Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008

Abstract

Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Fig 1

Histopathological analyses of cynomolgus monkeys naturally infected with CDV in the 2008 outbreak. Tissue sections obtained from cynomolgus monkey 11 were examined by hematoxylin and eosin staining (HE) and immunohistochemistry (IHC) using anti-CDV-NP monoclonal antibody. Giant cell pneumonia (A) and CDV antigen in the syncytial pneumocytes (B) were seen in the lung. Lymphocyte depletion (C) and CDV antigen in the mononuclear cells (D) were observed in the lymph node. Focal and slight microglia cell infiltration (E) and CDV antigen in neurons and glia cells (F) were observed in the cerebrum. HE, original magnification ×20; IHC, ×40.

Fig 2

Double immunofluorescence staining of tissues of cynomolgus monkey 11 naturally infected with CDV in the 2008 outbreak. Tissue sections obtained from cynomolgus monkey 11 were examined by double immunofluorescence staining with anti-CDV-NP antibody and rabbit anti-cytokeratin, rabbit anti-neuron-specific β III tubulin, rabbit anti-CD3, or goat anti-nectin4 antibody. CDV-NP antigens were detected in the bronchi, bronchiole, lymph node, and brain. Some CDV-NP antigen-positive cells were positive for cytokeratin, nectin4, or CD3. A few CDV-NP antigen-positive neurons were positive for βIII tubulin.

Fig 3

Phylogenetic tree of CDV based on the CYN07-dV sequence. Phylogenetic analysis of the H protein of the CDV showed that the CYN07-dV was closely related to the CDV strains isolated from rhesus monkeys in China. Scale bars indicate phylogenetic distance between CDV isolates.

Fig 4

Changes in body weight, rectal temperature, and the numbers of white blood cells, lymphocytes, and CDV-infected PBMCs and neutralization antibody level in the experimentally CDV-infected monkeys. (A) Body weight; (B) rectal temperature; (C) white blood cell count; (D) lymphocytes count; (E) CDV-infected PBMC count; (F) neutralizing antibodies against CDV.

Fig 5

Histopathological analyses of cynomolgus monkeys experimentally infected with CDV. Tissue sections obtained from cynomolgus monkey 4965 were examined by hematoxylin and eosin staining and immunohistochemistry using anti-CDV-NP monoclonal antibody. Giant cells (A) with CDV antigen (B) were observed in the alveolar area in the lung. Lymphocyte depletion (C) and CDV antigen-positive cells were observed in the follicular area (D) in the lymph node of CYN07-dV-infected monkey 4965. HE, ×20; IHC, ×40.

Fig 6

CYN07-dV utilizes macaca SLAM and macaca nectin4 as receptors. (A) Growth kinetics of CYN07-dV in Vero.DogSLAMtag, Vero/hSLAM, Vero/macSLAM, Vero/dNectin4, Vero/macNectin4, and Vero cells. The cells were infected with the virus at an MOI of 0.01, and titers at the indicated points were shown. (B) Induction of syncytium upon transfection with a mixture of plasmids expressing enhanced green fluorescent protein (EGFP) and CDV F with or without a plasmid expressing CDV H. (C) Infectivity of VSV pseudotype bearing H and F proteins of CYN07-dV (VSVΔG*-F-dVH) or that bearing the F protein (VSVΔG*-F) in Vero.DogSLAMtag, Vero/hSLAM, Vero/macSLAM, Vero/dNectin4, Vero/macNectin4, and Vero cells.