Pathogenicity of a novel bovine adenovirus type 3 with a natural deletion partial fiber gene in BALB/c mice

Abstract

Objective: A novel Bovine adenovirus type 3 (BAdV-3) with a natural deletion partial fiber gene was isolated in 2020 and named BO/YB24/17/CH. The objective of this study was to understand the pathogenicity of this virus.

Methods: Thiry-two 3-week-old BALB/c mice were divided into three experimental groups and a control group. Mice in the experimental groups were intranasally inoculated with virus, and mice in the control group were inoculated with MDBK cell supernatant. Mice were weighed and clinically examined daily for appearance of any clinical signs. Three infected mice and one control mouse were euthanized at 1, 3, 5, 7, 9, 11, 13, and 15 days after inoculation. Tissue samples were collected for histopathological examination, immunohistochemical staining, and detection of the replication dynamics of virus.

Results: All infected mice developed mild clinical signs such as lethargy, weight loss, loss of appetite, and a rough hair coat, and gross lesions were observed as pulmonary punctate hemorrhage, lobular atrophy and splenomegaly. Histopathological examination revealed thickening of alveolar septa and mildly dilated splenic nodules and blurred red-white medullary demarcation in the spleen. Immunohistochemical results further confirmed that the production of the above lesions was due to viral infection. Importantly, unlike previously reported BAdV-3 detection only in the lungs and trachea, this isolate could be detected in multiple organs such as the heart, liver, spleen, kidney, and even blood by virus isolation and titration and real-time PCR methods.

Clinical significance: This study provides further insight into the pathogenicity of the fiber region deletion strain BO/YB24/17/CH in BALB/c mice, which provides a reference for the prevention and control of BAdV-3 as well as the development of vaccines.

Keywords: BALB/c mice; bovine adenovirus type 3; deleted strain; fiber gene; pathogenicity.

Figure 1

Structural diagram of fiber protein of BO/YB24/17/CH strain. Dashed lines indicated amino acid deletion at position 192~270 of BO/YB24/17/CH in shaft region. The black box indicated the unique amino acid mutation of BO/YB24/17/CH compared to all available complete 9 BAdV-3 fiber sequences.

Figure 2

Changes in body weight gain and food intake of mice at different times after inoculation. (A) Daily weight gain. (B) Feed intake. Values are mean ± SD. *P < 0.05 (by one-way ANOVA).

Figure 3

Gross lesions in BALB/c mice after infection with BO/YB24/17/CH strain. (A) Lung congestion (the 3th day after infection). (B) Lung hyperemia with sporadic petechial, indicated with black arrow (the 11th day after infection). (C) Pulmonary hemorrhage and lobular atrophy, indicated with blue arrow (the 15th day after infection). (D) Lung of the control group. (E) Spleen swelling (the 5th day after infection). (F) The spleen of the control group.

Figure 4

Lung, Spleen and Trachea sections of mice after hematoxylin and eosin (H&E) staining. (A) 11 days after infection, the lungs of mice showed () marked congestion and hemorrhage, and () alveolar septal thickening (400×). (B) Normal control lung sections from mock-inoculated mice did not show observable histological changes (400×). (C) 5 days after infection, the spleens of mice showed () blurred red-white medullary demarcation (400×). (D) Normal control spleen sections from mock-inoculated mice did not show observable histological changes (400×). (E) 13 days after infection, the tracheas of mice showed () detached epithelial cells (400×). (F) Normal control trachea sections from mock-inoculated mice showing no observable histological changes (400×).

Figure 5

Immunohistochemistry of lung, Spleen and Trachea sections of mice (400×). Representative histological sections from lung tissue of mice at 3 days PI. (A) lung tissue of control. (B) Spleen tissue at 5 days PI. (C) Spleen tissue of control. (D) Trachea tissue at 13 days PI. (E) Trachea tissue of control. (F) were stained with immunohistochemical (IHC) using antibody specific for BAdV-3. Positive signals were indicated with black arrows.

Figure 6

Results of viral titers (TCID₅₀) and viral loads in various organs and blood at different infection time. Virus titration and viral load changes in the hearts (A), livers (B), spleens (C), lungs (D), kidneys (E), trachea (F), and blood (G).