Swine influenza is a highly contagious and widely distributed disease that generates important economic losses in the pig industry. Nowadays, one of the most extended strategy used to control Swine influenza viruses (SIVs) is the trivalent vaccine application, which formulation contains the most frequently circulating SIV subtypes H1N1, H1N2 and H3N2. These vaccines do not provide sterilizing immunity against the virus, potentially favoring viral evolutionary dynamics. To better understand the main mechanisms that shape viral evolution, in this work, the SIV intra-host diversity was analyzed in samples collected from both, vaccinated and non-vaccinated animals challenged with H1N1 influenza A virus. In the present study 276 single nucleotide variants were found within 28 whole SIV genomes obtained by next generation sequencing. Differences in nucleotide variants between groups were established and the impact of each substitution found was hypothesized according to previous literature. Substitutions were allocated along all influenza genetic segments, while the most relevant non-synonymous substitutions were allocated in the NS1 protein on samples collected only from vaccinated animals. These substitutions could affect both, mRNA viral translation and pathogenesis. Moreover, new viral variants were found in both vaccinated and non-vaccinated pigs, showing relevant substitutions in the HA, NA and NP proteins that may be contributing to evasion of host immune system, virulence and host adaptation. Overall, results of the present study suggest that SIV is continuously evolving despite vaccine application, therefore new substitutions may increase viral fitness under field conditions.

1. Variation analysis of Swine influenza virus (SIV) H1N1
sequences in experimentally infected vaccinated and non-
vaccinated pigs
Á. López-Valiñas1,2 , M. Sisteré-Oró1,2, S. López-Serrano1,2, L. Baioni3, C. Chiapponi3, A. Darji1,2, J. Segalés2,4,5, L. Ganges1,2 and J.I. Núñez1,2.
1 Centre de Recerca en Sanitat Animal (CReSA), Institut de Recerca en Tecnologies Agroalimentaries (IRTA), 08193 Barcelona, Spain. 2 OIE Collaborating Centre for the Research and Control of Emerging and
Re-Emerging Swine Diseases in Europe (IRTA-CReSA), 08193 Barcelona, Spain. 3OIE Reference Laboratory for Swine Influenza Virus, Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-
Romagna, 25124 Brescia, Italy. 4 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. 5OIE Reference Laboratory for Classical Swine Fever
Virus, IRTA-CReSA, 08193 Barcelona, Spain.
Swine influenza is a highly contagious and widely distributed disease that
generates important economic losses in the pig industry. Nowadays, one of the
most extended strategy used to control Swine influenza viruses (SIVs) is the
trivalent vaccine application, which formulation contains the most frequently
circulating SIV subtypes H1N1, H1N2 and H3N2. These vaccines do not
provide sterilizing immunity against the virus, potentially favoring viral
evolutionary dynamics. To better understand the main mechanisms that shape
viral evolution, in this work, the SIV intra-host diversity was analyzed in
samples collected from both, vaccinated and non-vaccinated animals
challenged with H1N1 influenza A virus.
❖ NS1 substitutions (Figure 5) could affect both, mRNA viral translation and
pathogenesis.
❖ Four substitutions found in HA protein could allow the virus avoid the immune
system (Figure 5):
❖ D200N and V233I allocated close to the Ca antigenic site.
❖ I513V and V521M allocated in the transmembrane region of the stalk
domain. It could affect the antigenic exposure.
Viral replication was observed in both vaccinated and nonvaccinated pigs,
although it was lower in vaccinated ones.
In the present study, 28 whole SIV genomes were obtained by next generation
sequencing (Figure 2). In total, 276 single nucleotide variants (SNV) were found
(Figure 3).
In vaccinated animals the number of nonsynonymous variants is greater than
synonymous ones, therefore natural selection pressure may be involved in
driving the SIV evolution. By contrary, in nonvaccinated animals purifying
selection may be acting. Hence, SIV could increase its fitness in both
scenarios, favoring viral maintenance in field.
Substitutions were allocated along all influenza genetic segments (Figure 4).
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%. * (chi-squared; p = 0.03123)
Figure 4. Genome segment distribution and number of synonymous and nonsynonymous SNVs found
from sequenced samples from vaccinated and nonvaccinated animals. (a) Substitutions with an allele
frequency greater than 1%. (b) Substitutions with an allele frequency greater than 5%
Figure 5. Location of all substitutions (allele frequency > 5%) described in this study in NS1, NP, HA,
and NA proteins. Substitutions highlighted in blue and green were found in vaccinated and
nonvaccinated animals respectively. Orange substitutions were simultaneously found in both.
Figure 2. Coverage of Illumina sequencing reads mapped against A/Swine/Spain/01/2010(H1N1) used
for challenge. Sequencing profiles of sequenced samples from vaccinated, nonvaccinated animals and
inoculum are plotted in different tones of blues, greens and red, respectively.
D67N
R81S
E179A
E65G
G161E
a) NS1
RNA Binding
domain
Effector
domain
Linker region
c) HA
I513V
V521M
V233I
I278V
D200N
Head
domain
Stalk
domain
S354N
V379I
b) NA Headdomain
K97R
A122E
G281V
D289E
A232T
E243G
d) NP
Body domain
Head domain
a) b)
1. Viral RNA extraction.
2. SIV detection RTq-PCR M segment.
3. Whole SIV Genome Amplification.
If 8 segments are amplified
4. NGS by Nextera-XT.
5. Bioinformatic workflow for Mapping and Variant Calling.
Material and methods
❖ SIV Detection, Sequenciation and Variant Calling
Introduction and objective
Results and discussion
Figure 1. Experimental design. Fifteen 6-weeks-old domestic pigs free from SIV were selected for
this study. Vaccinated animals were immunized twice, at 0 days post vaccination (dpv) and 21 dpv.
All animals were challenged at 42 dpv.
SIV Inoulumn
A/Swine/Spain/01/2010 (H1N1)
BALF
Lung
Nasal
Turbinate
Nasal Swab
RESPIPORC FLU3, IDT®
Sample collection
By two administration routes:
- Intranasal (1 mL per nostril)
- Endotracheal (5 mL)
106 TCID50
42 dpv
1 2
HA
E67G
HA
T345H
5
❖ Experimental Design
3
4
4
a) b)