Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus)

Abstract

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70-90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer (Moschussifanicus). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

Keywords: Alpine musk deer (Moschus sifanicus); China; isolation; rabbit hemorrhagic disease virus (RHDV).

Figure 1

Macroscopic lesions observed in diseased Alpine musk deer. A dead Alpine musk deer (A) and common signs of hyperemia, hemorrhage, and necrosis observed in the lung (B), stomach and intestine (C), spleen (D), liver (E), and trachea (F).

Figure 2

Histopathological lesions observed in diseased Alpine musk deer. Lung (A): moderate, diffuse congestion in alveolar capillary and blood vessels, extensive hemosiderin deposition, and dilation of bronchioles; liver (B): vacuolar degeneration of hepatocytes in the central zone of the lobule, karyopyknosis and karyolysis in some hepatocytes, and mild sinusoidal dilation and congestion; spleen (C): disorganization of the splenic architecture, extensive necrosis and atrophy of the white pulp with severe lymphocyte depletion, and moderate hemorrhage in the parenchyma; kidney (D): congestion of interstitial blood vessels, microthrombosis in the glomerular capillaries, and degeneration and karyopyknosis in numerous tubular epithelial cells. (H&E, 100×).

Figure 3

Detection of RHDV VP60 signals in the livers of the dead Alpine musk deer by immunohistochemistry. Incubation with the primary antibodies against VP60 (A), positive signals are indicated by arrow; incubation without the primary antibody as the control (B).

Figure 4

Electron micrograph of calicivirus-like particles from the liver homogenate of diseased Alpine musk deer. Bar: 50 nm.

Figure 5

Evolutionary relationships of the new RHDV isolate and reference strains based on the nucleotide sequences of the full-length genome (A) and VP60 (B). The evolutionary history was inferred using the Neighbor-Joining method implemented in MEGA7 and tested using 1000 bootstrap replicates. Only bootstrap values equal to or greater than 70 are shown. Each sequence is labeled with its GenBank accession number and name beside the branch, with an additional black dot for the new and black triangles for the Chinese strains.

Figure 6

Macroscopic signs observed in rabbits inoculated with the RHDV strain GS/YZ. Lung (A): pulmonary hyperemia, and carnification. Liver (C): friable, fatty, and discolored liver. Kidney (E): necrotic foci on the kidney. The lung (B), liver (D), and kidney (F) of uninfected rabbits.

Figure 7

Histopathological changes in rabbits infected with the RHDV strain GS/YZ. Lung (A): moderate, diffuse congestion in the alveolar capillary and blood vessels, extensive hemosiderin deposition, and dilation of bronchioles. Liver (B): vacuolar degeneration of hepatocytes in the central zone of the lobule, karyopyknosis and karyolysis in some hepatocytes, and mild sinusoidal dilation and congestion. Spleen (C): disorganization of the splenic architecture, extensive necrosis and atrophy of the white pulp with severe lymphocyte depletion, and moderate hemorrhage in the parenchyma. Kidney (D): congestion of interstitial blood vessels, microthrombosis in the glomerular capillaries, and degeneration and karyopyknosis in numerous tubular epithelial cells [33,34].

Figure 8

Detection of RHDV VP60 signals in the livers of the rabbits inoculated with the tissue homogenates of the dead Alpine musk deer by immunohistochemistry. Incubation with the primary antibodies against VP60 (A); incubation without the primary antibody as the control (B).