Identification and Characterization of Swine Influenza Virus H1N1 Variants Generated in Vaccinated and Nonvaccinated, Challenged Pigs

Abstract

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

Keywords: hemagglutinin (HA); neuraminidase (NA); next-generation sequencing; nonstructural protein (NS); nucleoprotein (NP); single nucleotide variants (SNVs); swine influenza virus (SIV).

Figure 1

Kinetics of the IgG antibody levels against the NP detected by ELISA in sera after vaccination and viral challenge. IgG antibodies measured as percentage of competition at first vaccination (0 dpv), 21, 42 dpv (day of challenge) and at 2, 5, and 10 dpi are depicted. Points above the dashed red line (competition > 45%) are considered positive, whereas points under the dashed grey line (competition < 50%) are considered negative. Values between the two lines are considered doubtful. Groups of vaccinated and nonvaccinated animals are shown in blue and green, respectively.

Figure 2

Coverage per genomic segment and sample from both experimental groups and the challenge inoculum. (a) Representation of the coverage of Illumina sequencing reads mapped against A/Swine/Spain/01/2010(H1N1) used for challenge. (a1) Sequencing profiles of sequenced samples from vaccinated animals and inoculum plotted in different tones of blue and red, respectively. (a2) Sequencing profile of sequenced samples from nonvaccinated animals plotted in different tones of green. (b) Representation of the mean read coverage heat map per segment. The identification of the animal from which the samples come and the kind of collected sample are specified on the figures, with the nasal swab collected at the indicated day being the samples with dpi.

Figure 3

Total number of synonymous and nonsynonymous SNVs found from vaccinated and nonvaccinated animals. (a) Substitutions with an allele frequency greater than 1%. (b) Substitutions with an allele frequency greater than 5%. * p < 0.05.

Figure 4

Genome segment distribution and number of synonymous and nonsynonymous SNVs found from sequenced samples from vaccinated and nonvaccinated animals. (left) Substitutions with an allele frequency greater than 1%. (right) Substitutions with an allele frequency greater than 5%.

Figure 5

Nucleotide diversity (π) in the viral population at different time points. (a) Nucleotide diversity bars of inoculum sequence. (b) Box plot of viral population nucleotide diversity from all collected samples at 2, 3, and 5 dpi from vaccinated and nonvaccinated groups. Whiskers represent variability outside the lower and upper quartiles of each represented box. * p < 0.05.

Figure 6

Location of all substitutions (allele frequency > 5%) described in this study in NS1, NP, HA, and NA proteins. (a) NS1 protein (PDB accession no. 4OPH) [61] RNA Binding (RBD), Linker region, and effector domain (ED) are highlighted in pink, green, and cyan, respectively. (b) NP trimer protein (PDB accession no. 2IQH) head domain is indicated in cyan, whereas body domain is highlighted in pink [64]. (c) HA trimer is also represented (PDB accession no. 3LZG); cyan, pink, and blue domains represent HA1, HA2, and Receptor binding domains, respectively. In orange, the CA antigenic site is highlighted [65]. I513V and V521M substitutions are not in the limit of the crystallographed structure. (d) NA tetramer (PDB accession no. 4B7Q) protein [66]. Finally, highlighted substitutions in blue were found in vaccinated animals, those in green were found in nonvaccinated animals, and those in orange in both. The PyMOL Molecular Graphics System, Version 4.3 was used to visualize the protein structures.