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.

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/11^A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11^A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11^A138S 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/11^A138S-infected pigs compared to sOH/04 while a MHCII^lowCD163^neg population was increased. The hVIC/11^A138S 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 overexpression 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. Author summary: Human-to-swine FLUAV spillover events are relatively common and have led to the establishment of multiple evolutionarily distinct virus lineages in the swine population worldwide, leading to important economic losses in the pork industry. This also increases the risk of reassortment and emergence of novel FLUAV that may retain the ability to infect and transmit in humans, an event exemplified by the H1N1 pandemic in 2009. However, the molecular mechanisms driving FLUAV adaptation to pigs following the introduction of a human virus are still not fully understood. In this study we found that a point mutation in a human-origin HA improved infection and transmissibility in pigs characterized by an enhanced lung replication and the disruption of lung-resident immune cell populations. This relationship between improved replication and infectivity of lung-resident cells provides insights into host barriers limiting human FLUAV adaptation to pigs and the complexities associated with the host immune response that influenza A viruses must overcome to become established in the swine population. [ABSTRACT FROM AUTHOR]