seemed proportional to previously observed replication, with sOH/04 reaching significantly higher titers than hVIC/11 at 48 and 72 hpi. Infection of PAMs was significantly reduced only when both sialic acid and SLA-DR were depleted by a neuraminidase treatment coupled with an anti-SLA-DR antibody incubation, in a dose-dependent manner \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Incubation with a control antibody did not affect replication in deacetylated PAMs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e).\u003c/b\u003e These results suggest that FLUAV can infect porcine alveolar macrophages in absence of sialic acid, and these infections might be dependent on MHCII availability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient expression of MHCII in deacetylated cells enables FLUAV entry and replication.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm that FLUAV uses MHCII as an entry receptor, we transfected HEK-293T cells with plasmids encoding the alpha and beta chain of either HLA-DR or SLA-DR. HEK-293T cells were selected due to their high transfection efficiency \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Transient expression of MHCII was demonstrated by immunofluorescence analysis \u003cb\u003e(Supplementary Fig.\u0026nbsp;2a)\u003c/b\u003e. Subsequently, transfected cells were deacetylated using exogenous neuraminidase \u003cb\u003e(Supplementary Fig.\u0026nbsp;2b).\u003c/b\u003e MHCII\u003csup\u003e+\u003c/sup\u003e HEK-293T cells depleted or not of SAs were inoculated with sOH/04 and hVIC/11. Under sialic acid-depleted conditions, FLUAV only infected MHCII-expressing cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. This was further confirmed by flow cytometry \u003cb\u003e(Supplementary Fig.\u0026nbsp;3a).\u003c/b\u003e Moreover, removal of SAs completely prevented FLUAV infection when cells were transfected with an empty plasmid \u003cb\u003e(Supplementary Fig.\u0026nbsp;3a-b)\u003c/b\u003e. Levels of FLUAV-positive cells among those bearing sialic acid were similar regardless of MHCII expression \u003cb\u003e(Supplementary Fig.\u0026nbsp;3c)\u003c/b\u003e. When only MHCII-expressing cells were considered \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u003cb\u003eand c)\u003c/b\u003e, we found a clear trend in which hVIC/11 preferably infected HLA-DR-expressing cells. Similarly, transient expression of SLA-DR in deacetylated cells significantly increased the affinity of sOH/04 to higher levels than when HLA-DR was present. Quantitatively, hVIC/11 infected 17.9% of HLA-DR\u003csup\u003e+\u003c/sup\u003e cells vs 5.1%of SLA-DR-expressing cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. On this line, sOH/04 preferentially infected SLA-DR\u003csup\u003e+\u003c/sup\u003e cells in which 22.7% of cells were infected, contrasting with the 6.7% of infected cells when HLA-DR was present \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Control infections performed in untreated cells or deacetylated cells transfected with an empty plasmid supported that the transfection process alone did not allow FLUAV infection of non-sialilated cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Alignment of the HLA-DR and SLA-DR alpha and beta chains showed multiple amino acid differences between the two homologue genes that could account for this effect \u003cb\u003e(Supplementary Fig.\u0026nbsp;4)\u003c/b\u003e. As a negative control we used the 2009 pandemic H1N1 A/California/04/2009 virus (pH1N1), a subtype previously shown to not use MHCII as an entry receptor \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. pH1N1 successfully infected untreated HEK-293T cells but failed to infect deacetylated HEK-293T \u003cb\u003e(Supplementary Fig.\u0026nbsp;5a)\u003c/b\u003e, even when HLA-DR or SLA-DR were expressed \u003cb\u003e(Supplementary Fig.\u0026nbsp;5b and c)\u003c/b\u003e. After recognition of sialic acid, FLUAV is internalized in endosomes that are further acidified, allowing the virus particle disassembly and genome release into the cytoplasm \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, we evaluated if FLUAV follows a similar route after interacting with MHCII. Prevention of endosomal acidification by ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e and bafilomycin A1 \u003csup\u003e22\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e strongly reduced FLUAV infection of deacetylated MHCII\u003csup\u003e+\u003c/sup\u003e HEK-293T cells suggesting that upon interaction with MHCII, FLUAV follows a similar endocytic pathway as when SA is used as a receptor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHEK-293T cells allow entry of FLUAV but they do not support efficient FLUAV replication. Therefore, to assess whether MHCII allows replication of FLUAV in deacetylated airway epithelial cells, we used A549 cells due to their high transfection efficiency, high permissibility to FLUAV replication, and as a representative of the primary target cells of FLUAV in mammalian hosts. Treatment with neuraminidase efficiently removed sialic acid from the cell surface \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cb\u003eSupplementary Fig.