Collaborators at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) and I have been working to investigate characteristics of avian influenza epidemics.  We have recently published a paper in Transboundary and Emerging Diseases titled “Spatiotemporal reconstruction and transmission dynamics during the 2016-17 H5N8 highly pathogenic avian influenza epidemic in Italy”. The focus of this paper was to use integrated analyses of genetic sequences and associated metadata to learn about how the epidemic dispersed spatially and the host types involved in such movements.

We used the dates and locations associated with viruses isolated from 83 domestic cases and from 12 wild birds collected by IZSve field epidemiologists to simultaneously reconstruct virus phylogeny and the movements and dates associated with branches of the phylogeny. Among other features, this revealed a highly skewed distribution of movements with many shorter localised movements interspersed with occasional long distance dispersal movements.

This map shows a reconstruction of the spread of the genetic clade ‘Italy-B’ that was mostly restricted to the north-west of Italy (ancestor starred, red rings = wild bird), though also experienced dispersal to Eastern Italy followed by a long-distance southwards movement later in the epidemic:

Reconstructing host type using evolutionary distances

We also wanted to reconstruct host type across the phylogeny in order to differentiate clusters of domestic-to-domestic transmission from incursions across the wildlife-domestic interface. This was challenging because of different sampling intensities in domestic and wild birds: a high proportion of domestic cases should be represented in the data whereas in contrast, the epidemic in wild birds was probably greatly undersampled. This meant that approaches such as ancestral state reconstruction would likely overestimate the proportion of internal nodes associated with domestic birds.

To estimate the likelihood of unobserved or cryptic wild bird-mediated transmission across the phylogeny we used an approach based on evolutionary distances – branch lengths of time-scaled phylogenies estimated using a molecular clock model in BEAST.

1. For each domestic case we first identified a putative domestic ancestor as the phylogenetically closest case from an earlier date. For a cluster of domestic cases below, blue arrows identify putative domestic ancestors. 
2. Phylogenetic (patristic) distances between domestic cases and putative domestic ancestors were calculated by summing the lengths of branches separating them.
3. Values within the observed range of these phylogenetic distances were considered as possible thresholds. Moving from tips to the root, internal nodes were used to infer host type at internal nodes. Internal nodes with both descendants being domestic cases with distances to putative domestic ancestors below the threshold were considered domestic, otherwise wild bird was inferred.
4. Using distances estimated across a range of phylogenies, the range of potential thresholds at which internal nodes were recorded as wild bird or domestic  was recorded. This is shown for a section of the outbreak phylogeny below.
5. The transmission type associated with each branch of the viral phylogeny could therefore be estimated as being either 1) wild bird-to-wild bird, 2) wild bird-to-domestic, or 3) domestic-to-domestic.

Using this approach, domestic-to-domestic transmission was estimated to be restricted to the second half of the epidemic under most possible threshold values, something which matched conclusions from epidemiological investigations. Using a threshold value that corroborated epidemiological investigations, we looked at the geographical distances estimated for branches associated with each transmission type.

Where large geographic distances were estimated to have been covered in branches of the tree where evolutionary distances indicated a cluster of domestic-to-domestic spread (i.e. domestic cases on nearby dates separated by small genetic distances), we hypothesised that such instances may be characteristic of human-mediated movements. This approach highlighted branches linking a cluster of domestic cases associated with travel to or from a live bird market.