Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors

Abstract

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Fig 1

Replication kinetics of CYN07-hV in various cells. Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, Vero/dNectin4, Vero/macNectin4, Vero/hNectin4, and parental Vero cells were infected with CYN07-hV at an MOI of 0.01, and the titers were determined at the indicated time points. Filled circles, squares, triangles, and diamonds indicate the growth kinetics in Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, and parental Vero cells, respectively. Open circles, squares, and triangles indicate the growth kinetics in Vero/dNectin4, Vero/macNectin4, and Vero/hNectin4 cells, respectively. The data shown are the means of three independent assays, with the error bars representing the standard deviations. d.p.i., days postinfection.

Fig 2

Syncytium induction in various cells upon infection with CYN07-hV and CYN07-dV. Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, Vero/dNectin4, Vero/macNectin4, Vero/hNectin4, and Vero cells were infected with CYN07-hV and CYN07-dV at an MOI of 0.01, cultured, and observed under a phase-contrast microscope. Syncytia induced in Vero.DogSLAMtag, Vero/macSLAM, and Vero/hSLAM cells at 24 h p.i., in Vero/dNectin4, Vero/macNectin4, Vero/hNectin4 cells at 36 h p.i., and in Vero cells at 36 h p.i. are shown.

Fig 3

Cell-to-cell fusion induction in cells expressing the CDV CYN07-dV or CYN07-hV H and F proteins. Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, Vero/dNectin4, Vero/macNectin4, and Vero/hNectin4 cells were transfected with a mixture of plasmids encoding enhanced green fluorescent protein (EGFP) (pEGFP-C1) and CDV F (pGAGGS-CYN-F) and H (pCAGGS-CYN-hV-H or pCAGGS-CYN-dV-H) proteins, cultured, and observed using a fluorescence microscope. (A, C) Fluorescence microscopic images of SLAM-expressing cells at 14, 24, and 36 h posttransfection (A) and those of nectin-4-expressing cells (C) at 24, 36, and 72 h posttransfection are shown. (B, D) The percentages of fluorescence areas in the total microscopic field were measured by using imageJ software (version 1.36b, NIH). White and black bars indicate data for experiments using pCAGGS-CYN-hV-H and pCAGGS-CYN-dV-H, respectively. Gray bars indicate data without the H-encoding plasmid. The data shown are the means of three independent assays (36 h posttransfection for SLAM-expressing cells [B] and 72 h posttransfection for nectin-4-expressing cells [D]), with the error bars representing the standard deviations.

Fig 4

Infectivities of pseudotyped VSVs bearing the CDV H and F proteins. Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, Vero/dNectin4, Vero/macNectin4, Vero/hNectin4, and parental Vero cells were infected with EGFP-expressing VSVs pseudotyped with the H and F proteins of CYN07-hV and CYN07-dV (VSVΔG*-F-hVH and VSVΔG*-F-dVH, respectively) and with the F protein alone (VSVΔG*-F). The numbers of cells expressing EGFP were counted at 24 h p.i., and the infectivity titers of each pseudotype in the cells were calculated. The data shown are the means of three independent assays, with the error bars representing the standard deviations.

Fig 5

Effects of the P541S substitution on the cell-to-cell fusion-supporting function of different CDV H proteins. Vero.DogSLAMtag, Vero/hSLAM, and parental Vero cells were transfected with a mixture of plasmids encoding EGFP (pEGFP-C1), CDV F protein (pGAGGS-CYN-F), and different H proteins as follows: H proteins of the CYN07-hV, MSA5, and Ac96I strains and those possessing the P541S mutation (the H protein of CYN07-dV possessing the P541S mutation corresponds to the CYN07-hV H protein). The cells were then cultured and observed using a fluorescence microscope. Cell fusion induced at 36 h posttransfection is shown.

Fig 6

Syncytium induction in various cells upon infection with CYN07-macV. Vero.DogSLAMtag, Vero/macSLAM, Vero/hSLAM, Vero/dNectin4, Vero/macNectin4, Vero/hNectin4, and Vero cells were infected with CYN07-macV at an MOI of 0.01, cultured, and observed under a phase-contrast microscope. Syncytia induced at 36 h p.i. are shown.