Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy

Abstract

Background: Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas.

Objective: To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi).

Methods: Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively.

Findings: Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi).

Main findings: Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Fig. 1:. experimental kinetics of the study with star monkey infected with the virus of the experimental kinetics of the study with star monkey infected with the yellow fever virus (YFV). Non-human primates (NHP) (Saimiri spp.) were infected with 0.5 mL of the yellow fever virus intradermally infective dose = 1 x 10⁶ plaque forming units (PFU)/mL and one animal was kept until the end of the experiment as a negative control (NC). One animal was sacrificed on pre-established days post-infection (dpi) for the collection of blood, brain, lymph node, heart, lung, liver, stomach and kidney samples.

Fig. 2:. gross photographs changes observed in squirrel monkeys (Saimiri spp.) infected by the yellow fever virus (YFV). (A) Ventral back of the abdomen with bleeding points at 7 days post-infection (dpi). (B) Diffuse yellow liver with haemorrhagic spots and subcapsular multifocal petechiae at 7 dpi. (C, D) Heavy bleeding throughout the liver of the animal that succumbed to 6 dpi. (E) Liver diffusely yellowed at 20 dpi. (F) Normal liver. (G) Brain with congestion and haemorrhage. (H) Skull lid with subcutaneous hematoma at 6 dpi (arrow). (I) Normal brain. (J) Marked pulmonary congestion (left). (K, L) Spleen with rounded edges and whitish and/or dark red areas (arrows). (M) Normal spleen. (N) Stomach with contents (blood/digested) in the lumen. (O) Normal stomach. NC: negative control.

Fig. 3:. detection of the genome, viral antigens and neutralising antibodies in monkeys’ squirrels (Saimiri spp.) infected with yellow fever virus (YFV). (A) Detection of viral RNA by RT-qPCR. (B) Detection of neutralising anti-YFV antibodies. (C) Detection of viral antigens by immunohistochemistry. LD₅₀: lethal dose; LNI: logarithmic neutralisation index.

Fig. 4:. detection of viral antigens in the organs of the squirrel monkeys (Saimiri spp.) infected with yellow fever virus (YFV) that died at 6 days post-infection (dpi). (A) In the brain, immunostaining was predominant in glial cells in the neural parenchyma (black arrow) and neurons (red arrow). (B) YFV tropism by infecting hepatocytes (black arrow) was obvious at 6 dpi. (C) In the kidney, the immunostaining was marked in the renal tubules (black arrow). (D, F) In the spleen, lymph node and lung, immunostaining was enhanced in the mononuclear cells. (G, H) Myocardial and stomach cells were the preferred target of YFV at 6 dpi (black arrow). 400X (20 µm). IHC: immunohistochemistry; NC: negative control.

Fig. 5:. anatomopathological changes observed in the organs of the squirrel monkeys (Saimiri spp.) infected with yellow fever virus (YFV), which died at 6 days post-infection (dpi). (A) Thickening of the meninges (black circle) with oedema, congestion and haemorrhage blue circle. Oedema in the neural parenchyma was characteristic (yellow circle). (B) In the liver, tissue damage was characterised by centrilobular and midzonal haemorrhage (black circle) and lytic necrosis (blue and yellow circle). (C) In the kidney, there were haemorrhagic points in Bowman’s capsule (black circle) and tubular necrosis was a relevant finding (blue and yellow circle). (D) The deleterious effects on tissue in the spleen are manifested mainly in haemorrhagic foci (black, blue and yellow circle). (E) In the lymph node, bleeding was evidenced in the centre of the cortical region (black, yellow and blue circle). (F) Pulmonary parenchyma showed congestion and haemorrhage (black, blue and yellow circle). (G) In the heart, nuclear pyknosis in myocardial cells was impressive (black, blue and yellow circle). (H) In the stomach with inflammatory infiltrate (6 dpi) (black circle) and moderate numbers of acidophilic cells with nuclear pyknosis (blue and yellow circle). H&E: 400X (20 μm). NC: negative control.