\u0026nbsp;6a)\u003c/b\u003e but the neuraminidase treatment alone did not fully prevent FLUAV replication \u003cb\u003e(Supplementary Fig.\u0026nbsp;6b and c).\u003c/b\u003e Replication was only abolished when cells were also incubated with SNA and MALII lectins in addition to the peanut agglutinin \u003cb\u003e(PNA, Supplementary Fig.\u0026nbsp;6b and c)\u003c/b\u003e to block any remaining SAs. A549 cells expressing either HLA-DR or SLA-DR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e were inoculated with hVIC/11 and sOH/04. Deacetylation by neuraminidase treatment followed by lectins and PNA treatment prevented H3N2 FLUAV replication. However, MHCII expression granted replication in a species-specific manner: HLA-DR expression allowed replication of hVIC/11 in non-sialylated cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e while SLA-DR expression enabled replication of sOH/04 to similar levels as the untreated control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Interestingly, sOH/04 also replicated in HLA-DR\u003csup\u003e+\u003c/sup\u003e cells, although titers at all timepoint were lower compared to infection in SLA-DR\u003csup\u003e+\u003c/sup\u003e cells. MHCII expression in presence of sialic acid did not boost replication of the viruses \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u003cb\u003eand d)\u003c/b\u003e. We then tested whether anti-MHCII antibodies inhibit FLUAV replication in deacetylated MHCII\u003csup\u003e+\u003c/sup\u003e A549 cells. The anti-HLA-DR antibody strongly inhibited hVIC/11 infection of deacetylated HLA-DR\u003csup\u003e+\u003c/sup\u003e cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, while the anti-SLA-DR antibody significantly reduced sOH/04 titers in SLA-DR\u003csup\u003e+\u003c/sup\u003e cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Finally, we evaluated if infection of MHCII\u003csup\u003e+\u003c/sup\u003e cells can induce cell death. hVIC/11 replication led to a significant increase in HLA-DR\u003csup\u003e+\u003c/sup\u003e cells mortality at 48 hpi \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e while sOH/04 replication resulted in significant increase in cell death in SLA-DR\u003csup\u003e+\u003c/sup\u003e cells, although it also affected HLA-DR\u003csup\u003e+\u003c/sup\u003e cells\u0026rsquo; viability \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u003cb\u003e).\u003c/b\u003e Altogether, these results show that MHCII can be used as an entry receptor, and MHCII-mediated entry of FLUAV in cells lacking sialic acid leads to productive infection and cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMutations near the sialic acid receptor binding site of H3N2 FLUAV HA alter species-specificity of MHCII-mediated entry.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFLUAV interacts with terminal sialic acids on the susceptible cell surface via the RBS on the HA head domain \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Previously, we showed that adaptation of hVIC/11 to pigs selected for three mutations within the HA RBS: A138S, V186G, and F193Y (H3 numbering), which resulted in increased replication in SA-bearing swine cells \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. All these residues are located in the surrounding of the sialic acid-binding pocket, although they do not directly interact with sialic acid \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Furthermore, the A138S amino acid change significantly increases affinity for PAMs \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Therefore, we evaluated the ability of mutants hVIC/11-A138S, hVIC/11-V186G, and hVIC/11-F193Y to utilize either HLA-DR or SLA-DR as a receptor \u003cem\u003ein vitro\u003c/em\u003e. Infection of deacetylated MHCII\u003csup\u003e+\u003c/sup\u003e HEK-293T cells revealed a stronger preference for SLA-DR over HLA-DR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb \u003cb\u003eand c).\u003c/b\u003e When only MHCII\u003csup\u003e+\u003c/sup\u003e cells were considered, a similar trend was observed, with all three mutant viruses using both HLA-DR and SLA-DR as entry receptors, but with a statistically significant higher preference for the swine homologue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Quantification of FLUAV-positive cells among total cells (regardless of MHCII expression) supported that infection of deacetylated cells is completely abolished even when cells are transfected with an empty plasmid \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e).