The magnitude of the 2013–2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013–2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

The large number of human-to-human transmissions of EBOV Makona during the 2013–2016 EVD epidemic provided greater opportunity for EBOV to adapt to the human host than in any previous outbreak. As expected for an RNA virus, monitoring over the course of the EVD epidemic revealed mutations throughout the genome of EBOV Makona (). However, except for the mucin-like domain in the glycoprotein (GP), which is under diversifying selection by the host humoral immune system, most of the EBOV genome exhibited purifying selection (). Although most nonsynonymous mutations detected during the epidemic probably had little effect on viral fitness, it is possible that some of these mutations proliferated because they conferred an advantage to the virus. One such candidate is the clade-defining A82V substitution in EBOV Makona GP, which emerged at a time in the epidemic just before the number of EVD cases increased exponentially. This mutation is particularly intriguing because it is located in the receptor-binding domain of EBOV GP. Here we describe our efforts to determine whether GP-A82V conferred a replication advantage to the virus.

Although sociological and epidemiological factors were central to the 2013–2016 epidemic’s unprecedented scale (), researchers have also examined the possibility that genetic changes unique to EBOV Makona played a role. To date, experiments with EBOV Makona have not detected evidence for increased replication phenotypes. Studies in primates and in immunodeficient mice, for example, failed to detect increased virulence with EBOV Makona compared to EBOV Mayinga, the 1976 reference isolate (). Likewise, the EBOV Makona immunomodulatory proteins VP35 and VP24 inhibited interferon signaling to the same extent as the analogous proteins encoded by EBOV from previous outbreaks (). These studies, however, examined only the early reference EBOV Makona isolate from Kissidougou, Guinea (C-15), and did not address possible changes in the virus over the course of the epidemic.

What factors might have led to the emergence of Ebola in West Africa?.

Ebola virus (EBOV) is an enveloped filovirus with a 19-kb, negative-sense, single-stranded RNA genome that causes sporadic outbreaks of lethal hemorrhagic fever in humans (). EBOV was identified as a human pathogen in 1976 (). The high case-fatality rate and self-limited nature of EVD outbreaks suggest that EVD is a zoonosis (). Detection of anti-EBOV antibodies and EBOV RNA in several fruit bat species from central Africa makes them leading candidates for the animal reservoir (). Historically, Ebola virus disease (EVD) outbreaks have been geographically limited and resolved after at most a few hundred cases (). In contrast, the epidemic caused by the EBOV Makona variant was much larger: it began in Guinea in 2013 (), spread to Sierra Leone and Liberia in 2014, and infected more than 28,000 people before it was controlled in 2016 ().

Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus.

To better understand the association between GP-A82V and mortality, we modeled the fatality rates for both the ancestral and GP-A82V genotypes over a range of viral loads (transformed C(t) values) ( Figure 6 F). After correcting for differences in C(t) values, the adjusted odds ratio for GP-A82V remained above 1, but with a wide confidence interval (OR = 2.09, 95% CI [0.94, 4.64]). When we added multiple potential confounding factors, including geographic variation in base fatality rate and access to health care, to the model, they had little effect on the conclusion. We found only modest evidence of geographic variation ( Figure S1 ), while including the cumulative number of EVD cases per region (a proxy for EVD-associated burdens on the health care infrastructure) failed to improve model fit ( Figure S1 ). Taken together, these data suggest an increased risk of death in people infected with GP-A82V, but we cannot rule out that there is no independent effect of the mutation or demonstrate that the association is causal.

We fit a model that allowed each location to have its own base fatality rate (i.e., its own intercept) and also its own slope for the cumulative cases (cumCases) variable, in order to account for heterogeneity in how locations responded to demand for healthcare. We find that the variability in the data and possibly the relatively small number of observations lead to very broad estimates of the model parameters. While there seem to be differences in base fatality rate between Forecariah and Nzerekore for example, the overlap of confidence intervals precludes definitive conclusions.

