Pathogenesis and Immune Response of Ebinur Lake Virus: A Newly Identified Orthobunyavirus That Exhibited Strong Virulence in Mice

Abstract

Orthobunyaviruses are a group of viruses with significant public and veterinary health importance. These viruses are mainly transmitted through mosquito-, midge-, and tick-vectors, and are endemic to various regions of the world. Ebinur Lake virus (EBIV), a newly identified member of Orthobunyavirus, was isolated from Culex mosquitoes in Northwest China. In the present study, we aimed to characterize the pathogenesis and host immune responses of EBIV in BALB/c mice, as an animal model. Herein, we determined that BALB/c mice are highly susceptible to EBIV infection. The infected mice exhibited evident clinical signs including weight loss, mild encephalitis, and death. High mortality of mice was observed even with inoculation of one plaque-forming unit (PFU) of EBIV, and the infected mice succumbed to death within 5-9 days. After EBIV challenge, rapid viremic dissemination was detected in the peripheral tissues and the central nervous system, with prominent histopathologic changes observed in liver, spleen, thymus, and brain. Blood constituents' analysis of EBIV infected mice exhibited leukopenia, thrombocytopenia, and significantly elevated ALT, LDH-L, and CK. Further, EBIV infection induced obvious cytokines changes in serum, spleen, and brain in mice. Collectively, our data describe the first study that systematically examines the pathogenesis of EBIV and induced immune response in an immunocompetent standard mouse model, expanding our knowledge of this virus, which may pose a threat to One Health.

Keywords: BALB/c mouse; Ebinur Lake virus; Orthobunyavirus; immune response; pathogenesis.

Figure 1

Clinical illness and survival curve during Ebinur Lake virus (EBIV) infection in male and female BALB/c mice. (A) Behavioral changes. (B) Survival curve. Adult mice (n ≥ 10 per group) were challenged intraperitoneally (i.p) with dose from 10⁻⁴ to 10⁵ plaque-forming unit (PFU). (C) Weight change over 6 days when challenged with dose of 10 PFU.

Figure 2

Viral load in different organs of EBIV infected female BALB/c mice inoculated by i.p with 10 PFU. Viral titer was measured and quantified in serum and organs from day 1 to 5 post-infection by plaque assay (n = 5). Viral titers for the Serum (A), Liver (B), Spleen (C), Kidney (D), Lung (E), Intestines (F), Brain (G), and Thymus (H) are shown.

Figure 3

Gross examination of organs in female BALB/c mice following EBIV infection. Representative pictures of organs in mock-infected mice (A)+(C-a) and EBIV-infected mice (B)+(C-b). Appearance of liver color of EBIV-infected mice lighter brown compared to that of mock-infected mice (black arrow). Spleen (white arrow) and thymus (a,b) of EBIV-infected mice achieved a significant reduction in size compared to those of mock-infected mice. The intestine was almost empty (green arrow), and accompanied by severe congestion (red arrow).

Figure 4

Immunohistochemical (IHC) findings in female BALB/c mice following EBIV-infection at 5 days post-infection (d.p.i). Original magnification was 400× in tissues. Infected animals exhibited positive immunostaining (brown) for EBIV with increased significant IOD differences in the Liver, intestine, brain, spleen, thymus, and Peyer’s patch sections. Viral antigen can be found scattered in Kupffer cells of liver sections (purple arrow), gliocytes of brain sections (blue arrow), and neutrophils of the intestine (green arrow), spleen (red arrow), thymus (orange arrow), and Peyer’s patch (black arrow) sections. Significance was determined by comparing to the control. Error bars represent SD. The two-tailed p values are indicated as follows: ^**p ≤ 0.01.

Figure 5

Histopathologic examination in EBIV-infected female BALB/c mice by i.p route at 5 d.p.i. Original magnification was 200× or 400×. Liver: showed hepatocellular edema (yellow arrow) and disordered arrangement (green arrow). Intestine: showed damaged structure (gray arrow) and exfoliated epithelial cells (red arrow) in the intestinal villi, increased neutrophils (blue arrow), and cell necrosis (brown arrow) in the mucosal layer. Brain: showed inflammatory cells, including neutrophils, monocytes, macrophages, and lymphocytes, infiltrated around the meningeal blood vessels (purple arrow). Spleen: showed lymphocyte necrosis (nuclear fragmentation; black arrow). Thymus: showed focal necrosis of lymphocytes (dark gray arrow). Peyer’s patch: showed interstitial edema, loosely arranged cells, and lymphocyte necrosis (light yellow arrow).

Figure 6

Transmission electron microscopy analysis of tissues infected with 10 PFU of EBIV i.p at 5 d.p.i in female mice. Original magnification was 1700× or 7800×. Liver: lipid droplets in some hepatocytes increased (yellow arrow) and viral particles were found in the Kupffer cell of liver sinus. Brain: the capillary endothelial cells (BCECs) swelled (blue arrow) and viral particles were detected (red arrow). Spleen: lymphocytes were necrotic (brown arrow) and viral particles were seen in the granulocyte (green arrow).

Figure 7

Hematologic abnormalities induced by EBIV infection with 10 PFU in female mice. BALB/c mice were inoculated by i.p route (n = 5) and 10 mock-infected animals as control, Each symbol represents one animal. (A) Total White blood cells (WBCs). (B) Lymphocyte. (C) Neutrophil. (D) Monocyte. (E) Eosinophil. (F) Basophil. Composite graph showing average absolute counts (G) and average population proportions (H) of lymphocytes, neutrophils, monocytes, eosinophils, and basophils. (I) Total red blood cells (RBCs). (J) Hemoglobin (Hb). (K) Hematocrit. (L) Platelets.

Figure 8

Cytokine abnormalities induced by EBIV infection in female mice. (A) IL-2, IL-4, IFN-γ, TNF-α, IL-10, and IL-5 levels were determined in serum (A) and in different tissues, spleen (B), brain (C), and liver (D) of mock and EBIV-infected mice. All cytokine concentrations in tissues were normalized to the mass of the respective homogenized tissue. Significance was determined by comparing to the control. Error bars represent SD. The two-tailed p values are indicated as follows: ^*p ≤ 0.05; ^**p ≤ 0.01.