\u003c/b\u003e Evaluation of the replication capacity of the viruses in MHCII-transfected A549 cells, revealed an increase of the viral titers overtime in both HLA-DR- and SLA-DR-expressing deacetylated cells, with no differences observed between the MHCII from both species \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-h\u003cb\u003e)\u003c/b\u003e. This contrasts with hVIC/11 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e which failed to replicate in deacetylated, SLA-DR-expressing cells. Overall, these data demonstrate that the introduction of swine-adapting point mutations near the receptor binding site of hVIC/11 increase the affinity of the virus for SLA-DR. This is consistent with previous reports suggesting that these mutations represent an advantage for the virus in the swine host \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, we tested if these mutations could also affect the SA-binding preference of the virus. By performing a solid-phase SA receptor-binding assay, we observed that hVIC/11-A138S and hVIC/11-F193Y exhibited higher affinity for α2,6 SA compared to hVIC/11 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b,d\u003cb\u003e)\u003c/b\u003e although no changes were seen for α2,3 binding. Contrary, hVIC/11-V186G \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e had a reduced binding to both α2,3 and α2,6 compared to all the mutants, including the parental hVIC/11. Taken together, these results demonstrate that mutations in the proximity of the sialic acid RBS can have a dual effect and impact MHCII recognition and SA binding at the same time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent methodological advances have increased our understanding on the tropism and receptor-specificity of FLUAV beyond the sialic acid linkage to galactose, including the description of numerous non-canonical entry receptors \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, these newly identified molecules often rely on sialic acid to act as a functional receptor \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, with the exception of a subset of phosphoglycans \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Bat-derived H17 and H18 and duck-derived H19 HAs were recently shown to be unable to bind sialic acid and instead use MHCII as a sialic acid-free entry mechanism \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Interestingly, MHCII was suggested to promote entry of H2N2 FLUAV into dendritic cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which was further recently demonstrated \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this report we found that MHCII acts as a functional SA-independent entry receptor for two prototypic H3N2 viruses from human- and swine-origin and that binding is likely mediated by the vicinity of the HA RBS. To our knowledge, this is the first report for MHCII-mediated infection of group 2 HAs. Moreover, affinity for different MHCII homologues seems to correlate with the host from which the virus originated, implying a role of the MHCII complex in the adaptation process of FLUAV to a new host.\u003c/p\u003e \u003cp\u003eInfection of deacetylated PAMs revealed that the swine-adapted sOH/04 was still able to replicate but replication was inhibited by blocking SLA-DR. Findings were similar for the human-seasonal hVIC/11, although replication was lower. Previous literature has shown that human and swine alveolar macrophages exhibit similar levels of α2,3 and α2,6 SA \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Hence, it is likely that the observed differences in affinity for these swine cells correlate with the ability of the viruses to recognize the SLA-DR more than the affinity for host-specific SA conformations. Moreover, our findings suggest that binding to MHCII in addition to SAs increases the affinity of FLUAV to PAMs but not in an additive effect. A recent report showed that replication of a seasonal H3N2 was not boosted by HLA-DR expression in the presence of sialic acid \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This is in line with our results in which neither HLA-DR nor SLA-DR enhanced viral replication when SA was present. We hypothesize that MHCII acts as an alternative host-specific receptor, increasing the availability of receptors when there is a potential sialic acid mismatch. Further, since our previous findings revealed that MHCII-mediated FLUAV infection of macrophages can induce cell death\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, this could be a mechanism used by FLUAV to deplete AMs in the lungs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and reduce their antiviral activity \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e; hence, facilitating virus spread in the respiratory tract. Future studies beyond the scope of this research will be needed to confirm this hypothesis. Further, the mechanism in which MHCII-mediated entry occurs and how it differs from the canonical sialic acid-triggered entry remains unknown. However, our data showed that MHCII-mediated entry and infection was disrupted by NH\u003csub\u003e4\u003c/sub\u003eCl and bafilomycin A1. These compounds prevent endosomal acidification \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and acid lysosome formation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, respectively, and inhibit FLUAV infection by preventing fusion with the endosome membrane and viral particle disassembly \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Hence, our data suggest that upon MHCII-mediated internalization, FLUAV follows a similar trafficking pathway as occurs with sialic acid-triggered entry.