Open Access
Abstract
An
explosive epidemic occurred in Madeira Island (Portugal) from October
2012 to February 2013. Published data showed that dengue virus type 1
introduced from South America was the incriminated virus. We aim to
determine the origin of the strain introduced to Madeira by travellers
returning to Europe. Using phylogeographic analysis and complete
envelope sequences we have demonstrated that the most probable origin of
the strain is Venezuela.
Keywords
- Dengue fever;
- dengue virus 1;
- Madeira;
- South America;
- travel infection
After more than 80 years without dengue fever in Europe, in 2010 autochthonous transmission was reported in France and Croatia [1]. In October 2012, a cluster of autochthonous cases was reported in Madeira, Portugal [2]. The outbreak in Madeira evolved rapidly because of the abundance of the competent vector Aedes aegypti [3]
and the totally naive population, resulting in over 2168 cases from 26
October 2012 to 3 March 2013. Until 3 February 2013, at least 78 cases
in 13 other European countries were reported as introductions via
travellers departing from Madeira [4]. The causative virus was identified as dengue virus type 1 (DENV-1) [5].
A
recent study investigating Madeira air travel interconnectivity to
track the possible origin of DENV-1 introduction in Europe indicated
that from 22 948 air travellers to Madeira in 2012 arriving from 29
dengue endemic countries, almost 90% of them came from Venezuela and
Brazil [6].
In addition, in 2012 DENV-1 partial capsid pre-membrane sequences were
obtained from two Finnish travellers (399 and 396 nucleotides,
respectively) returning from Madeira to Finland with dengue fever [7].
These sequences were similar to that of an autochthonous case from
Madeira and grouped phylogenetically with South American lineages within
the America/Africa genotype. Analysis of partial E sequences from
Belgian travellers reported similar findings [7] and [8]. These findings suggest a unique introduction from a South American country to Madeira.
Here
we report for the first time the full envelope dengue sequence data
from travellers who had acquired dengue fever in Madeira and compared
them with recent sequences obtained from travellers returning from South
America to find the most probable source of the epidemic.
As part of a collaborative study to monitor dengue virus importation into Europe through travellers (Dengue tools project, www.denguetools.net),
we collected acute positive samples (as detected by RT-PCR or
Non-structural protein 1 antigen test (NS1-Ag)), from the beginning of
2012 from patients seen by each of the 17 Tropical Medicine centres
enrolled in the study in 11 European countries [9]
onwards. Positive samples were sent to the reference laboratory
(Instituto de Salud Carlos III, Madrid, Spain) for strain isolation and
characterization. Samples from travellers returning to Europe from
Madeira or South America were selected for comparing virus sequences at
the Envelope (E) gene and its junction with the NS1 gene. The ethics review board of both hospitals approved this study.
Briefly, RNAs were extracted with a Qiamp viral RNA extraction kit (Qiagen, Hilden, Germany), and the complete E gene (1485 nucleotides) and the E/NS1 junction (441 nucleotides) of DENV-1 strains were RT-PCR amplified [10]. The amplification products were purified and sequenced using a set of 15 primers (11 internal and two external primers for E, plus two external primers for E/NS1).
In total, we obtained the sequences from three new isolates: one from a
German traveller returning from Madeira in 2012, and two from Spanish
travellers returning from Venezuela in 2010 and 2012. These sequences
were aligned with 140 other DENV-1 sequences retrieved from GenBank, and
chosen to represent the different virus genotypes and different
geographical origins and time of detection for each genotype. Using this
sequence data set, a Bayesian Markov Chain Monte Carlo (MCMC)
phylogenetic tree was inferred with BEAST v1.7.5 [11], incorporating the best-fitted nucleotide substitution model as determined by jModelTest 2.1.3 [12].