The Mucin-Like Domain of the Ebola Glycoprotein Does Not Impact Virulence or Pathogenicity in Ferrets

Abstract

Background

Ebola virus (EBOV) is considered among the most dangerous viruses with case fatality rates approaching 90% depending on the outbreak. While several viral proteins (VPs) including VP24, VP35, and the soluble glycoprotein are understood to contribute to virulence, less is known of the contribution of the highly variable mucin-like domain (MLD) of EBOV. Early studies have defined a potential role in immune evasion of the MLD by providing a glycan shield to critical glycoprotein residues tied to viral entry. Nonetheless, little is known as to what direct role the MLD plays in acute EBOV disease (EVD).

Methods

We generated an infectious EBOV clone that lacks the MLD and assessed its virulence in ferrets compared with wild-type (WT) virus.

Results

No differences in growth kinetics were observed in vitro, nor were there any differences in time to death, viremia, or clinical picture in ferrets infected with recombinant EBOV (rEBOV)–WT or rEBOV-Δmucin.

Conclusions

The EBOV MLD does not play a critical role in acute pathogenesis of EVD in ferrets.

Ebola virus (EBOV) is considered among the most dangerous viruses, with case fatality rates approaching 90% depending on the outbreak [1]. While several virulence factors such as viral protein (VPs) VP35 and VP24 have been identified to interfere with innate immune function, the viral glycoprotein has been implicated as a viral determinant of EBOV pathogenicity [2]. Early observations by Yang et al demonstrated ex vivo cytotoxic effects of endothelial cells induced by EBOV glycoprotein (GP) in human, nonhuman primate (NHP), and porcine blood vessels [3]. The loss of endothelial cells and resulting vascular permeability induced by the EBOV glycoprotein was mapped to the serine-threonine–rich, mucin-like domain (MLD) contained within the GP1 subunit of the VP [3]. Serial euthanasia studies of EBOV in NHPs demonstrated endothelial cell infection late in the course of disease, suggesting that virally induced endothelial toxicity may not be central to pathogenesis [4]; yet no in vivo studies have been performed to directly investigate the role of the GP1 MLD as a determinant of viral pathogenesis in an animal model.

The MLD of the viral GP is thought to be a major contributor to evasion of adaptive immunity development [5, 6]. This region is understood to be one of the most variable regions of the EBOV GP where it is believed to contribute to evasion of the host immune response or delay in the host immune response. Indeed, existing evidence suggest the MLD to function as a glycosylated shield that blocks the viral GP from antibody neutralization [7, 8].

The MLDs from different species of the genus Ebolavirus are considerably divergent, which implies this region may also play a conserved role in the varied virulence contributed by each species [9]. Removal of the MLD has supported the protective role of this region as seen when recombinant viruses expressing EBOV GP or those from other Ebolavirus species lacking the MLD can readily be neutralized using serum from convalescent plasma specimens from survivors of EBOV infection [10], suggesting that this region may not only contribute to altered virulence but is also a mechanism for understanding cross-neutralization across Ebolavirus species. Because of how closely they recapitulate human disease, NHPs have been invaluable for dissecting the events leading to death after EBOV infection [4, 11, 12]. Unfortunately, NHPs are also very demanding in terms of maximum containment space requirements and have become increasingly difficult to source due to international demand. Small animal models have proven critical for the development of medical countermeasures such as vaccines and therapeutics, and in some cases can also help to address questions related to filovirus pathogenesis [13]. Unfortunately, most rodent models of EBOV infection such as mice, hamsters, and guinea pigs require adaptation, which results in genomic changes that may also impact the course of disease [13]. Recently, a ferret model was developed that was well suited to study genomic contributions to pathogenesis as no adaptation was needed in order to produce lethal disease similar to that seen in humans and NHP [14, 15]. This study sought to understand the role of EBOV GP1 MLD in contributing to virulence by generating a full-length infectious clone lacking the MLD and comparing its virulence to a wild-type (WT) infectious clone in the ferret model [14], using comparable doses to those used in NHP studies.