We examined associations between viral load (as assessed by C(t) values), risk of death, and GP-82 allele and found that both viral load and possession of GP-A82V were significantly associated with higher mortality. Lower C(t) values (corresponding to higher viral loads) were highly predictive of negative outcome ( Figure 6 C), consistent with previous reports (). GP-A82V-infected individuals had slightly higher viral loads (lower C(t) value, Figure 6 D), but the difference was not statistically significant (Student’s t test: mean A = 20.07, mean V = 19.68, 95% CI for difference in the means [−0.69, 1.44]; p value = 0.49). Infection by virus with GP-A82V was associated with a significantly higher risk of death (raw odds ratio: 2.64, 95% CI [1.29, 5.38]; Figure 6 E).

To determine whether the GP-A82V mutation had detectable effect on viremia or mortality, we obtained EBOV genotype data from 56 locations in Guinea, Liberia, and Sierra Leone () where mortality and viral load information was also available. This data recapitulated the skew (82V > 82A) that was seen in EBOV GP genotype prevalence per country depicted in Figure 1 Figure 6 A). Further analysis was restricted to those cases for which data were available regarding all four parameters: viral load, mortality, sampling location, and viral genotype (). All but 15 of these 194 cases occurred in Guinea, so the analysis was further restricted to Guinea, with samples from 19 districts. After paring down the data, geographic differences in the prevalence of viral genotypes remained ( Figure 6 B).

(F) Depiction of the correlation between GP genotype and mortality, based on results of a binomial generalized linear model using C(t) values and GP genotype as covariates to predict case fatality rates over a range of viral loads (depicted by transformed C(t) values). C(t) values were transformed by subtracting the mean, dividing by two standard deviations and flipping the sign such that the value 0 in the graph corresponds to the average C(t) value and the transformed variable reflects viral load.

Plots in (C) and (D) show mean with the box covering from the second to third quartile (25%–75%) of samples, and the bars marking the 5% and 95% quantiles. Dots represent samples outside of the 95% probability region.

(D) Viral load information (as determined by C(t) values) in individuals infected with EBOV encoding either ancestral or A82V GP. This analysis used all observations for which C(t) values were measured (A82: n = 97; V82: n = 216).

(C) Association between patient viral load (as determined by C(t) values) and EVD-associated mortality. This analysis used all observations for which C(t) values were measured (n = 313).

(A and B) Spatial distribution of GP genotypes for all available EBOV Makona sequencing data (A) or Guinean isolates linked to information regarding clinical outcome and viral load (B). The data included in (B) were used in subsequent modeling analyses.

US Army Medical Research Institute of Infectious Diseases National Institutes of Health Integrated Research Facility–Frederick Ebola Response Team 2014–2015 Monitoring of Ebola virus Makona evolution through establishment of advanced genomic capability in Liberia.

Homology modeling was used to examine how EBOV GP-A82V and NPC1 loop 2 domain orthologs from the mammalian species tested here—as well as from bat species that are potential EBOV reservoirs—may alter virus-host interactions ( Figure 5 ). The EBOV GP-A82V mutation is located on the α1 helix that directly interacts with NPC1 loop 2 ( Figure 5 A), though residue 82 is located on the back side of the helix. In terms of how V82 is likely to affect the viral GP, our modeling suggests that the GP-A82V mutation will have little impact on the α1 helix backbone itself. Rather, the additional alkane branches in the side chain of valine compared to alanine may differentially impact neighboring amino acids such as the arginine at residue 85 ( Figures 5 B and 5C). R85 extends into a charged pocket present on the opposite side of the α1 helix to where NPC1 interaction occurs and the nitrogens of the terminal guanidinium group are likely to form ionic interactions with the sidechains of E178, Y109, and the backbone hydroxyl group of A76. On the host side, several amino acid differences in the NPC1 loop 2 domain among mammals are likely to impact filovirus susceptibility in a species-specific manner (). Specifically, a lysine is found at residue 499 in primates while all other species examined possess a non-polar residue here and several species, including mouse and dog, possess a tyrosine at 504 in place of phenylalanine ( Figures 5 D and 5E).

