Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions

Abstract

Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFN-α and IFN-β, IFN-γ was demonstrated to control filovirus infection in bat cells but not in human cells, suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which were detected in human cells ectopically expressing bat IFN-α and IFN-β. As bat IFN-γ induced the type I IFN pathway, its antiviral effect is likely to be partially induced via cross talk.IMPORTANCE Bats serve as reservoirs for multiple emerging viruses, including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg or Ebola viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution.

Keywords: Ebola virus; Marburg virus; accidental host; bat; immune evasion; interferon-inhibiting domain; interferons; natural host.

FIG 1

Innate immune genes of Egyptian rousette bats demonstrate only a limited sequence identity to human counterparts. A phylogenetic neighbor-joining tree of IFN-γ mRNA is shown. Bootstrap values (1,000 replicates) are present at the nodes, and sequence generated during this study is shown in red.

FIG 2

Bat type I IFNs induce innate immune responses in both bat and human cells, while bat type II IFN induces responses only in bat cells. Uninfected cells were transfected with plasmids expressing IFNs as indicated under each plot. Shown are fold changes of selected transcripts 24 h posttransfection compared to cells transfected with the empty plasmid vector. Red arrows indicate the transfected genes whose concentration reflects both RNA resulting from gene expression and the residual plasmid DNA that could not be removed efficiently by DNase I treatment. Mean values with SD based on triplicate samples are shown. (A) RO6EJ cells; (B) RoNi/7 cells; (C and E) HepG2 cells; (D and F) 293T cells.

FIG 3

Susceptibility of Egyptian rousette and human cells to wt and mutated EBOV and effects of overexpressed bat IFNs. Cell monolayers in six-well plates were transfected with either empty vector (mock) or plasmids expressing IFN-α, IFN-β, or IFN-γ of Egyptian rousette bats, and 24 h later they were infected with the indicated viruses at an MOI of 2 PFU/cell. After a 1-h-long adsorption at 37°C, cells were washed 3 times with PBS and fresh medium was added to the wells. The photographs were taken 24 h postinfection using a fluorescence microscope with a 20× objective.

FIG 4

Susceptibility of human 293T cells to wt and mutated EBOV, effects of overexpressed bat IFNs, and induction of innate immune response genes. The experiment was performed as described in the legend to Fig. 3. (A) Cell monolayers at 24 h postinfection. The images were taken using a fluorescence microscope with a 20× objective. (B) Relative copy numbers of wt EBOV genome determined by qRT-PCR. Mean values ± SD based on triplicate samples are shown. (C) Expression of innate immune response genes after infection with wt EBOV and EBOV mutants.

FIG 5

Virus loads in the infected rousette and human cells at 24 and 48 h postinfection at an MOI of 2 PFU/ml. The experiment was performed as described in the legend to Fig. 3. Mean values ± SD based on triplicate samples are shown.

FIG 6

Virus loads in the infected rousette and human cells at 24 and 48 h postinfection at an MOI of 0.1 PFU/cell. The experiment was performed as described in the legend to Fig. 3, except that the MOI was different. Mean values ± SD based on triplicate samples are shown.

FIG 7

Induction of innate immune response to wt EBOV, MARV-Uga(h), and EBOV mutants with disabled IID in rousette and human cells. See the legends to Fig. 2 and 3 for explanations of the experimental procedure; cells were transfected with an empty plasmid vector rather than plasmids expressing IFNs. The vertical axis shows fold changes of gene expression compared to the level for noninfected cells. Mean values ± SD based on triplicate samples are shown.

FIG 8

Expression of ISG56 in rousette and human cells infected with NDV, two strains of EBOV, and two strains of MARV. See the legend to Fig. 3 for an explanation of the experimental procedure. The vertical axis shows fold changes of gene expression compared to the level for mock-infected cells. Mean values ± SD based on triplicate samples are shown.

FIG 9

Transcriptome analysis of RO6EJ and RoNi/7 cells infected with wild-type EBOV, MARV, or EBOV mutants with disabled IID. See the legend for Fig. 2 for an explanation of the experimental procedure. The map includes genes involved in innate immune responses which demonstrated differential expression compared to uninfected cells (mean values from two independent samples are shown). The scale bar shows fold changes of gene expression.

FIG 10

Human type I but not type II IFNs suppress filovirus infection in human cells. Monolayers in six-well plates were transfected with human IFNs or with the empty plasmid vector (mock), and 24 h later they were infected with the indicated viruses at an MOI of 2 PFU/cell. See the legend to Fig. 3 for an explanation of the experimental procedure. Mean values ± SD based on triplicate samples are shown.