Passage of human-origin influenza A virus in swine tracheal epithelial cells selects for adaptive mutations in the hemagglutinin gene

Abstract

Frequent spillover of influenza A viruses from humans to swine contributes to the increasing diversity of influenza viruses circulating in pigs. Although these events are common, little is known about the adaptation processes that take place when viruses jump between the two species. We examined the changes that occurred during serial passages of a reassortant H3N2 virus (VIC11pTRIG) containing human seasonal surface genes (Hemagglutinin and Neuraminidase) and a swine-adapted internal gene constellation in differentiated primary swine tracheal epithelial cells (pSTECs). The VIC11pTRIG reassortant virus was serially passaged 8 times in pSTECs and compared to a control swine-adapted strain (OH/04p) containing the same internal gene constellation. Viral RNA from passages 0 (inoculum), 1, 3, 4-8 were sequenced via next generation or Sanger sequencing. Hemagglutinin diversity was highest at passage 3. Two amino acid mutations in the Hemagglutinin protein (N165K and N216K) were fixed at passages 7 and 5, respectively. These changes were associated with increased fitness of the virus in pSTECs compared to the original parental strain. Our results suggest that the adaptation of human seasonal H3N2 to swine cells may lead to the selection of HA mutations located near the receptor binding site. These mutations may result in increased fitness of human-origin H3N2 strains to adapt in swine.

Fig 1. Viral titers during serial passages of reassortant VIC11pTRIG and OH/04p influenza viruses in primary swine tracheal epithelial cells (pSTECs).

A total of 8 serial passages were conducted in pSTECs. (A) Titers of passage 1 to passage 8 (P1-P8) of VIC11pTRIG virus. (B) Titers of P1-P8 of OH/04p virus. Titers were calculated via the Reed-Muench method. Values are shown as mean TCID₅₀/ml titers ± standard error of the mean. Dotted line represents the limit of detection. Columns which does not share letters are considered significantly different(p < 0.05).

Fig 2. Location of mutations identified on the HA of Vic11pTRIG virus.

Dominant mutations identified in this study on the HA of VIC11pTRIG virus are highlighted on the structure of the human A/Victoria/361/2011 hemagglutinin. Red, residues N165K and N216K. The 190 helix, 130 loop, and 220 loop of the receptor binding site (RBS) are shown.

Fig 3. In vitro replication kinetics of mutants containing N165K and/or N216K substitutions in the HA of VIC11pTRIG in primary swine tracheal epithelial cells (pSTECs) and MDCK cells.

Viral growth kinetics of mutant viruses (VIC11pTRIG_ N165K, VIC11pTRIG_N216K, VIC11pTRIG_N165K/N216K), VIC11pTRIG, and OH/04p in (A) pSTECs and (B) MDCK cells. pSTECs and MDCK cells were infected at an MOI of 0.05, and supernatants were collected at 0, 12, 24, 48, and 72 h post infection (hpi). Viral titers were quantified by qRT-PCR and titers calculated by TCID₅₀ equivalency. Values are shown as mean TCID₅₀/ml equivalent titers ± standard error of the mean. Dotted lines indicate limit of detection. Asterisks indicate statistical significance (p < 0.05).

Fig 4. Amino acid substitutions at position 165 and 216 on the HA from H3N2 viruses isolated from pigs globally between years 2000-2025.

The relative proportion of mutations between residues 160 and 170 position and 211 and 221 are shown in the figure, staggered for every 7−8 years. Variations in position 165 in swine H3N2 FLUAVs isolated between 2000-2025 in (A) North America and (B) Asia, Europe, Oceania, and South America. Variations in position 216 in swine H3N2 FLUAVs isolated between 2000-2025 in (C) North America and (D) Asia, Europe, Oceania, and South America. Panels generated using WebLogo 3.