(D and E) NPC1 loop 2 amino acid (D) sequence alignments and (E) target-template homology models of cell species used in this study (rodents, green; other mammals, cyan; Figure 4 ) and bats (purple) compared to primates (orange). Highlighted in (C) are the amino acid differences in loop 2 from the target species compared to the template, humans, and their location compared to the GP-A82V variant.

(B and C) Zoom of the (B) ancestral GP-A82 (blue) and (C) derived GP-V82 (red) variants in relation to proximal amino acid side chains. The distance between R85 and E178 is close enough (i.e., <4 Å) where a salt bridge is possible.

(A) Structural overview of EBOV GP2 (yellow) and cleaved GP (GPcl, light blue) bound to human NPC1 (orange), as experimentally derived by. The EBOV GP-A82V substitution (red) occurs in the α1 helix of proteolytically cleaved GP that interacts with NPC1 loop 2.

Based on the NPC1 sequence alignments, we anticipated that infections using EBOV GP-A82V variants in non-human primate cells would result in a similar enhancement to that seen in human cells. To test this prediction, we transduced cells from several primate species, as well as carnivores and rodents, with GFP-expressing lentiviral vectors pseudotyped with the EBOV GPs. Indeed, cells from non-human primates were more susceptible to infection by GP-A82V ( Figure 4 C). In contrast, cells from rodents and carnivores showed no difference in infectivity between the ancestral EBOV GP and GP-A82V. These results demonstrate that GP-A82V provides primate-specific enhancement of infectivity.

EBOV interacting loop 2 of NPC1 is almost perfectly conserved in primates ( Figure 4 B). Only two species, the New World monkey Callithrix jacchus and the lemur Microcebus murinus, harbor amino acid differences from those found in human NPC1: K499I in Callithrix jacchus and K499V in Microcebus murinus. Outside of primates, nearly all species have a non-polar amino acid at residue 499, the exception being rabbits (Oryctolagus cuniculus), which have a glutamine. Several species harbor amino acid changes in residue 502. Previous studies have shown that a D-to-F substitution at residue 502 disrupts interaction between EBOV GP and NPC1 in straw colored fruit bats (Eidolon helvum) (), and it is likely that similar substitutions would prevent EBOV infection in killer whales (Orcinus orca), pigs (Sus scrofa), Jamaican fruit bats (Artibeus jamaicensis), and possibly the common vampire bat (Desmodus rotundus).

Next, we determined whether GP-A82V was more infectious for cells of any permissive species or whether the increased infectivity was a human-specific adaptation. NPC1 is the main cellular receptor for EBOV (), and the EBOV GP-interacting region of NPC1 has been well defined (). Because the EBOV interacting loop 2 of NPC1 is positioned in close proximity to EBOV GP-82, we assessed species-specific amino acid differences within this portion of NPC1 from 49 mammalian species ( Figures 4 A and 4B , Table S3 ).

Data are means ± SEM (n = 3), with data points representing infections using independent viral stocks.p < 0.01; repeated-measures ANOVA with Dunnett’s post-test comparing to ancestral EBOV GP. See also Table S3

(C) Relative infectivity data for EBOV GP containing the A82V substitution in relation to the ancestral GP in four primate cell lines and five cell lines from other mammalian species.

(B) Alignment of NPC1 sequences in and around the second NPC1 interacting loop from a subset of mammalian species used for (A).

(A) Plot of the amino acid conservation in mammals across the region of NPC1 that interacts with EBOV GP. Shaded regions indicate EBOV-interacting residues of NPC1.

