Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs

Abstract

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-β. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Fig 1. In vitro characterization of the hVIC/11^A138S virus.

(A) Prevalence of the S138 residue in swine and human H3N2 FLUAV isolates reported from 1992 to 2022. Sequences were obtained from GISAID and aligned using ClustalW. (B) Representative electron microscopy pictures of sOH/04, hVIC/11, and hVIC/11^A138S. Scale bar = 100 nm (C) Plaque morphology produced by sOH/04, hVIC/11, and hVIC/11^A138S in MDCK cells at 37 and 39°C. (D) Plaque sizes produced by the viruses at 37 and 39°C. Two independent experiments were performed in triplicates. Values represent the mean ± standard error of the mean (SEM). Statistical analysis was performed by two-way ANOVA. **p<0.005.

Fig 2. A138S increases HA thermal stability, binding for α2,6 receptors, and NA activity.

(A) Thermal stability of sOH/04 (blue), hVIC/11 (yellow), and hVIC/11^A138S (red) was determined by incubating them at different temperatures for 1 hour. Data were fitted to a dose-response-inhibition non-linear fit. Receptor-binding affinity of sOH/04, hVIC/11, and hVIC/11^A138S for 3’SLN (B) or 6’SLN (C) was assessed by incubating the viruses with different concentrations of 3’SLN or 6’SLN. (D) NA activity was determined by normalizing the viruses at 10⁴ PFU/well in the presence of 100 μM MUNANA. Fluorescence was measured every 60 seconds for 1 hour and data was fitted to a linear regression model. (E) NA activity of the viruses was determined by normalizing based on NA activity. Viruses were incubated at different MUNANA concentrations for 1 hour and kinetic parameters (K_M and V_max) were determined by fitting the data to the Michaelis-Menten equation. For all assays, two experiments were performed in triplicates. Values represent the mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***p<0.0005.

Fig 3. A138S improves infection of the lower respiratory tract of pigs.

(A) Viral titers in different anatomical sections of the lungs of seeder pigs at 5 dpi (n = 3) normalized to 1μg total RNA. All statistical analyses were performed by two-way ANOVA. Values represent the mean ± SEM. *p<0.05, **p<0.005. (B) Influenza immunofluorescence staining in the respiratory tract of seeder pigs. Hemagglutinin (red) was detected using a polyclonal multi-H3 antibody and cell nuclei (blue) were stained with DAPI. The scale bar represents 50 μm.

Fig 4. A138S does not affect histopathological findings in tracheas and lung lobes of infected pigs.

Representative photomicrographs of trachea and lung sections of seeder pigs at 5 dpi, H&E, 10X. Mock group showing normal tissue sections. hVIC/11 group, trachea: epithelium was diffusely sloughing into the lumen (arrow) with a mild-moderate suppurative inflammation in the submucosal glands (asterisk). Lung: mild-moderate degree of suppurative bronchitis and bronchiolitis were present (asterisks). hVIC/11^A138S group, trachea: mild-moderate lymphohistiocytic inflammation expanded the lamina propria and effaced the mucosal epithelium. Lung: mild suppurative and catarrhal bronchitis and bronchiolitis (asterisks) were present with mild lymphohistiocytic cuffing of the airway. sOH/04 group, trachea: mild lymphohistiocytic tracheitis (arrow) was present. Lung: marked suppurative inflammation in the airways (asterisks)and adjacent alveoli.

Fig 5. Distinct patterns of immune response are triggered by the viruses in different lung anatomical sections of pigs.

Relative mRNA levels of pro-inflammatory cytokines, interferon-stimulated genes, and patter-recognition receptors. RNA was normalized to 1μg and gene expression was assessed by qPCR and normalized to RLP-19 expression in the (A) right cranial lobe, (B) left cranial lobe, (C) right caudal lobe, (D) left caudal lobe, and (E) accessory lobe. Values are shown as log₂ fold induction of the mean between the seeder pigs (n = 3) of each group at 5dpi. Fold induction of each group was normalized to the non-infected negative control group.

Fig 6. Distinct antigen-presenting cells, macrophages, and dendritic cells abundance induced after infection with influenza viruses in different lung anatomical sections of pigs.

(A) Single-cell suspensions were labeled with anti MHCII, CD163, and CD172a antibodies and then analyzed by multi-color flow cytometry. Live cells from singlets were filtered and used to assess MHC II, CD163, and CD172a content. From the MHCII^high CD163^pos (antigen-presenting cells) population in lung tissue samples, cDC1 cells appear as CD172a^negCD163^neg, cDC2 are CD172a^posCD163^neg, moDC are CD172a^posCD163^low, moMϕ are CD172a^posCD163^int, and PiAMs are CD172aposCD163^high. Abundance of APC, cDC1, cDC2, moDC, moMϕ, and PiAMs in the (A) right cranial lobe, (B) left cranial lobe, (C) right caudal lobe, (D) left caudal lobe, and (E) accessory lobe was quantified among total live cells (APC) or MHC^highCD163^pos cells (cDC1, cDC2, moDC, moMϕ, and PiAMs). Values represent the mean ± SEM for seeder pigs (n = 3) in each group. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

Fig 7. hVIC/11^A138S infection reduces the abundance of PAMs in BALF.

(A) Total cell count in BALF samples from mock, hVIC/11, hVIC/11^A138S, and sOH/04-infected pigs. (B) Representative histograms showing PAMs abundance (cell count, MHCII^highCD163^pos) in BALF samples. BALF samples were analyzed by multi-color flow cytometry and the MHCII content of the populations was quantified among total live cells. (C) Variation of PAMs abundance in mock, sOH/04, hVIC/11, and hVIC/11^A138S-infected pigs quantified by flow cytometry. Values represent the mean ± SEM for seeder pigs (n = 3) per group. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.005.

Fig 8. FLUAV infection of PAMs affects the expression of GM-CSF but not PPARγ expression and promotes apoptosis of alveolar macrophages.

(A) PAMs collected from BALF samples of seeders at 5 dpi were stained for HA detection and analyzed by multi-color flow cytometry to detect the amount of MHCII^highCD163^pos (PAMs) FLUAV -positive cells. Average histograms normalized to mode showing FLUAV -positive PAMs compared to the mock group for (B) hVIC/11, (C) hVIC/11^A138S, and (D) sOH/04-infected seeder pigs (n = 3) per group. From the comparison of FLUAV-positive cells to the mock group, (E) the percentage of FLUAV-positive cells was determined using the Overton % method. (F) Relative mRNA levels of GM-CSF and PPARγ assessed by qPCR and normalized to RLP-19 expression in BALF samples. Values are shown as log₂ fold induction of the mean between the seeder pigs (n = 3) of each group. (G) Flow cytometry analysis of apoptotic cells (Live/Dead^negAnnexin V^pos) at 12 hpi. (H) Growth kinetics of sOH/04 (blue), hVIC/11 (yellow), and hVIC/11^A138S (red) in 3D4/21 cells at 37°C. Two independent experiments were performed in triplicates each time. Values represent the mean ± SEM. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.