Genotyping of dengue virus from infected tissue samples embedded in paraffin

Abstract

Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history.

Keywords: Colombia; Dengue virus; Genotype; Paraffin-embedded tissues.

Fig. 1

Morphological changes observed in tissues from selected cases. a Hepatic tissue. Note the loss of the radial arrangement of the hepatic plaques with pale eosinophilic staining and pyknotic or absent nuclei; Cv-Central vein, Hc-Hepatic cells, N-necrosis, H-Hemorrhage, Kh-Kupffer cells hyperplasia, E-Esteaosis, Pi-Portal tract infiltrate. b Splenic tissue. The image mainly shows an enlargement of the white pulp and congestion of the venous sinuses; Ln-Lymphoid node, Wp-White pulp, Hw-Hyperplasia white pulp, C-Congestion, Rp-Reactive plasmacytosis. c Pulmonary tissue. Note the septum thickening and serous fluid in the alveolus (Edema).; As-Alveolar septum, A-Alveoli, E, Edema and d Cerebral tissue. The image shows a decrease in neuronal size due to retraction of the cytoplasm, with pyknosis and hyperchromasia of the nucleus; Nc-Neuronal cell, Hx-Hipoxia, Hematoxylin and Eosin coloration (H&E)

Fig. 2

Amplification products of the pairs of primers used to obtain fragments of the DENV-2 E gene. a Expected amplification products, b amplification products obtained for some samples (S20, S21, S4, S10). S letter corresponds to the coding assigned to the samples of 2% agarose gels stained with SYBR safe, as shown in Additional file 1: Table S1. M1–M4 abbreviations of the primer mixture are described in Table 1

Fig. 3

Phylogenetic analysis of DENV-2 based on reference sequences of each genotype and lineage