Nipah virus uses leukocytes for efficient dissemination within a host

Abstract

Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.

Fig. 1.

NiV replication in human leukocytes. (A) Kinetics of the expression of NiV N, M, F, G, and L genes in PBLs, DC, macrophages, and U373 cells infected with NiV after analysis by RT-qPCR. (B) NiV-EGFP replication in human cells 48 h p.i. observed under light and fluorescence microscopes. (C) Kinetics of NiV production by human leukocytes. The results are from 1 representative experiment out of 2 to 5 performed with different blood donors.

Fig. 2.

Expression of cellular receptors for NiV by human leukocytes. (A and B) Expression of ephrinB2 (A) and ephrinB3 (B) mRNAs in human leukocytes. The results are presented as copy numbers of ephrinB2 or ephrinB3/μg of RNA, normalized with GAPDH; the average was calculated for 3 to 5 donors for each cell type. MACRO, macrophage; MONO, monocyte. (C) Transduction of human leukocytes with GFP-encoding MLV-based pseudotyped virus bearing either NiV glycoproteins G and F or VSV attachment protein G (MOI = 1) and observed under light (left) and fluorescence (right) microscopes at 72 h p.i. (D) Cells were purified from human blood, DC, and macrophages generated from monocytes and put in contact with NiV for 1 h, washed twice, harvested for RNA isolation and determination of the cell-retained NiV genome material by RT-qPCR, and analyzed as described in Materials and Methods.

Fig. 3.

Transinfection capacity of human leukocytes. (A) Experimental protocol. PBLs, DC, or monocytes were infected with NiV-EGFP at either 37°C or 4°C in the presence or absence of EIPA, an inhibitor of NiV entry; washed; and returned to culture. (B) Light and fluorescence observation after 4 days of coculture of NiV-infected DC or PBLs (10⁵ cells/well) for 24 h with endothelial HPMEC as cell indicators. (C) Kinetics of cell-free NiV production by PBLs and DC infected at 4°C or 37°C in the presence or absence of EIPA. Supernatants were taken at different time points and titrated by plaque assay. (D) Amounts of infectious NiV associated with PBLs, DC, or monocytes collected at 24 or 96 h p.i. and measured by infectious-center assay after 72 h of coculture with Vero cells.

Fig. 4.

Characterization of lymphocyte-mediated transinfection. (A) Experimental protocol; lymphocytes were treated or not with trypsin, pronase, EGTA, or EphB4-Fc and then infected with NiV, washed, and further cultured for 24 to 120 h. Lymphocyte-bound NiV was additionally analyzed after treatment with pronase or anti-NiV antibodies. (B) Stability of NiV in culture. Lymphocytes (10⁶) were incubated with NiV (10⁶ PFU) for 1 h and then washed twice before further incubation in 2 ml of medium (grey bars). As a control, the same amount of virus was directly incubated in 2 ml of medium in the absence of any cells (black bars). The virus titer was determined after the indicated periods of culture by plaque essay using a Vero cell monolayer. The data are expressed as percentages of the initial titer determined after 1 h of incubation of either free virus or cell-bound virus. (C) Levels of infectious NiV bound to PBLs, purified CD3⁺ T cells, and CD19⁺ B cells after 24 h and 96 h of incubation as measured by infectious-center assay. (D) Lymphocytes were infected as in panel B, and CHO cells were infected as an adherent cell monolayer and detached with trypsin 24 h after infection. Cell-bound infectious virus was determined after coculture with a Vero cell monolayer. The data are expressed as PFU/ml and presented as averages of 3 different experiments using lymphocytes from different donors. (E) Sensitivity of NiV binding ability of PBLs to pretreatment with either pronase, trypsin, EGTA, or soluble EphB4-Fc. (F) Sensitivity of NiV bound to PBLs to stripping with pronase or neutralization by anti-NiV MAbs. The results in panels D to F are expressed as percentages of inhibition compared to untreated lymphocytes. The vertical bars indicate standard deviations.

Fig. 5.

Transinfection of NiV ex vivo and in vivo. (A and B) Hamster splenocytes are not permissive to infection in vitro with rNiV-EGFP, as observed under light (A) and fluorescence (B) microscopes at 4 days p.i. (C to I) Hamsters were infected with 10³ PFU of rNiV-EGFP, and either blood or spleen leukocytes were taken and analyzed. (C) The titer of NiV bound to blood cells taken from infected hamsters was measured by incubating the cells with a Vero cell monolayer for 4 days. The results are presented as numbers of PFU obtained from 1 to 4 animals for each time point, and the error bars correspond to standard deviations. (D to G) Spleens were taken from rNiV-EGFP-infected hamsters 4 days p.i., mononuclear leukocytes were isolated, and 10⁶ cells were cultured either alone (D and E) or with Vero cells (F and G) and observed by fluorescence microscope by 4 days p.i. (H) In parallel, 25 × 10⁶ purified spleen leukocytes isolated either from rNiV-EGFP-infected hamsters (circles) or from noninfected hamsters (triangles) were injected i.p. into naïve animals. Control animals received 10³ PFU of rNiV-EGFP i.p. (squares), and all animals were followed for signs of infection for 2 weeks. The results are expressed as percentages of surviving animals in each group.

Fig. 6.

Model of NiV dissemination within a host. (1) NiV initially enters the host using an ill-defined crossing pathway and subsequently infects local DC within the epithelium of the digestive or respiratory tract. (2) Infected DC migrate via lymphatic vessels to draining lymphoid organs. (3) There, they produce NiV particles, which can bind to surrounding lymphocytes. (4) Lymphocytes enter blood vessels, where they continue interacting and rolling over the endothelium. This intimate and dynamic contact favors the transfer of the lymphocyte virus load to the endothelial cells (E.C.), which initiate virus amplification and infection of the underlying smooth muscle cells (S.M.), a characteristic histopathological feature of NiV infection (20). (5) From the infected vessels, NiV invades underneath tissues and organs that are permissive to the infection. Such a mechanism may explain virus passage through the blood brain barrier and the development of fatal encephalitis.