The potential for respiratory droplet-transmissible A/H5N1 influenza virus to evolve in a mammalian host

Abstract

Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Fig. 1

Phylogenetic trees of the A/H5N1 HA1 nucleotide sequences. The sequences are split into three trees: 2,022 avian H5 sequences from East and South-East (E and SE) Asia (top row); 1,097 avian H5 sequences from Europe, the Middle East, and Africa (middle row); and 385 human H5 sequences (bottom row). Each sequence is color coded by the minimum number of nucleotide mutations required: to obtain the four amino acid substitutions in HA in the Herfst et al-set (column 1), to obtain the four amino acid substitutions in the Imai et al-set (column 2), to disrupt the N-linked glycosylation sequon spanning positions 154–156 in HA (column 3), and to obtain E627K in the PB2 segment of the corresponding virus in these HA trees (column 4). In columns 1 and 2, blue indicates five nucleotide changes, green four, and orange three. In columns 3 and 4 yellow indicates viruses that require one mutation and pink zero mutations. Gray indicates PB2 not sequenced. Clades as defined by (35) are marked to the right of the branches, the red portion of the vertical clade-identification lines indicates strains sampled in 2010 or 2011. The viruses indicated by black arrows are two nucleotides from the Imai et al-set. The virus indicated in A by the brown arrow has the H103Y substitution, the virus indicated in B by the brown arrow has the T315I substitution. The blue circle indicates A/Indonesia/5/2005, the red circle indicates A/Vietnam/1203/2004, the starting viruses used by (1) and (2) respectively. The initial trees were constructed with PhyML version 3.0 (36), with A/Chicken/Scotland/1959 as the root, using GTR+I+Γ4 [determined by Model Test (37)] as the evolutionary model. GARLI version 0.96 (38) was run on the best tree from PhyML for one million generations to optimize tree topology and branch lengths. Fig. S1 shows “zoom-able” versions of these trees to show detail.

Fig. 2

Expected proportions and absolute numbers of respiratory droplet transmissible A/H5N1 virions within a host initially infected by strains that require five (blue), four (green), three (orange), two (red), or one (purple) mutation(s) to become respiratory droplet transmissible, calculated from the deterministic model. A. Expected proportion of respiratory droplet transmissible A/H5N1 viruses in the total virus population over time in the random mutation case (when all mutations are fitness neutral). B. The absolute number of respiratory droplet transmissible A/H5N1 viruses in a host; the intersections with the grey line indicate the point when at least one virus in each host is expected to have the required mutations. The change in slope is due to the transition in the virus population from exponential expansion to constant size.

Fig. 3

Factors that increase the proportion of respiratory droplet transmissible A/H5N1 virus based on starting viruses that require five (blue), four (green), three (orange), two (red), or one (purple) mutation(s) to become respiratory droplet transmissible. A. The effect of hill-climb and all-or-nothing positive selection compared to random mutation alone. B. The effect of avian–mammal transmission of partially adapted virus (100 viruses start the infection, one of which has a mutation) and the effect of alternate substitutions with 10 functionally equivalent sites for a virus requiring five mutations (blue), nine requiring four (green), eight requiring three (orange), seven requiring two (red), six requiring one (purple); both with hill-climb selection, as compared to hill-climb selection alone. C. The effect of two of the required mutations being individually deleterious (i.e. for these two specific mutations either mutation alone reduces the replicative fitness of the virus to zero) and the effect of complete order dependence of acquiring mutations, both with hill-climb selection as compared to hill-climb selection alone.

Fig. 4

Proportion of respiratory droplet transmissible A/H5N1 virus in a long infection with virus replication for 14 days in the presence of hill-climb selection. Bold lines show results from a probability-based deterministic model of virus mutation, the pale region (composed of lines) shows 10,000 stochastic model simulations for each starting virus. Starting viruses require either five (blue), four (green), three (orange) mutations to become respiratory droplet transmissible. For the stochastic simulations the lines start when the first virion that has the required mutations appears.

Fig. 4

Proportion of respiratory droplet transmissible A/H5N1 virus in a long infection with virus replication for 14 days in the presence of hill-climb selection. Bold lines show results from a probability-based deterministic model of virus mutation, the pale region (composed of lines) shows 10,000 stochastic model simulations for each starting virus. Starting viruses require either five (blue), four (green), three (orange) mutations to become respiratory droplet transmissible. For the stochastic simulations the lines start when the first virion that has the required mutations appears.