\u003c/p\u003e \u003cp\u003eSimilar to H17, H18, and H19 viruses that can use a wide range of MHCII homologues from different bat species, humans, swine, and chickens \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, the H3N2 strains used here were shown to utilize both HLA-DR or SLA-DR as entry receptors in HEK-293T cells. As observed in PAMs, this promiscuity may have implications for the amplified tropism of H3N2 viruses that would facilitate host jumps. However, the human-adapted hVIC/11 virus exhibited preference for HLA-DR, and similarly, the swine-adapted sOH/04 preferentially used SLA-DR. The same pattern was observed in deacetylated A549 cells. Such host-specificity could have implications on the host range of influenza viruses but the ability to still bind to MHCII from other species, even if less efficiently, could play a role in facilitating the adaptation to a new host.\u003c/p\u003e \u003cp\u003eIn a previous report we found that the A138S mutation in a hVIC/11 HA increased affinity for PAMs \u003cem\u003ein vivo\u003c/em\u003e but did not reach the same level of affinity as the swine-adapted sOH/04 \u003csup\u003e16\u003c/sup\u003e. Other mutations near the RBS (V186G and F193Y) were also found after transmission in pigs and resulted in a fitness advantage for the viruses in swine respiratory cells \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Here, introduction of these single point mutations in the HA, near the RBS of hVIC/11, modified the MHCII recognition pattern of the virus and allowed utilization of both HLA-DR and SLA-DR. Although computational analysis suggests that the H18 binds to MHCII via the head domain \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, the specific site on the HA protein responsible for MHCII recognition by H17-H19 remains unknown. Our data are in agreement with these observations and suggest that the interaction between MHCII and H3 is mediated by the HA sialic acid RBS or its surroundings, as the residues evaluated here do not directly interact with sialic acid but may affect conformation of the pocket \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Interestingly, a recent report found that antibodies targeting the H2 RBS can also inhibit MHCII binding \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which support that residues near the HA RBS play a role in MHCII binding. Furthermore, we found that all three mutations also affected SA binding, demonstrating that mutations in the HA RBS can have a dual impact on MHCII and sialic acid recognition. However, it is possible that residues 138, 186, and 193 do not directly interact with MHCII but simply induce conformational changes in the RBS that could indirectly favor or prevent recognition of MHCII. Crystallization of the HA-MHCII complex in future studies will be critical to understand the molecular basis of this interaction.\u003c/p\u003e \u003cp\u003eOverall, our results demonstrate that H3N2 viruses can use the MHCII complex as an alternative entry receptor, resulting in a dual receptor preference (sialic acid and MHCII), and binding to MHCII is likely mediated by the sialic acid RBS of the HA. Similar to SA, affinity of the viruses for MHCII seems to be host-specific and adaptation of human viruses to pigs selects for mutations in the HA that enhance affinity for SLA-DR. Interestingly, during early stages of adaptation, the virus is still able to utilize both HLA-DR and SLA-DR, enabling the virus to expand its host range. Further, mutations occurring in the vicinity of the H3 RBS can affect the affinity of the HA for both SAs and MHCII receptors simultaneously. This is particularly important as MHCII might act as a tropism determinant that selectively promotes infection of antigen-presenting cells and ultimately favors virus replication in the lungs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eEthics statement\u003c/strong\u003e \u003cp\u003e Animal studies described in this report were approved by Institutional Animal Care and Use Committee (IACUC) at the University of Georgia (protocol A2019 03-031-Y3-A9). Animals were maintained under biosafety level 2 conditions according to the Guide for the Care and Use of Agricultural Animals in Research and Teaching. At the end of the study, animals were euthanized following the American Veterinary Medical Association (AVMA) guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCells and viruses\u003c/strong\u003e \u003cp\u003eMadin-Darby canine kidney (MDCK), human lung carcinoma (A549), and human embryonic 293T (HEK-293T) cells were maintained in Dulbecco\u0026rsquo;s Modified Eagles Medium (DMEM, Sigma-Aldrich, St Louis, MO) supplemented with 2mM 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 incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eFLUAV viruses in this study were generated using an 8-plasmid reverse genetic system as previously described \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e using an isogenic backbone. HA sequences were confirmed by Sanger sequencing and viral stocks were made in MDCK cells in opti-MEM containing 1 \u0026micro;g/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemicals, Lakewood, NJ) at 37\u0026deg;C. Viral titers were determined by TCID\u003csub\u003e50\u003c/sub\u003e using