A fatal case of dengue hemorrhagic fever associated with dengue virus 4 (DENV-4) in Brazil: genomic and histopathological findings

Abstract

Dengue infection is the most prevalent arthropod-borne viral disease in subtropical and tropical regions, whose primary vector is Aedes aegypti mosquitoes. The mechanisms of dengue virus (DENV) pathogenesis are little understood because we have no good disease models. Only humans develop symptoms (dengue fever, DF, or dengue hemorrhagic fever, DHF) and research has been limited to studies involving patients. Samples from serum, brain, cerebellum, heart, lungs, liver, and kidneys from a 13-year-old male patient that died with hemorrhagic manifestations were sent for differential diagnosis at Adolfo Lutz, using both classical virological methods (RT-qPCR, virus isolation, ELISA, and hemagglutination inhibition test) and immunohistochemistry (IHQ). A DENV serotype 4 was detected by a DENV multiplex RT-qPCR, and the C6/36 cell supernatant was used for NGS using Minion. Lesions were described in the heart, liver, lung, and kidney with positive IHQ in endothelial cells of the brain, cerebellum, heart, and kidney, and also in hepatocytes and Kuppfer cells. A whole genome was obtained, revealing a DENV-4 genotype II, with no evidence of secondary dengue infection.

Keywords: Dengue virus; Immunohistochemistry; Phylogeny; Whole-genome sequencing.

Fig. 1

A Photomicrography of the liver. Hepatocellular necrosis and apoptosis with Councilman-Rocha Lima resembling bodies. Hematoxylin and eosin (× 400). B Photomicrography of the lung. Marked intra-alveolar hemorrhage and edema. Hematoxylin and eosin (× 100). C Photomicrography of the kidney. Mutifocal acute tubular necrosis. Hematoxylin and eosin (× 100). D Photomicrography of the liver. Kupffer and endothelial cells have cytoplasmic immunolabeling for dengue virus antigen. IHC (× 400). E Photomicrography of the kidney. Dengue virus antigen in mesangial and endothelial cells of the glomerulus. IHC (× 400)

Fig. 2

ML Phylogenetic tree of DENV-4 genotype II isolate SPH329894 (depicted in red). Branches are colored according the country. Sequences of DENV-4 whole genomes were aligned using MAFFT. Best model and ML tree were generated using IQ-Tree [22]. The tree was constructed using FigTree v.1.4.3 with an automatic scale and mid-point root. Scale in nucleotide substitutions per site. Numbers at nodes indicate bootstrap values. Sylvatic DENV-4 was used as outgroup. Sequences are represented by Genbank accession numbers