METHODS

Recovery of Infectious Clones

The infectious clone plasmid, pTM-T7G-Ebo-Rib [16], which contains a T7 RNA polymerase promotor, a complementary DNA (cDNA) encoding the full-length genome of EBOV (Mayinga strain), and a ribozyme sequence, was used to recovered WT EBOV by reverse genetics. The WT plasmid was modified to lack nucleotides 6987–7439 in the cDNA genome corresponding to most of the MLD domain of the GP. Infectious viruses were generated using an established reverse genetics system [17, 18].

In Vitro Infection Kinetics

Human hepatoma (Huh) 7 cells were grown in 6-well cell culture dishes in Dulbecco's modified Eagle's medium (Gibco) 10% fetal bovine serum, 1× Glutamax (Invitrogen, Carlsbad, California), 50 U/mL penicillin (Thermo Fisher Scientific), and 50 μg/mL streptomycin (Thermo Fisher Scientific). Three wells of cells were infected at a targeted multiplicity of 0.1 for 1 hour, then washed gently 5 times with serum-free media to remove any unadhered virus. Two milliliters of complete media was added back and supernatants were collected at 24, 48, 72, and 96 hours postinfection and frozen at −80°C for subsequent titration as described in methods for detection of viremia.

Ethics Approval and Institutional Oversight

Animal studies were performed at the Galveston National Laboratory, University of Texas Medical Branch at Galveston (UTMB) and were approved by the UTMB Institutional Animal Care and Use Committee (IACUC). This facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Animal Challenge, Disease Monitoring, and Biological Sampling

Six female ferrets weighing 0.75–1 kg were housed 3 per cage separated by virus isolate. Ferrets were anesthetized by inhalational isoflurane to effect. Prior to challenge, transponder chips (BioMedic Data Systems) were subcutaneously implanted for identification and temperature monitoring. Subjects were challenged intranasally with 1000 plaque-forming units (PFU) of either the wild-type recombinant EBOV (rEBOV-WT; n = 3) or rEBOV-Δmucin (n = 3). Whole blood, ethylenediaminetetraacetic acid (EDTA) plasma, and citrated plasma samples were collected from superior vena cava for hematology, serum biochemistry, and viremia determination on day 0, day 3, and at time of euthanasia. Clinical signs, weights, and transponder-mediated temperatures were recorded daily up to the point of euthanasia. Clinical scores were determined on a scale of 0–12 based on coat appearance, social behavior, and provoked behavior. Animals scoring ≥9 were euthanized per UTMB IACUC protocol criteria.

Gross pathology findings were documented and portions of select tissues were aseptically removed and frozen at −70°C for virus infectivity assays. Portions of select tissues were also fixed in formalin and processed for histology and immunohistochemistry.

Hematologic and Serum Biochemical Analysis

Total white blood cell counts, white blood cell differentials, granulocyte counts, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing EDTA, using a laser-based hematologic analyzer (Hemavet). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, glucose, cholesterol, total protein, total bilirubin, blood urea nitrogen, creatinine, and C-reactive protein by using a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis).

Detection of Viremia

Determination of infectious EBOV in plasma, spleen, liver, kidney, adrenal, pancreas, lung, and brain were made using conventional plaque assays of plasma or tissue homogenates. In brief, for tissues 10% homogenates were prepared in Eagle's minimum essential media containing 5% bovine serum albumin, then clarified by centrifugation. Homogenates and plasma were then assayed in increasing 10-fold dilutions through adsorption to Vero E6 cell monolayers in duplicate wells (200 μL) and stained with 5% neutral red containing phosphate-buffered saline before counting. The limit of detection was 25 PFU/mL of plasma and 250 PFU/g of tissue.