the Reed and Muench method \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePorcine alveolar macrophages isolation\u003c/strong\u003e \u003cp\u003eSwine alveolar macrophages were collected and isolated as previously described \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e with minor modifications. Three-weeks-old cross-bred pigs were obtained from Midwest Research Swine Inc (Glencoe, MN). Animals were housed under biosafety level 2 conditions at the University of Georgia. After a 7-days acclimatation period, animals were confirmed negative for anti-FLUAV antibodies by competitive ELISA. For euthanasia, pigs were anesthetized with a cocktail of ketamine (6 mg/kg), xylazine (3 mg/kg), and telazol (6 mg/kg), and an intravenous pentobarbital overdose (Euthasol, 87 mg/kg). Lungs were aseptically collected, and alveolar macrophages were harvested by rinsing the lungs twice with calcium-free phosphate buffered saline (PBS). Samples were pelleted at 1,500 rpm for 10 minutes at 4\u0026deg;C and washed twice with calcium-free PBS. Subsequently, the pellet was resuspended in RPMI-1640 media (Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS, 1% antibiotics/antimycotic, 2 mM L-glutamine, and 1% non-essential amino acids. Cells were passed through a 40 \u0026micro;m cell strainer and then were incubated for 2 hours at 37\u0026deg;C in 10-cm petri dishes. After incubation, non-adherent cells were discarded, and PAMs were transferred to a new petri dish and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e using the media described above.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue immunofluorescence\u003c/b\u003e: Right cranial lobe samples were collected from infected (sOH/04) and non-infected pigs in neutral-buffered formalin and paraffin embedded in a previous study\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For SLA-DR and HA detection, samples were deparaffinized and stained as previously described \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e with minor modification. Briefly, lung sections were deparaffinized and rehydrated followed by an antigen retrieval step in citrate buffer (10 mM sodium citrate, pH 6.0) for 40 minutes. Samples were permeabilized with 0.3% Triton-X100 (Sigma-Aldrich, St Louis, MO) and blocked with 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, St Louis, MO) for 1 hour. FLUAV was detected using an anti-Multi-Hemagglutinin (H3N2) polyclonal antibody (eEnzyme, Gaithersburg, MD) while an anti-pig SLA class II DR clone 2E9/13 (Bio-Rad, Hercules, CA) for MHCII detection was used. Samples were then incubated with a secondary Alexa 594-conjugated anti-rabbit antibody in a 1:1,000 dilution for HA detection and an Alexa 488-conjugated anti-mice antibody in a 1:1000 dilution for SLA-DR. After a 1-hour incubation, tissue samples were stained with 0.5 \u0026micro;g/mL 4\u0026rsquo;,6-diamine-2- phenylindole (DAPI, Sigma-Aldrich, St Louis, MO) for 15 minutes. Finally, samples were mounted on glass slides with mounting media (Vector Laboratories, Newark, CA) and imaged using a ZEISS LSM 900 confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSialic acid depletion\u003c/b\u003e: α2,3 and α2,6-SA were removed from the cell surface by incubating cells with 100 mU/mL of neuraminidase from \u003cem\u003eClostridium perfringens\u003c/em\u003e (Sigma-Aldrich, St Louis, MO) for 24 hours prior to infection. After infection, fresh neuraminidase was added, and new neuraminidase was added every 24 hours post infection. To prevent binding to uncleaved SA, cells were also supplemented with 15 \u0026micro;g/mL of \u003cem\u003eSambucus Nigra\u003c/em\u003e Lectin (SNA, Vector Laboratories, Newark, CA) and \u003cem\u003eMaackia Amurensis\u003c/em\u003e Lectin II (MAL II, Vector Laboratories, Newark, CA). Finally, cells were supplemented with 20 \u0026micro;g/mL of Peanut Agglutinin (PNA, Sigma-Aldrich, St Louis, MO) to prevent any unspecific binding to exposed β-1,3-\u003cem\u003eN\u003c/em\u003e-acetylgalactosamine. Sialic acid depletion was confirmed by immunofluorescence using fluorescein-conjugated SNA (Vector Laboratories, Newark, CA) and biotinylated MALII (Vector Laboratories, Newark, CA).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePAMs viral growth kinetics\u003c/strong\u003e \u003cp\u003eIsolated PAMs were seeded in a 12-well plate at a density of 1.5x10\u003csup\u003e5\u003c/sup\u003e cells/well. Similarly, A549 cells were seeded in a 6-well plate at a density of 5x10\u003csup\u003e5\u003c/sup\u003e cells/well. All cells were incubated at 37\u0026deg;C overnight and then infected at an MOI of 0.01 for 1 hour at 37\u0026deg;C. After infection, plates were washed three times with PBS and cells were supplemented with Opti-MEM (ThermoFisher Scientific, Waltham, MA) containing 0.1 \u0026micro;g/mL of TPCK-treated trypsin. Supernatant aliquots were collected at 0, 12, 24, 48, and 72 h post-infection and kept at -80\u0026deg;C until processing. Viral RNA was extracted using the MagMax-96 AI/ND viral RNA (ThermoFisher Scientific, Waltham, MA) isolation kit according to manufacturer\u0026rsquo;s instructions. Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly MA) and specific primers targeting the M segment of the virus \u003cb\u003e(Supplementary Table\u0026nbsp;1)\u003c/b\u003e. FLUAV titers were determined by comparison to a TCID\u003csub\u003e50\u003c/sub\u003eeq standard curve of an exact virus match of known titer previously determined using the Reed and Muench method.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHLA-DR and SLA-DR antibody blocking\u003c/strong\u003e \u003cp\u003ePAMs were seeded in 12-well plates at a density of 1.5x10\u003csup\u003e5\u003c/sup\u003e cells/well while A549 cells were plated in a 6-well plate at a density of 5x10\u003csup\u003e5\u003c/sup\u003e cells/well. Plates were incubated overnight at 37\u0026deg;C. The next day, cells were incubated for 1 hour at 4\u0026deg;C with an anti-SLA-DR (Bio-Rad, Hercules, CA) or an anti-HLA-DR (ThermoFisher Scientific, Waltham, MA) antibody at concentrations ranging from 1 to 10 \u0026micro;g/mL or an anti-GFP antibody as a control (Sigma-Aldrich, St Louis, MO). After incubation cells were infected at an MOI of 0.1 for 1 hour at 37\u0026deg;C in the presence of the indicated antibody concentrations. Finally, cells were washed three times with PBS and supplemented with Opti-MEM containing 0.1 \u0026micro;g/mL of TPCK-treated trypsin and the desired antibody concentrations. Samples were collected at 48 hpi for further virus titration.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmids\u003c/b\u003e: HLA-DRA (GenBank accession number: NM_019111.5), HLA-DRB1 (GenBank accession number: NM_002124.4), SLA-DRA (GenBank accession number: MF498819.1), and SLA-DRB1 (GenBank accession number: MF498811.1) gene fragments were synthetized (Twist Bioscience, San Francisco, CA) and amplified by PCR to introduce EcoRI and BglII restriction sites using the primers listed in \u003cb\u003eSupplementary Table\u0026nbsp;1.\u003c/b\u003e pCAGGS plasmid and MHCII fragments were digested using EcoRI and BglII restriction enzymes (New England Biolabs, Ipswich, MA) according to the manufacturer\u0026rsquo;s instructions for 1 hour at 37\u0026deg;C for further ligation and transformation. Plasmids sequences were confirmed by whole plasmid sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMHCII expression in A549 and HEK-293T cells\u003c/b\u003e: Cells were seeded at a density of 8x10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates and were incubated at 37\u0026deg;C until a 70% confluence was reached. MHCII subunits were transfected using equimolar quantities of either HLA-DRA and HLA-DRB1 or SLA-DRA and SLA-DRB1 and TransiT LT1 (Mirus Bio, Madison, WI) in a 1:2 ratio DNA:TransiT. HLA-DR and SLA-DR complexes expression was assessed by flow cytometry and immunofluorescence as described below. At 24 hours post transfection, cells were deacetylated as described above and non-sialylated cells were infected at 48 hours post transfection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMHCII-mediated entry assessment\u003c/b\u003e: HEK-293T and A549 cells were seeded and transfected as described above. At 24 hours post transfection, cells were deacetylated with 100 mU/mL of neuraminidase from \u003cem\u003eClostridium perfringens\u003c/em\u003e for 24 hours at 37\u0026deg;C. Non-deacetylated cells were left untouched until 48 hours post transfection. 1 hour before infection, deacetylated cells were incubated with 15 \u0026micro;g/mL of SNA, 15 \u0026micro;g/mL of MALII, and 20 \u0026micro;g/mL of PNA. At 48 hours post transfection, cells were infected at an MOI of 0.1 for 1 hour at 37\u0026deg;C. After incubation, virus-containing supernatant was discarded, and fresh media supplemented with 0.1 \u0026micro;g/mL TPCK-treated trypsin and 100 mU/mL of neuraminidase was added. Neuraminidase was not included in the non-deacetylated control groups. Finally, infections were incubated for 24 hours at 37\u0026deg;C and cells were then processed for either flow cytometry or immunofluorescence.\u003c/p\u003e \u003cp\u003e\u003cb\u003eFlow cytometry analysis\u003c/b\u003e: 24 hours after infection as described above, supernatants from A549- and HEK-293T-infected cells were collected, and cells were trypsinized and centrifuged at 1,500 rpm for 5 minutes. Cells were resuspended in PBS supplemented with 5% FBS and stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer\u0026rsquo;s instructions. Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by a 15-minute permeabilization with 0.3% Triton-X (Sigma-Aldrich, St Louis, MO). Afterwards, cells were washed three times and incubated for 1 hour with the following primary antibodies: Alexa 647-conjugated anti-SLA-DR clone 2E9/13 (Bio-Rad, Hercules, CA), anti-HLA-DR clone LN3 (ThermoFisher Scientific, Waltham, MA) at a 1:200 dilution in PBS and an anti-H3 HA antibody (stem specific antibody) clone CR8020 at a 1:500 dilution. Following incubation, cells were washed three times with PBS. For simultaneous detection of FLUAV and MHCII, samples were incubated for 1 hour with an Alexa 488-conjugated anti-human secondary antibody and an APC-conjugated anti-mice secondary antibody respectively (ThermoFisher Scientific, Waltham, MA), both in a 1:1,000 dilution. Finally, cells were washed three times with PBS and analyzed using a NovoCyte Quanteon flow cytometer system (Agilent, Santa Clara, CA) using the gating strategy exemplified in \u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e. Data was analyzed using FlowJo version 10.8.2 (FlowJo, Ashland, OR).