The EBOV matrix protein VP40 is sufficient to drive assembly and budding of filamentous virus-like particles (VLPs) from the producer cell plasma membrane (). VP40 is also sufficient for incorporation of EBOV GP into the virion membrane, such that the resulting particles are capable of bona fide receptor-mediated entry into target cell cytoplasm. To facilitate quantitation of EBOV GP-mediated entry, EBOV VP40 fused to β-lactamase was used to generate VLPs. These VLPs were used to infect target cells, and subsequently target cells were loaded with a fluorogenic substrate that is trapped within the target cell cytoplasm. The substrate can be cleaved only if the GP successfully attaches, binds, and triggers fusion, releasing β-lactamase-VP40 into the cytoplasm (). This experimental system does not require transcription of reporter genes, and therefore more directly tests whether the EBOV GP mutants increase the ability of EBOV VP40 cores to enter into the target cell cytoplasm. β-lactamase-containing VLPs bearing ancestral or mutant EBOV GPs were incubated with U2OS target cells, and then cells were loaded with the fluorogenic substrate. GP-A82V- or GP-A82V/T230A-bearing particles exhibited a significant increase in entry as compared with the ancestral EBOV GP ( Figure 3 ). In contrast, a decrease in entry was measured with the T230A and D637G mutant GPs.

Virus-like particles (VLPs) were generated with EBOV VP40-β-lactamase fusion protein and either the ancestral EBOV GP or one of the indicated mutants. U2OS cells were incubated with VLP-containing supernatant for 2 hr at 4°C, then for 2 hr at 37°C, and then loaded with CCF4-AM overnight at 11°C. Cleavage of CCF4-AM was measured using a fluorescent plate reader with 400/30 excitation and 460/40 emission filters. Signal for cleaved CCF4-AM signal for all EBOV GPs was compared to that observed with the ancestral EBOV GP. Data are means ± SEM (n = 6 viral infections) from a representative experiment. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; N.S. p > 0.05; one-way ANOVA with Burnett’s post-test comparing with ancestral EBOV GP.

Amino acid substitutions introduced into the EBOV GP may alter rates of protein production and processing, protein stability, or protein incorporation into virions. To determine whether any of these properties were changed by the EBOV GP mutants, the amount of ancestral versus mutant GP present in our EBOV GP-pseudotyped lentiviruses was compared. EBOV GP-pseudotyped lentiviruses were enriched by acceleration through a 25% sucrose cushion, and virion-associated proteins were analyzed by western blotting, using polyclonal antiserum against a V5 epitope tag that was appended to the carboxyl terminus of the GP. GP is cleaved by furin into two components, GP1 and GP2, and our western blot showed two distinct bands. One band was 70 kDa, consistent in size with full-length GP, either uncleaved by furin or GP1+GP2 covalently bound by disulfide linkages ( Figure 2 D). The second band was 25 kDa, consistent with the furin-processed GP2 product. GP1 + GP2 band intensity was compared to the intensity of the lentivirion capsid core (p24). All GPs showed equivalent levels of incorporation into particles, indicating that the enhanced infectivity observed in human cells with the GP-A82V-containing EBOV GPs was not due to differences in protein synthesis, processing, or GP incorporation into lentiviral particles.

Dendritic cells (DCs) are targets for EBOV replication in vivo, at both early and later stages of infection (). We therefore looked at the effect of the GP mutants on infection of this critical cell type ( Figure 2 C). Human monocyte-derived DCs were generated from eight blood donors. These cells were challenged with ancestral and with mutant EBOV GP-pseudotyped lentiviral vectors. As compared to the ancestral GP, A82V-containing GPs had significantly enhanced infectivity, while individually the T230A and D637G substitutions either had no effect or had less infectivity.

To determine whether the observed infectivity differences were particular to U2OS cells, similar transductions were performed with HEK293 cells as target cells. In HEK293 cells, a 2-fold increase in infectivity was observed with lentiviral particles pseudotyped with GP-A82V, GP-A82V/T230A, or GP-A82V/T230A/D637G ( Figure 2 B). Once again, no statistically significant changes were observed with transductions using either of the single mutants GP-T230A or GP-D637G.