Preparation of Histopathological Materials

Tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. Slides went through heat antigen retrieval in a steamer at 95°C for 20 minutes in Sigma citrate buffer, pH 6.0, 10× (Sigma-Aldrich, St Louis, Missouri). To block endogenous peroxidase activity, slides were treated with 3% hydrogen peroxide and rinsed in distilled water. The tissue sections were processed for immunohistochemistry (IHC) using the Thermo Autostainer 360 (ThermoFisher, Kalamazoo, Michigan). Sequential 15-minute incubations with avidin D and biotin solutions (Vector Laboratories, Burlingame, California; #SP-2001) were performed to block endogenous biotin reactivity. Specific anti-Ebola Zaire VP40 immunoreactivity was detected using an anti-Ebola Zaire VP40 primary antibody (Integrated Bio Therapeutics, Rockville, Maryland) at a 1:4000 dilution for 60 minutes. Secondary antibody used was biotinylated goat antirabbit IgG (Vector Laboratories; #BA-1000) at 1:200 for 30 minutes followed by Vector horseradish peroxidase streptavidin, ready to use (Vector Laboratories; #SA-5704) for 30 minutes. Slides were developed with Dako DAB chromogen (Dako, Carpinteria, California; #K3468) for 5 minutes and counterstained with hematoxylin for 45 seconds.

Statistical Analysis

Conducting animal studies in a biosafety level 4 facility severely restricts the number of animal subjects, the volume of biological samples that can be obtained, and the ability to repeat assays independently and, thus, limits the power of statistical analyses. Consequently, data are presented as the mean calculated from replicate samples, not replicate assays, and error bars represent the standard deviation across replicates. When feasible, statistical analysis (eg, comparisons of survival and viral burden) were made using the GraphPad Prism 9.4.1 program and the analytical packages contained within. Animal numbers per group were minimized to 3 per group in order to utilize the minimum number of animals to create a signal of variation to help guide future experiments.

RESULTS

Virus Recovery and In Vitro Kinetics

The highly glycosylated MLD spans residues 313–501 of the GP. Attempts to generate an infectious clone with the entire MLD deleted failed after 3 attempts. Adding back amino acid residues of the MLD resulted in the generation of an infectious clone lacking the majority of the MLD (a 151 amino acid deletion corresponding to nucleotides 6987–7439 [amino acids 316–467] in the GP) along with an infectious clone of the original 1976 isolate of EBOV, variant Mayinga. Both infectious clones were generated by established reverse genetics approaches previously described [17, 18]. Plaque purified infectious clones were expanded with a single additional passage in Vero-76 cells for use in subsequent in vitro and in vivo studies. A head-to-head growth curve analysis was completed in an interferon-competent liver cell line (Huh7) but showed no significant differences over the course of 96 hours (Figure 1A).

Figure 1.

In vitro and in vivo characterization of Ebola virus (EBOV) and the EBOV mucin-like domain (MLD). A, Growth kinetics of wild-type recombinant EBOV (rEBOV-WT) and rEBOV-MLD in HUH-7 cells. B, Survival of ferrets infected with rEBOV-WT and rEBOV-Δmucin. C, Plasma viremia from ferrets infected with rEBOV-WT and rEBOV-Δmucin. D, Infectious virus burden in tissues from ferrets taken at time of euthanasia from animals infected with rEBOV-WT and rEBOV-Δmucin. *P ≤ .05. Abbreviations: Fr, frontal lobe; hpi, hours postinfection; ns, not significant; PFU, plaque-forming units; rEBOV-WT, wild-type recombinant Ebola virus.

