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. 2021 Sep 29;13(10):1954.
doi: 10.3390/v13101954.

Morphological Aspects and Viremia Analysis of BALB/c Murine Model Experimentally Infected with Dengue Virus Serotype 4

Arthur da Costa Rasinhas  1 , Fernanda Cunha Jácome  1 , Gabriela Cardoso Caldas  1 , Ana Luisa Teixeira de Almeida  1 , Marcos Alexandre Nunes da Silva  1 , Daniel Dias Coutinho de Souza  1 , Amanda Carlos Paulino  1 , Derick Mendes Bandeira  1 , Raphael Leonardo  1 , Priscila Conrado Guerra Nunes  2 , Ronaldo Mohana-Borges  3 , Ortrud Monika Barth  1 , Flavia Barreto Dos Santos  2 , Debora Ferreira Barreto Vieira  1
Affiliations

Morphological Aspects and Viremia Analysis of BALB/c Murine Model Experimentally Infected with Dengue Virus Serotype 4

Arthur da Costa Rasinhas et al. Viruses. .

Abstract

Ever since its brief introduction in the Brazilian territory in 1981, dengue virus serotype 4 (DENV-4) remained absent from the national epidemiological scenario for almost 25 years. The emergence of DENV-4 in 2010 resulted in epidemics in most Brazilian states. DENV-4, however, remains one of the least studied among the four DENV serotypes. Despite being known as a mild serotype, DENV-4 is associated with severe cases and deaths and deserves to be investigated; however, the lack of suitable experimental animal models is a limiting factor for pathogenesis studies. Here, we aimed to investigate the susceptibility and potential tropism of DENV-4 for liver, lung and heart of an immunocompetent mice model, and to evaluate and investigate the resulting morphological and ultrastructural alterations upon viral infection. BALB/c mice were inoculated intravenously with non-neuroadapted doses of DENV-4 isolated from a human case. The histopathological analysis of liver revealed typical alterations of DENV, such as microsteatosis, edema and vascular congestion, while in lung, widespread areas of hemorrhage and interstitial pneumonia were observed. While milder alterations were present in heart, characterized by limited hemorrhage and discrete presence of inflammatory infiltrate, the disorganization of the structure of the intercalated disc is of particular interest. DENV-4 RNA was detected in liver, lung, heart and serum of BALB/c mice through qRT-PCR, while the NS3 viral protein was observed in all of the aforementioned organs through immunohistochemistry. These findings indicate the susceptibility of the model to the serotype and further reinforce the usefulness of BALB/c mice in studying the many alterations caused by DENV.

Keywords: BALB/c mice; DENV-4; heart; histopathology; liver; lung; ultrastructure.

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Conflict of interest statement

The authors declare no conflict of interest exists.

Figures

Figure 1
Figure 1
(A) Temperature values at pre-infection time (T0) and 72 h post-infection (T1) of negative control (NC) and DENV-4-infected BALB/c mice (INF). (B) Temperature variation between T0 and T1 (Δϴ) of negative control (NC) and DENV-4-infected BALB/c mice (INF).
Figure 2
Figure 2
Liver samples of BALB/c mice stained with hematoxylin and eosin and observed in bright field microscope. (A) Negative control sample. Portal vein branch (PVB); hepatic artery branch (HAB); biliary duct (BD); hepatocytes (H). (B–E) DENV-4-infected samples. Inflammatory infiltrate (IC); vascular congestion (VC); edema (*); ballooning degeneration of hepatocytes (dashed outline); erythrocytes within the tissue (Eri); dilation of sinusoid capillaries (arrows). Magnification: (A) = 200×; (B) = 200×, (C) = 200×; (D) = 200×; (E) = 200×.
Figure 3
Figure 3
Liver samples of BALB/c mice observed in transmission electron microscope. (A) Negative control sample. Hepatocyte (H); nucleus (N). (B–F) DENV-4-infected samples. Microvesicular steatosis (dashed outline); mononuclear inflammatory cells (MIC) presenting filopodia (arrows); capillary (Cap); hepatocytes (H); activated platelet (P); erythrocyte; vascular congestion (VC); pyknoctic nucleus (PN). Magnification: (A) = 5000×; (B) = 8000×, (C) = 12,000×; (D) = 10,000×; (E) = 12,000×; (F) = 12,000×.
Figure 4
Figure 4
Lung samples of BALB/c mice stained with hematoxylin and eosin and observed in bright field microscope. (A,B) Negative control samples. Alveolar sac (AS); alveolus (Alv); interalveolar septum (IS). (C,D) DENV-4-infected samples. Areas of hemorrhage (H); thickening of the interalveolar septa (*); bronchiole (Brc). Magnification: (A) = 100×; (B) = 400×, (C) = 50×; (D) = 100×.
Figure 5
Figure 5
Lung samples of BALB/c mice observed in transmission electron microscope. (A) Negative control sample. Type II pneumocyte (PII); endothelial cell (EC); alveolus (Alv); capillary (Cap); erythrocyte (Eri). (B–D) DENV-4-infected samples. Type I Pneumocyte (PI); mononuclear inflammatory cell (MIC); polymorphonuclear cell (PIC); platelet (P); filopodia (arrows). Magnification: (A) = 6000×; (B) = 6000×, (C) = 12,000×; (D) = 6000×.
Figure 6
Figure 6
Heart samples of BALB/c mice stained with hematoxylin and eosin and observed in bright field microscope. (A,B) Negative control samples. Cardiac Fibers (CF); cardiomyocytes (Ca). (C,D) DENV-4-infected samples. Inflammatory infiltrate (IC); area of hemorrhage (H). Magnification: (A) = 50×; (B) = 200×, (C) = 200×; (D) = 200×.
Figure 7
Figure 7
Heart samples of BALB/c mice observed in transmission electron microscope. (A) Negative control sample. Cardiac fibers (CF); nucleus (N); intercalated disc (ID). (B–F) DENV-4-infected samples. Dengue virus-like particles (arrows); intercalated disc (ID); mononuclear inflammatory cell (MIC); platelet aggregation (red arrows); platelet adhered to the erythrocyte (E), endothelium (P); capillary (C); intercalated disk disorganization (dashed outline). Magnification: (A) = 5000×; (B) = 30,000×, (C) = 15,000×; (D) = 12,000×; (E) = 10,000×; (F) = 20,000×.
Figure 8
Figure 8
Liver samples of BALB/c mice counterstained with Harris hematoxylin and observed in bright field microscope. (A) Negative control sample. Hepatocytes (H); erythrocytes showing no peroxidase reaction (arrows). (B,C) DENV-4-infected samples. Portal vein branch (PVB); hepatocytes (H); areas of DENV-4 antigen detection (arrows). Magnification (A) = 100×; (B) = 200×, (C) = 400×.
Figure 9
Figure 9
Lung and heart samples of BALB/c mice, counterstained with Harris hematoxylin and observed in bright field microscope, showing detection of the NS3 protein. (A) Negative control lung sample. Alveolar sac (AS); alveolus (Alv); erythrocytes showing no peroxidase reaction (arrow). (B) Negative control heart sample. Cardiomyocyte (Ca); erythrocytes showing no peroxidase reaction (arrows). (C) DENV-4-infected lung sample. Pneumocytes (P); areas of DENV-4 antigen detection (arrow). (D) DENV-4-infected heart sample. Areas of DENV-4 antigen detection (arrow). Magnification (A) = 50×; (B) = 50×, (C) = 400×; (D) = 400×.
Figure 10
Figure 10
Individual titers of DENV-4 RNA detected in liver, lung, heart and serum of infected BALB/c mice, expressed in logarithmic values.
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