The naturally occurring GP mutants A82V, A82V/T230A, and A82V/T230A/D637G were evaluated for their ability to infect cells by placing them in a mammalian expression plasmid containing the gene encoding EBOV Makona GP. Plasmids were constructed for the ancestral sequence and for each mutant combination. Additionally, the single mutants GP-T230A and GP-D637G were constructed, although these mutations were never observed independently of GP-A82V during the epidemic. The expression plasmids were used to generate EBOV GP-pseudotyped lentiviral virion particles bearing a GFP reporter gene. Equal volumes of each supernatant were used to transduce the human osteosarcoma cell line U2OS after confirming that each construct generated comparable amounts of particles by measuring reverse transcriptase activity in the supernatant. 72 hr later, the transduction efficiency for each GP was assessed by measuring the percent GFP-positive cells by flow cytometry. Lentiviruses bearing GP-A82V, GP-A82V/T230A, or GP-A82V/T230A/D637G produced 4-fold more GFP-positive cells than did particles bearing the ancestral GP ( Figure 2 A). In contrast, pseudotypes bearing either of the single mutants GP-T230A or GP-D637G produced GFP-positive cells at a rate similar to the ancestral GP.

In (A) and (B), each data point represents a normalized transduction using lentiviral stocks derived from independent transfections. In (C), data points represent independent experiments with four independent viral stocks and eight different human donors. ∗ p < 0.05; ∗∗ p < 0.001; ∗∗∗ p < 0.001; repeated-measures ANOVA with Dunnett’s post-test comparing to ancestral EBOV GP.

(D) Western blots (top) of enriched lentiviral particles pseudotyped with C-terminally V5-tagged EBOV GPs probed with anti-V5 and anti-p24 antibodies. Bar graph showing EBOV GP1 + 2 signal intensity relative to that observed for the corresponding lentiviral capsid (p24).

(A–C) Lentiviral virions bearing a GFP transgene and pseudotyped with ancestral EBOV Makona GP or the indicated GP variants were produced by transfection of HEK293 cells and used to transduce U2OS cells (A), HEK293 cells (B), or MDDCs (C). GFP-positive cells resulting from transduction with variant GPs were quantified by flow cytometry and normalized to the ancestral GP using lentivirions produced in parallel. Shown are means ± SEM.

We detected 114 additional amino acid substitutions in the EBOV Makona GP population, though none of these approached the prevalence of GP-A82V. The majority of these were not pursued further because they were located in the mucin-like domain, which is dispensable for entry into target cells (). Instead, we focused on a sub-lineage of GP-A82V with an A-to-G change at nucleotide 6,726 that results in a GP-A82V/T230A double mutant ( Figure 1 A). Aside from GP-A82V, T230A was the most frequently observed non-synonymous mutation (2.5%) outside of the mucin-like domain. Four isolates from the GP-A82V/T230A sub-lineage possessed an additional A-to-G substitution at nucleotide 7,947 that resulted in the triple mutant, GP-A82V/T230A/D637G. These mutants were studied further, as described below.

The two lineages were plotted according to date and country of sampling ( Figures 1 B–1E). GP-A82V was first sampled in Guinea in March 2014. It was next sampled in May 2014 in Sierra Leone. In June 2014 the GP-A82V lineage was again sampled in Guinea. The number of EVD cases caused by GP-A82V in Liberia and Sierra Leone grew rapidly in the summer and fall of 2014 with no apparent contribution from the first, ancestral lineage. In the fall of 2014, EBOV was re-introduced into Guinea from Liberia and Sierra Leone, resulting in a burst of additional cases ().

To get the broadest possible perspective on the EBOV sequence evolution that took place during the 2013–2016 EVD epidemic, we generated a phylogenetic tree using 1,489 EBOV Makona sequences for which near-complete genomes were available ( Tables S1 and S2 ). The tree demonstrated two distinct lineages ( Figure 1 A). The first lineage consisted of 86 sequences and was largely confined to the region around Conakry, Guinea. The second lineage formed a monophyletic clade comprised of 1,403 genomes sampled between March 2014 and August 2015. The second lineage included EBOV from all countries affected by the epidemic and was largely defined by two mutations: a non-synonymous C-to-T substitution at nucleotide 6,283, resulting in the GP-A82V substitution, and a synonymous T-to-C substitution at nucleotide 1,849, which encodes amino acid D460 of the viral NP. In addition to being present in the vast majority of EBOV infections during the 2013–2016 epidemic, the GP-A82V mutant was of interest because residue 82 is located at the receptor binding interface ().