Ferret Challenge

In a narrowly focused study to initially assess the virulence of rEBOV-Δmucin in vivo, 3 ferrets were challenged intranasally with 1000 PFU of while a positive control group of 3 ferrets were challenge in parallel with rEBOV-WT. All 6 ferrets enrolled in the study met euthanasia criteria between days 4 and 5 after challenge (Figure 1B). With the exception of liver (P = .013, paired t test) and kidney (P = .031, paired t test), there were no discernable differences in any parameter measured in viral burden (Figure 1C and 1D) or hematological or serum biochemistry. All animals demonstrated hallmark features of EBOV disease (EVD) including marked lymphocytopenia, neutrophilia, thrombocytopenia, hypoalbuminemia, and elevated liver enzyme levels with no differences between the rEBOV-WT and rEBOV-Δmucin groups (data not shown).

Pathology

All ferrets enrolled in the study displayed gross lesions consistent with EVD. All ferrets had mild to moderate petechial rashes (Figure 2D and 2J), hepatitis (Figure 2A and 2G), lymphadenomegaly, splenomegaly, pulmonary congestion, and meningeal congestion. Mucosal and serosal reddening of the proximal duodenum were noted in 3 of the 6 ferrets (2 rEBOV-WT and 1 rEBOV-Δmucin). Histology and IHC supported the gross findings of EVD. All ferrets had centrilobular vacuolar degeneration and necrosis with sinusoidal mixed leukocytosis and rarely vasculitis (Figure 2B and 2H). EBOV antigen was detected via IHC of hepatocytes, endothelium, and rarely biliary duct epithelium (Figure 2C and 2I). Marked destruction of splenic germinal centers with lymphocytolysis and hemorrhage was evident along with rare multifocal necrosis and poorly arranged fibrin within the red pulp (Figure 2E and 2K). IHC-positive mononuclear cells and endothelium were noted throughout the red and white pulp of the spleen (Figure 2F and 2L). Similar necro-inflammatory lesions were noted in adrenal glands in which mixed inflammatory infiltrates and degeneration with occasional necrosis were noted in the cortex and medulla. Rarely, vasculitis and partially occluded vessels with fibrin were noted in the adrenal medulla of all ferrets.

Figure 2.

Representative gross pathology and histopathology of ferrets challenged with wild-type Ebola virus (EBOV) (A–F) and recombinant EBOV-Δmucin (G–L). A, Multifocal to coalescing hepatitis. B, Centrilobular vacuolar degeneration and necrotizing hepatitis (*) (hematoxylin and eosin staining [H&E], ×20 magnification). C, Immunohistochemistry (IHC) positive (brown) for anti-EBOV antigen of hepatocytes and endothelium (white arrow) (IHC, ×20). D, Petechial rash of the lower ventral abdomen (black arrows). E, Splenitis with lymphocytolysis of germinal centers (H&E, ×10). F, IHC positive (brown) for anti-EBOV antigen of mononuclear cells of the red and white pulp and endothelium (IHC, ×10). G, Multifocal to coalescing hepatitis. H, Centrilobular vacuolar degeneration and necrotizing hepatitis (*) and blood vessel (white arrow) (H&E, ×20). I, IHC positive (brown) for anti-EBOV antigen of hepatocytes and blood vessel endothelium (white arrow) (IHC, ×20). J, Petechial rash of the lower ventral abdomen (black arrows). K, Splenitis with lymphocytolysis of germinal centers and the periarteriolar sheaths (H&E, ×10). L, IHC positive (brown) for anti-EBOV antigen of mononuclear cells of the red and white pulp and endothelium (IHC, ×10). Full color version available online.

DISCUSSION

EBOV infection can cause widespread inflammation and cellular damage resulting in vascular abnormalities. Previous in vitro and ex vivo reports have demonstrated a role of the viral glycoprotein in mediating cellular toxicity as seen by endothelial cell death and increased vascular permeability [3]. The mechanism behind this cytotoxic phenotype can be partially attributed to the dysregulation of cellular adhesion molecules [19] and have been mapped to the MLD of the glycoprotein [3]. In addition, the MLD serves as an antigenic shield with a function to protect critical GP epitopes [8]. However, in our current in vivo studies, we provide experimental evidence suggestive of a limited role for the EBOV MLD to contribute to virulence in vivo. Using infectious clones with or without the EBOV MLD, we discerned no overt differences with respect to time to death, viremia, or clinical profile. However, our infectious clone lacks the majority of the MLD since we were unable to generate a virus lacking the complete MLD. Though unlikely, these remaining 3–4 amino acids on both end of the MLD could potentially play a role in viral pathogenesis.

