Serological evidence of zoonotic filovirus exposure among bushmeat hunters in Guinea

Abstract

Human Ebola virus (EBOV) outbreaks caused by persistent EBOV infection raises questions on the role of zoonotic spillover in filovirus epidemiology. To characterise filovirus zoonotic exposure, we collected cross-sectional serum samples from bushmeat hunters (n = 498) in Macenta Prefecture Guinea, adjacent to the index site of the 2013 EBOV-Makona spillover event. We identified distinct immune signatures (20/498, 4.0%) to multiple EBOV antigens (GP, NP, VP40) using stepwise ELISA and Western blot analysis and, live EBOV neutralisation (5/20; 25%). Using comparative serological data from PCR-confirmed survivors of the 2013-2016 EBOV outbreak, we demonstrated that most signatures (15/20) were not plausibly explained by prior EBOV-Makona exposure. Subsequent data-driven modelling of EBOV immunological outcomes to remote-sensing environmental data also revealed consistent associations with intact closed canopy forest. Together our findings suggest exposure to other closely related filoviruses prior to the 2013-2016 West Africa epidemic and highlight future surveillance priorities.

Fig. 1. Sampling locations of villages in Macenta Prefecture, Guinea.

Points represent centroids of sample villages coloured red if EBOV-affected (PCR-confirmed EBOV 2013–16) and blue if unaffected. Green squares represent approximate locations of EBOV index cases or EBOV RNA fragment isolation from bats (Miniopterus inflatus). White squares show locations of Bombali virus RNA fragment isolation from bats (Mops condylurus). Black marks indicate intermediate and large settlements as delineated by GRID3 as indicators of density. White lines indicate non-minor roads suitable for motorised vehicles extracted from OpenStreetMap to indicate accessibility. Figure generated using QGIS software.

Fig. 2. Individual-level responses of Macenta and EVD survivor serum samples using EBOV-GP ELISA.

Point colours indicate village status of participant (blue = unaffected, red = affected) and groups are based on fit of latent profile analysis. Box widths indicate group sizes proportional to √n, n = 498 (A). Comparison of EBOV-GP ELISA titre between Macenta samples in (A) and Guinean PCR-confirmed EVD survivors (n = 137) and their contacts (n = 90) sampled annually 2016-17. Note that the contact group includes a mix of symptomatic, asymptomatic and unaffected individuals (B). Centre = median. Box = interquartile range (ie. +/− 25% of the data either side of the median) i.e. 25th and 75th centiles. Whiskers = Q1 – 1.5*interquartile range and Q3 + 1.5* interquartile range.

Fig. 3. Analysis of serological responses of Macenta serum samples.

Outcomes are stratified by high (left panel) and intermediate (right panel) titre EBOV-GP ELISA groups red dots indicate a positive result and green dots indicate a negative result (A). Representative western blots from a triple positive (JB154) and triple negative (JB050) sample, (B) Neutralisation titre of serum samples against EBOV strain Mayinga (n = 62; geometric mean titre of serum dilution) and regressed against paired ELISA-GP result using a cubic spline (p = 0.004 versus linear) blue dots indicate those samples that belong to latent class A and red dots indicate those samples that do not belong to latent class A (C) and stratified by latent class group blue points = group A, red points = group B. Grey shading denotes the estimated standard error of a natural spine with a maximum of 3 degrees of freedom. These are the results of a latent profile / finite mixture model, which classifies the most likely groups assuming they all form a Gaussian distribution in the total population. D Note the WHO EBOV international standard has an approximate value of ELISA GP IUml⁻¹ Log2 of 2.5 and a Log₂ neutralisation GMT of ~10.

Fig. 4. Broad serological analysis of Macenta serum samples.

A Anti-filovirus total binding IgG antibody response as detected by Luminex-based multiplexed microsphere binding immunoassay for group A (n = 20). Median fluorescence intensity (MFI) values per glycoprotein (GP) antigen are shown for each individual (labelled by sample identification code). The World Health Organisation (WHO) Ebola Zaire (EBOV) standard and an ERVEBO® rVSV-ZEBOV vaccinee are included for comparison. B The average antigen-specific total binding IgG antibody response (median fluorescence intensity) for latent class group A against a multiplexed panel of filovirus antigens. Detected by Luminex-based multiplexed microsphere binding immunoassay.

Fig. 5. Spatial occurrence of serological outcomes.

Heatmap of outcomes by village and further stratified by affected status. Note that participant numbers varied by village, so plots do not convey prevalence (A). Maps showing locations of sampled villages (white points) overlaid with occurrence of at least one serological outcome (B). B was generated using QGIS software.