Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses

Abstract

The highly pathogenic avian influenza (HPAI) H5N1 virus lineage has undergone extensive genetic reassortment with viruses from different sources to produce numerous H5N1 genotypes, and also developed into multiple genetically distinct sublineages in China. From there, the virus has spread to over 60 countries. The ecological success of this virus in diverse species of both poultry and wild birds with frequent introduction to humans suggests that it is a likely source of the next human pandemic. Therefore, the evolutionary and ecological characteristics of its emergence from wild birds into poultry are of considerable interest. Here, we apply the latest analytical techniques to infer the early evolutionary dynamics of H5N1 virus in the population from which it emerged (wild birds and domestic poultry). By estimating the time of most recent common ancestors of each gene segment, we show that the H5N1 prototype virus was likely introduced from wild birds into poultry as a non-reassortant low pathogenic avian influenza H5N1 virus and was not generated by reassortment in poultry. In contrast, more recent H5N1 genotypes were generated locally in aquatic poultry after the prototype virus (A/goose/Guangdong/1/96) introduction occurred, i.e., they were not a result of additional emergence from wild birds. We show that the H5N1 virus was introduced into Indonesia and Vietnam 3-6 months prior to detection of the first outbreaks in those countries. Population dynamics analyses revealed a rapid increase in the genetic diversity of A/goose/Guangdong/1/96 lineage viruses from mid-1999 to early 2000. Our results suggest that the transmission of reassortant viruses through the mixed poultry population in farms and markets in China has selected HPAI H5N1 viruses that are well adapted to multiple hosts and reduced the interspecies transmission barrier of those viruses.

Figure 1. Dated phylogeny of the surface genes of H5N1 viruses isolated in Eurasia.

The HA gene (A) and N1 gene (B) trees scaled to time (horizontal axis) generated using the SRD06 codon model and uncorrelated relaxed clock model. Nodes correspond to mean TMRCAs and blue horizontal bars at nodes represent the 95% HPDs of TMRCAs. Red branches indicate Gs/GD lineage H5N1 viruses. Identical phylogenetic trees with virus names are shown in Figure S1. TMRCAs and HPDs for each of the nodes marked with Roman numerals are given in Table 2.

Figure 2. Dated phylogeny of the internal genes of viruses isolated in Asia.

The NP (A), PA (B) and PB2 (C) gene trees scaled to time (horizontal axis) generated using the SRD06 codon model and uncorrelated relaxed clock model. Nodes correspond to mean TMRCAs and blue horizontal bars at nodes represent the 95% HPDs of TMRCAs. Red branches indicate Gs/GD lineage H5N1 viruses. Identical phylogenetic trees with virus names are shown in Figure S2. TMRCAs and HPDs for each of the major H5N1 genotype internal genes are given in Table 3.

Figure 3. Diagram representing the emergence of major H5N1 reassortant viruses.

Virus particles outlined in black represent donor viruses (with mean TMRCAs above the particle) and those outlined in red represent characterized H5N1 genotypes placed at the year of first detection. Gene segments are ordered PB2, PB1, PA, HA, NP, NA, M and NS from top to bottom within the virus particle diagram. Arrows represent possible reassortment pathways of genotype development. The start of the black arrows (filled circles) indicate the earliest possible time of corresponding genotype generation. Colored arrows represent reassortment between existing H5N1 genotypes.

Figure 4. Dated phylogeny of the HA gene of H5N1 viruses isolated in Asia.

The tree is scaled to time (horizontal axis) and was generated using the SRD06 codon model and uncorrelated relaxed clock model. Nodes correspond to mean TMRCAs and blue horizontal bars at nodes represent the 95% HPDs of TMRCAs. TMRCAs and HPDs for each of the major H5N1 lineage are given in Table 4.

Figure 5. Population dynamics of genetic diversity of H5N1 viruses isolated from poultry in China.

Bayesian skyline plot of the HA gene (A) showing changes in genetic diversity of H5N1 viruses. A measure of genetic diversity is given on the y-axis with the 95% HPD shown in blue. The red dashed line indicates the mean TMRCA of the Gs/GD lineage; the blue dashed line represents the time of the first detected H5N1 outbreak China. HA gene tree (B) scaled to time (horizontal axis) generated using the SRD06 codon model and uncorrelated relaxed clock model. Nodes correspond to mean TMRCAs and blue horizontal bars at nodes represent the 95% HPDs of TMRCAs. Numbers to the right of the HA tree indicate H5N1 clades based on the World Health Organization nomenclature system . An identical phylogenetic tree with virus names is shown in Figure S3. The red vertical bar in both panels indicates the period of divergence of major H5N1 lineages in poultry.