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence staining\u003c/b\u003e: HEK-293T and A549 cells were plated in poly-D-Lysine-coated glass slides and incubated at 37\u0026deg;C until an 80% confluence was reached. Cells were transfected and deacetylated as described above. Cells were infected at an MOI of 0.01 and incubated for 24 hours at 37\u0026deg;C as described above. After infection, cells were fixed with 4% formaldehyde for 30 minutes at room temperature, followed by a 10-minute permeabilization with 0.3% Triton-X100 and a 1-hour blocking step using 3% bovine serum albumin (BSA, ThermoFisher Scientific, Waltham, MA) and 0.1% Triton-X100 in PBS. HLA-DR, SLA-DR, and FLUAV were stained using the specific antibodies described above in a 1:200 dilution for 1 hour at room temperature in 3% BSA and 0.1% Triton-X100 in PBS. After incubation, samples were washed three times with PBS and incubated with the secondary antibodies described above in a 1:1,000 dilution with 0.5 \u0026micro;g/mL 4\u0026rsquo;,6-diamine-2- phenylindole (DAPI, Sigma-Aldrich, St Louis, MO), 3% BSA, and 0.1% Triton-X100 in PBS for 1 hour. Finally, cells were washed 5 times with PBS and mounted on glass slides using mounting media. For sialic acid staining, samples were incubated with either biotin-conjugated or fluorescein-labelled SNA and biotin-conjugated MAL II lectins in a 1:250 dilution in PBS for 30 minutes prior to permeabilization. After incubation with lectins, cells were incubated for 30 minutes with a secondary Alexa 594-conjugated streptavidin in a 1:1,000 dilution. Samples were imaged using a ZEISS LSM 900 confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFLUAV entry blockage and cell viability assay\u003c/strong\u003e \u003cp\u003eHEK-293T cells were plated and transfected as described above. 1 hour before infection, cells were incubated with 20 mM of NH\u003csub\u003e4\u003c/sub\u003eCl (Sigma-Aldrich, St Louis, MO) or 10 nM Bafilomycin A1 (Cells Signaling Technologies, Danvers, MA). After incubation, cells were infected at an MOI of 0.1 as described above and kept at 37\u0026deg;C for 24 hours in presence of the desired inhibitor. At 24 hpi, cells were washed three times with PBS and total cellular RNA was extracted using the MagMax-96 AI/ND RNA (ThermoFisher Scientific, Waltham, MA) according to the manufacturer\u0026rsquo;s instructions. RNA samples were normalized to 1 \u0026micro;g and FLUAV M segment copy number was determined by RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly MA) and a standard curve of a M-cloned segment plasmid of known concentration.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eCellular viability was assessed using the CellTiter Glo Cell Viability kit (Promega, Madison, WI) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMHCII-mediated replication in A549 cells\u003c/strong\u003e \u003cp\u003eA549 cells were seeded and transfected as described above. At 24 hours post transfection, sialic acid was removed with 100 mU/mL of neuraminidase for 24 hours. Following incubation, deacetylated cells were further incubated with 15 \u0026micro;g/mL of SNA, 15 \u0026micro;g/mL of MALII, and 20 \u0026micro;g/mL of PNA for 1 hour before infection. Non-deacetylated control cells remained untouched until infection. At 48 hours post transfection, cells were infected at an MOI of 0.01 for 1 hour at 37\u0026deg;C. After incubation, supernatant was discarded and replaced with media containing 0.1 \u0026micro;g/mL TPCK-treated trypsin and 100 mU/mL of neuraminidase. Fresh neuraminidase was added every 24 hours. No neuraminidase was used in non-deacetylated cells. Time-points were collected at 0, 12, 24, 48, and 72 hours post infection and viral titers were determined by RT-qPCR as described above.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSolid-phase binding assay\u003c/b\u003e: Virus affinity for α2,3 and α2,6 SA was assessed as previously described \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Briefly, viruses were purified by ultracentrifugation at 28,000 rpm for 3 hours using a 20% sucrose cushion. Pelleted viruses were resuspended in TNE buffer (0.01M Tris, 0.001M EDTA, 0.1 M NaCl, pH 7.2). Samples were normalized to 128 HAU and adsorbed in fetuin-coated plates at 4\u0026deg;C overnight. Unbound virus was washed using 0.02% Tween-80 in PBS (washing buffer) and plates were blocked for 2 hours with deacetylated BSA (BSA-NA). After blocking, plates were washed three times with washing buffer and incubated with different concentrations of Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (α2,3 SA, Glycotech, Gaithersburg, MD) or Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (α2,6 SA, Glycotech, Gaithersburg, MD) in 0.02% Tween-80, 0.1% BSA-NA, and 2 \u0026micro;M oseltamivir in PBS (reaction buffer). After a 1-hour incubation, plates were washed five times with washing buffer and incubated with HRP-conjugated streptavidin (ThermoFisher Scientific, Waltham, MA) in a 1:1,000 dilution for 1 hour at 4\u0026deg;C. Finally, plates were washed 5 times with washing buffer and incubated with TMB (ThermoFisher Scientific, Waltham, MA) for 10 minutes. Reaction was stopped using a stop solution (ThermoFisher Scientific, Waltham, MA), and absorbance was measured at 450 nm using a Synergy HTX Multi-Mode Microplate Reader Agilent BioTek, Santa Clara, CA).