In vitro growth kinetics in an interferon-competent human liver cell line denoted no growth advantages over the course of 96 hours. Comparing viremia and viral burden in tissues noted only statistical differences in infectious virus levels in the liver and kidney with higher levels in animals infected with rEBOV-Δmucin; however, given that these differences were within a log of infectious virus content, we cannot rule out sampling bias tied to tissue portion selection at the time of necropsy contributing to the observed differences. Furthermore, while we did observe evidence of endothelial infection in ferrets that succumbed to challenge, we could not discern any overt differences between the experimental groups in terms of severity.

Given the rapid onset of disease and short time to death, it is unlikely that differences in endothelial infection between the WT and MLD deletion group were present. It is also unlikely that we would have observed differences in the development of potentially protective adaptive immune responses as the mean time to generate such responses is more on the order of 10–12 days in most systems. Thus, we can conclude that the remaining unaltered virulence factors such as VP24, VP35, or the various soluble or shed permutations of GP likely worked in concert to result in an unadulterated pathophysiology. Furthermore, while this study was narrowly focused to assess virulence differences at a single challenge dose, determination as to whether pathogenic differences might arise at lower challenge doses remains a question. While these findings do not discern a clear contribution of the EBOV MLD to lethality in acute disease, it may be that contributions of the MLD provide an added barrier to immunity later in disease once adaptive immunity begins to become established.

Recently, several studies have generated MLD deletion-based vaccine approaches in attempts to capitalize on increased access to critical entry mediating epitopes [20–23]; however, these approaches have yet to demonstrate protective efficacy in animal models. Interestingly, 2 different vaccination approaches, 1 DNA based [24] and 1 using a recombinant vesicular stomatitis virus (VSV)–vectored approach [25], independently demonstrated reduced protective efficacy in mice using MLD deletion-based glycoprotein antigens, suggesting a necessary role for the MLD to appropriately shape robust protective immunity. Indeed, both vaccine approaches approved for use in humans, Ervebo (VSV) and Zabdeno (Ad26.ZEBOV)/Mvabea (MVA-BN-Filo), employ an intact MLD within the encoded GP, further underscoring the value of this region. This work represents the first examination of the direct role of the MLD domain on pathogenicity of EBOV and may be critical when considering refinement of vaccine or therapeutic efficacy against EBOV and related filoviruses.

Notes

Author contributions. R. W. C., P. J. H., Y. K., and T. W. G. conceived and designed in vitro and animal challenge experiments. C. B. L. and C. E. M. recovered infectious clones used in this work. R. W. C. and V. B. performed the animal procedures and conducted clinical observations. C. B. L. and V. B. performed virological and clinical pathology assays. K. A. F. performed gross pathologic, histologic, and immunohistochemical analysis of the data. All authors analyzed the data. R. W. C. and P. J. H. wrote the manuscript. Y. K. and T. W. G. edited the manuscript. All authors had access to the data and approved the final version of the manuscript.

Acknowledgments. The authors thank the University of Texas Medical Branch at Galveston (UTMB) Animal Resource Center for husbandry support of laboratory animals. We also thank Natalie Dobias for assistance with histopathology preparations.

Disclaimer. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the University of Texas Medical Branch.

Financial support. This study was supported by funds provided by the Department of Microbiology and Immunology, UTMB (to T. W. G.) and the Systems Biology Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant number U19AI106772 to Y. K.).

Supplement sponsorship. This article appears as part of the supplement “10^th International Symposium on Filoviruses.”