Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa

Abstract

Usutu virus (USUV), one of the most neglected Old World encephalitic flaviviruses, causes epizootics among wild and captive birds and sporadic infection in humans. The dynamics of USUV spread and evolution in its natural hosts are unknown. Here, we present the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution. The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow. Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks. Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles.

Importance: Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts.

FIG 1

Bayesian maximum clade credibility (MCC) trees representing the time scale phylogeny, reconstruction of spatiotemporal spread pattern of USUV in Africa and Europe and the three major flyway (East Atlantic, Black Sea/Mediterranean, and East Africa/West Asian flyways; see color codes on the small map) networks (marked with gray lines onto the map) of migratory birds (66). The colored branches of MCC trees represent the most probable geographic location of their descendant nodes (see color codes). Bayesian posterior probabilities (≥90%) and 1,000 parallel maximum likelihood bootstrap replicates (≥70%) are indicated at the nodes (asterisks). The main lineages are indicated to the right of the tree. Time is reported in the axis below the tree and represents the year before the last sampling time (2014). Dotted lines on the maps between locations represent the temporal dynamics of USUV spatial diffusion with the most probable sources and target localities of branches in MCC trees. The distinct introductions within or between Africa and Europe are represented by different colored lines, while the estimated TMRCA (oldest possible year of introduction) of USUV strains from different countries are shown with 95% posterior time intervals in parentheses.

FIG 2

Maximum clade credibility trees of E (a) and NS5 (b) gene sequences colored by reconstructed host species (Markov jump process). Branches are colored by host and represent the transitions between different host species for the virus and the host of the common ancestor of all USUV strains.

FIG 3

Migration pattern of USUV between or within Africa and Europe. Viral migration patterns based on E (a) and NS5 (b) genes are indicated between the different regions and are proportional to the strength of the transmission rate (Bayes factor [BF]). The color of the connections indicates the origin and the direction of migration. Only connections with a BF of <3 are shown.

FIG 4

Schematic representation of the genome organization of USUV. Red arrows below indicate O-glycosylation sites, blue arrows N-glycosylation sites, and orange arrows C-glycosylation sites. A number indicating the position of amino acid mutations and the single-letter amino acid codes are used to denote the geography- and/or host-specific mutations along the polyprotein of the African and European USUV strains.

FIG 5

Structural location of the USUV mutations in different hosts from Africa and Europe depicted on the predicted USUV envelope glycoprotein structure. (A) The three-dimensional ribbon structure of a single monomer of the USUV envelope glycoprotein is shown with the corresponding three viral domains (domain I in blue, domain II in yellow, and domain III in green) and surface-exposed variable residues. (B and C) Structural mapping of the variable sites in the mosquito-, bird-, and human-derived USUV E protein on the surface rendition is shown from the side view and in a counterclockwise ~90° rotation. (D and E) The same model as in panel C in a counterclockwise ~90° plus ~90° rotation showing the external surface of the predicted USUV E monomer. All unique mutations in the singleton Africa 1 strain are shown in red and Europe 2-specific mutations in orange. Specific amino acid mutations found only in the African strains are indicated in cyan, and the Spanish-specific mutation is in green, while the unique mutation detected in the human USUV case from Italy is shown in blue. Amino acid V126M (magenta) is also indicated.