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 10 software. Data is presented as the mean \u0026plusmn; standard error of the mean of at least three independent experiments unless stated otherwise in the figure legend. Each data point represents the average of three technical replicates for a total of three biological replicates per experiment. P values were obtained by ordinary two-way ANOVA with Tukey\u0026rsquo;s multiple comparison test.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAll source data relevant for this study is available within the manuscript and has been provided as a source data file and supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the University Research Animal Resources personnel at the University of Georgia for assistance with animal care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u0026nbsp;\u003c/strong\u003eMCP, DSR, and CJC designed the experiments. MCP performed the cloning, growth kinetics, immunofluorescence, and MHCII expression experiments. MCP performed the cell viability assays. MCP and SC performed the flow cytometry experiments. MCP analyzed the data. MCP and DSR wrote the manuscript. CJC, AGS, and DRP edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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 to DRP and by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a National Institute of Allergy and Infectious Diseases (NIAID) funded Center for Influenza Research and Response (CEIRR, contract 75N93021C00014 to DRP and AG-S. 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 also funded by 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 expertise from the Georgia Advanced Computing Resource Center, a partnership between the University of Georgia\u0026rsquo;s Office of the Vice President for Research and the Office of the Vice President for Information Technology. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe A.G.-S. laboratory has received research support from Avimex, Dynavax, Pharmamar, 7Hills Pharma, ImmunityBio and Accurius, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, Virofend and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The other authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao, C. \u0026amp; Pu, J. 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However, some FLUAV subtypes (H17, H18, H19) were recently shown to use the major histocompatibility complex class II (MHCII) as an entry receptor instead of sialic acid (SA), challenging our knowledge about FLUAV tropism and interspecies transmission potential. Here we show that H3N2 viruses can use MHCII as an alternative entry receptor in a host-specific manner, and adaptation of human viruses to pigs increases affinity for the MHCII swine leukocyte antigen (SLA). By using two prototypic human-seasonal (hVIC/11) and swine-adapted (sOH/04) H3N2 viruses we found that expression of the human (HLA) but not the swine MHCII conferred replication of hVIC/11 in deacetylated, non-susceptible cells which ultimately led to cell death. Further, expression of SLA in deacetylated, non-susceptible cells conferred susceptibility to infection by sOH/04. Introduction of point mutations near the hVIC/11 HA receptor-binding site (RBS) allowed the use of both human and swine MHCII. Our findings revealed that MHCII can serve as a sialic acid-independent entry receptor to H3N2 FLUAV in a host-specific manner, expanding the cell tropism and host range of the virus, with potential implications for the viral pathogenesis and adaptation to a new species.\u003c/p\u003e","manuscriptTitle":"MHC class II is a functional receptor for H3N2 Influenza A viruses and mediates host-specificity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 09:37:09","doi":"10.21203/rs.3.rs-6278351/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d3bd030b-b632-444a-a90e-f3267cfa7ffe","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47901615,"name":"Biological sciences/Microbiology/Virology/Virus\u0026#x2013;host interactions"},{"id":47901616,"name":"Biological sciences/Microbiology/Virology/Influenza virus"}],"tags":[],"updatedAt":"2026-03-19T07:12:11+00:00","versionOfRecord":{"articleIdentity":"rs-6278351","link":"https://doi.org/10.1038/s41467-026-69267-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-10 05:00:00","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-05-02 09:37:09","video":"","vorDoi":"10.1038/s41467-026-69267-6","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69267-6","workflowStages":[]},"version":"v1","identity":"rs-6278351","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6278351","identity":"rs-6278351","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}