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

Abstract

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV 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 swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing 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, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, 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 swine IAV, under natural infection and vaccination pressure scenarios.

Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals

“…Notably, the proportion of nonsynonymous substitutions was always higher in vaccinated animals, maintaining this dynamic throughout all the variant allelic frequencies analyzed. This could be indicating that the immune pressure of the vaccinated pigs was additionally acting as positive selection force in the evolution of the virus ( Fitch et al., 1991 ; Li et al., 2011 ), as previously reported for H1N1 and H3N2 SIAV subtypes ( López-Valiñas et al., 2021 ; López-Valiñas et al., 2022 ; López-Valiñas et al., 2023 ). On the other hand, this proportion was around 50% for sequences from nonvaccinated animals suggesting that a neutral selection could be acting in this scenario ( Frost et al., 2018 ).…”

“…Hence, in the present study, although limited, the effect of the vaccine in viral shedding and clinical signs reduction was proved. However, viral replication was not prevented by vaccination, facilitating the virus to acquire novel nucleotide variants, as previously found ( Murcia et al., 2012 ; López-Valiñas et al., 2021 ; López-Valiñas et al., 2022 ; López-Valiñas et al., 2023 ).…”

“…In previous studies, substitutions were also found in vaccinated animals previously challenged with the H1N1 subtype ( López-Valiñas et al., 2021 ). However, substitutions were not reported in the H3N2 subtype ( López-Valiñas et al., 2022 ; López-Valiñas et al., 2023 ). Hence, it could be inferred that the plasticity of NP is different depending on the subtype, being greater in H1N1 and H1N2 ones, while H3N2 NP seems to be more conserved.…”

“…However, genetic differences between SIAV strains used in vaccine formulation and current circulating strains, make that the immunity generated by vaccine is not complete, allowing virus replication and evolution. Previous studies have shown different evolution patterns on H1N1 and H3N2 SIAV subtypes, that may be caused by different host immune pressures ( López-Valiñas et al., 2021 ; López-Valiñas et al., 2022 ; López-Valiñas et al., 2023 ). Nevertheless, the effect of vaccine application on H1N2 SIAV evolution through NGS technology is poorly studied.…”

“…However, due to the genomic distance between farm circulating and vaccine formulation strains, the protection generated during vaccination is not complete, allowing virus circulation, potentially favoring viral evolution ( Thacker and Janke, 2008 ; Ma and Richt, 2010 ). In previous studies, the viral evolution patterns of the SIAV H1N1 and H3N2 subtypes were analyzed in vaccinated and nonvaccinated pigs, showing different evolutionary patterns in both experimental groups ( López-Valiñas et al., 2021 ; López-Valiñas et al., 2022 ; López-Valiñas et al., 2023 ). However, the evolutionary pressure generated by the trivalent vaccine on SIAV H1N2 subtype is still poorly studied.…”