Evolution of an Eurasian avian-like influenza virus in naïve and vaccinated pigs

Abstract

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.

Figure 1. Layout of the transmission studies.

Seeder pigs (S) were experimentally infected with A/swine/England/453/2006 and are shown in pink. Naïve pigs (N) are shown in green and vaccinated (V) pigs are shown in red. Arrows indicate the direction of transmission among pairs. Numbers on each pig refer to unique identifiers. (B) Nucleotide position and absolute frequency of synonymous and nonsynonymous mutations relative to the reference sequence. Each panel is shown below the corresponding study.

Figure 2. The mutational spectra of intra-host EA-SIV populations are highly dynamic.

Median joining networks derived from representative individual pigs in the naïve (A) and vaccinated (B) studies. Each network was inferred by compiling sequences from multiple days. The number of sequences that constituted the consensus is indicated and circles are sized relative to their frequency in the data set. The identification number and the order of each pig the transmission chain is shown at the bottom of each panel. Colors indicate the day in which the sample was taken relative to the start of the study. Black dots along the branches indicate individual mutations relative to the sequence of the node from which they are derived. Cycles represent alternative potential evolutionary pathways.

Figure 3. Transmission of multiple variants and mixed infections are common during infection of EA-SIV in pigs.

Schematic representation of the number of shared mutations throughout the transmission studies in naïve (A) and vaccinated (B) pigs. Each circle represents a compiled data set for each pig (i.e. all the sequences derived from a pig along the course of infection), with the circle size being proportional to the mean pairwise distance of each data set. Seeder (S), naïve (N), and vaccinated (V) pigs are colored as shown in Figure 1. The infection route and relative position in the transmission chain is indicated on the left. The identification number of each pig is shown within each circle. The number of shared mutations between any two pigs is shown for in black boxes for each link in the chain. Grey arrows indicate that the consensus sequence of the viral population is the same as the reference whereas red arrows indicate the transmission of a fixed mutation (A696, see text). For full details of the shared mutations see Figure S3.