Genetic and structural characterization of dengue virus involved in the 2023 autochthonous outbreaks in central Italy

Abstract

Dengue virus (DENV) has been expanding its range to temperate areas that are not usually affected, where the spread of vectors has been facilitated by global trade and climate change. In Europe, there have been many cases of DENV imported from other regions in the past few years, leading to local outbreaks of DENV among people without travel history. Here we describe the epidemiological and molecular investigations of three transmission events locally acquired DENV infections caused by serotypes 1, 2 and 3, respectively, in the Latium Region from August to November 2023. Next-generation or Sanger sequencing was used to obtain the whole genomes, or the complete E-gene of the viruses, respectively. The structure of the DENV-1 and DENV-3 sequences was analysed to identify amino acid changes that were not found in the closest related sequences. The major cluster was supported by DENV-1 (originated in South America), with 42 autochthonous infections almost occurring in the eastern area of Rome, probably due to a single introduction followed by local sustained transmission. Seven DENV-1 subclusters have been identified by mutational and phylogenetic analysis. Structural analysis indicated changes whose meaning can be explained by the adaptation of the virus to human hosts and vectors and their interactions with antibodies and cell receptors.

Keywords: Dengue; arbovirus; genomic surveillance; molecular epidemiology; whole-genome sequencing.

Figure 1.

Algorithm for suspected DENV cases diagnosed in the Lazio region, Italy, from 23 August 2023–30 November 2023, following the first diagnosed autochthonous case.

Figure 2.

Panel A: Phylogenetic analysis based on the complete E gene and WGS of autochthonous and imported cases of DENV-1 and DENV-3. Sequences from autochthonous cases are highlighted in red, and those from imported cases are highlighted in blue. Bootstrap values > 80 are reported aside the corresponding nodes. Panel B: Enlarged detail of the phylogenetic tree of complete E gene sequences from autochthonous DENV-1 cases (red). The identified subclusters are indicated by capital letters. Bootstrap values > 80 are reported aside the corresponding nodes.

Figure 3.

Geographical distribution of DENV-1 sequenced cases. Each dot represents cases that occurred within an area of 1 square kilometre. Colours represent clusters identified by phylogenetic analysis. The epidemic curve shows the appearance of individual cases in chronological order based on the first appearance of symptoms. The cases are represented with the same colour as the cluster they belong to.

Figure 4.

Colour-coded multiple sequence alignment with Clustal Omega [18] in the region of the pS227P variant for the INMI-A19 case. Comparison of INMI sequence, reference and representative genome sequences with S227P, S227L and S227T variants are shown in panels A, B and C, respectively. The pS227P variant in the INMI-A19 case is highlighted with a green background. (D) Model of the DENV envelope. The surface is composed of a network of 360 E and M proteins. The variant pS227P variant in the E protein of DENV-1, found in INMI patient INMI-A19, is highlighted in red colour; (E) Model of the E-M tetramer complex in membrane. The two E proteins are coloured yellow and blue, while the two M ones are green and orange. The two Pro227 are shown in red, as in panel A.

Figure 5.

(A) Colour-coded multiple sequence alignment with Clustal Omega [18] in the region of the A313D variant for INMI-A11 case. Comparison of INMI sequence, reference and representative genome sequences with A313P, A313V and A313S variants are shown. (B) Model of the DENV E-M tetramer complex in membrane. The A313D variant in the E protein of DENV-1, found in case INMI-A11, is highlighted in red colour; (C) Enlargement of the D313 region showing the interface proximity to the residue with the polar lipid heads in the membrane.

Figure 6.

(A, B) Colour-coded multiple sequence alignment with Clustal Omega [18], for INMI-A15 and – A16 cases, in the region of the A203S and T471I variants are shown in panels A and B, respectively. (C) Model of the DENV E-M tetramer complex in the membrane. The A203S and T471I variants in the E protein of DENV-3 are highlighted in red colour. (D) Enlargement of the S203 region. (E) Enlargement of the I471 region, showing the proximity of the residue with the polar lipid heads in the inner viral membrane.