Vaccination against swine influenza virus (SIV) in pigs causes different drift evolutionary patterns upon SIV experimental infection and reduces the possibility of genomic reassortments

Influenza viruses (IVs) can infect wide variety of bird and mammal species. Its genome is characterized by having 8 RNA single stranded segments. The low proofreading activity of its polymerases and the genomic reassortment between different IVs subtypes allow virus to be continuously evolving, constituting a constant threat to human and animal health. In 2009, a pandemic of an Influenza A virus highlighted the importance of the swine host in IVs adaptation between humans and birds. The swine population and the incidence of swine influenza (SI) is constantly growing. In previous studies, despite vaccination, SI virus (SIV) growth and evolution was proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of SIV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent SIVs seeders. Nasal swab samples were daily recovered and BALF was also collected at necropsy day from each pig for SIV detection and whole genome sequencing. In total, 39 SIV whole genome sequences were obtained by NGS from samples collected from both experimental groups. Subsequently, genomic and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, we observed that the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the possibility of genomic reassortment events. In relation to SIV intra-host diversity, a total of 239 and 74 SNV were detected in the H1N1 and the H3N2 subtypes, respectively. Different proportion of synonymous and nonsynonymous substitutions was found, indicating that vaccine may be influencing the mechanism of natural, neutral, and purifying selection that play the main role in SIV evolution. SNV were detected along the whole SIV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural protein, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of SIV, under natural infection and vaccination pressure scenarios.