(B–E) Plots of the temporal sampling of EBOV GP genotypes during 2014 (left axis) and the cumulative number of EVD cases (right axis). Shown are data for all Mano River Union (MRU) countries (B), Guinea (C), Sierra Leone (D), and Liberia (E).

(A) Maximum likelihood phylogeny of 1,489 full-length Makona EBOV sequences. Branches are color coded according to country of sampling. Bars to the right side indicate GP sequence and arrowheads indicate when individual residue changes occurred. Scale bar indicates nucleotide substitutions/site.

GP-A82V Was First Detected Just Prior to the Exponential Increase in Cases and Rapidly Exceeded the Prevalence of the Ancestral Makona EBOV

Discussion

CDC, 2016 CDC (2016). Outbreaks Chronology: Ebola Virus Disease | Ebola Hemorrhagic Fever | CDC. http://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html (CDC). The devastating 2013–2016 EVD epidemic led to two orders of magnitude more cases than in previous EVD outbreaks (), providing greater opportunity for EBOV to undergo human adaptive changes than ever before. The A82V mutation in EBOV Makona GP arose early during the outbreak, nearly replacing the ancestral genotype, and was present in the viruses responsible for the majority of EVD cases during the epidemic ( Figure 1 ). Our data show that GP-A82V enhances infectivity in human and other primate cells but not in cells of other mammalian species ( Figures 2 and 4 ). Given that GP-A82V is located at the NPC1 binding interface, we are left with a strong suspicion that GP-A82V represents an EBOV adaptation to the human host. Human population data reveal a modest trend toward higher viremia with GP-A82V and a significant association with increased mortality ( Figure 6 ), raising the intriguing possibility that these phenotypes result from this mutation.

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Ball J.K. Human adaptation of Ebola virus during the West African outbreak. In a co-submitted manuscript, Urbanowicz and colleagues independently arrived at the conclusion that GP-A82V enhances viral infectivity in human cells (). In their hands, GP-A82V enhanced infectivity within the context of numerous GP sequences observed during the outbreak. Complementary to our findings, they showed that GP-A82V has decreased infectivity for a variety of bat cell lines, species with amino acid substitutions in NPC1 binding loop 2 that are similar to those in the non-primate species tested here ( Figures 4 and 5 ). Their data support the hypothesis that A82V is a primate-specific adaptation.

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Stuart D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions. The GP-A82V mutation is on the backside of the α1 helix that interacts with NPC1 and is therefore predicted to have little direct impact on binding to NPC1. However, there are likely to be differences in how the alanine and valine side chains pack against neighboring amino acids. Valine contains more alkane branches than alanine, which would place the side chain in closer proximity to GP-R85 and -W86. Steric hindrance between V82 and R85 may alter a predicted salt bridge between R85 and E178 (∼2.7 Å apart, Figure 5 ). In addition, the added “bulk” of the GP-A82V mutation may alter the α1 helix conformation, to provide better access for proximal resides, such as GP-V79, -P80, -T83, and -W86, to interact with NPC1 (). Compared to the human NPC1 loop 2 domain, the inclusion of hydrophobic side chains at site 499 (valine or isoleucine) in other mammals (non-primates) probably adds more bulk near the backbone, potentially restricting how the loop can adapt to new conformations. Primates also possess a phenylalanine at amino acid 504, while several other species have a tyrosine. Changes between phenylalanine and tyrosine are common, differing only by an ortho hydrogen (phenylalanine) or hydroxyl group (tyrosine) on the benzene ring, and both are generally non-reactive and rarely involved in protein function. F504, however, is predicted to make contact with up to eight residues on EBOV GP (measured by a distance of <4.5 Å []) and is potentially a critical site for filovirus susceptibility (). In addition to the effects the A82V substitution could impart to NPC1/GP interactions, it may also alter the propensity for EBOV GP to undergo “triggering” upon interaction with NPC1. In this context, the triggering process refers to the dramatic conformational changes undertaken by the EBOV GP resulting in the exposure of the viral fusion peptide on GP2, which is normally sequestered by GP1.