key: cord-1047788-zmh488ce
authors: Brook, Cara E.; Ranaivoson, Hafaliana C.; Broder, Christopher C.; Cunningham, Andrew A.; Héraud, Jean-Michel; Peel, Alison J.; Gibson, Louise; Wood, James L. N.; Metcalf, C. Jessica; Dobson, Andrew P.
title: Disentangling serology to elucidate henipa- and filovirus transmission in Madagascar fruit bats
date: 2019-04-15
journal: Journal of Animal Ecology
DOI: 10.1111/1365-2656.12985
sha: 060bec011b08d9c310b8141fa8b90dc420077ba9
doc_id: 1047788
cord_uid: zmh488ce

1. Bats are reservoirs for emerging human pathogens, including Hendra and Nipah henipaviruses and Ebola and Marburg filoviruses. These viruses demonstrate predictable patterns in seasonality and age structure across multiple systems; previous work suggests that they may circulate in Madagascar’s endemic fruit bats, which are widely consumed as human food. 2. We aimed to (a) document the extent of henipa- and filovirus exposure among Malagasy fruit bats, (b) explore seasonality in seroprevalence and serostatus in these bat populations and (c) compare mechanistic hypotheses for possible transmission dynamics underlying these data. 3. To this end, we amassed and analysed a unique dataset documenting longitudinal serological henipa-and filovirus dynamics in three Madagascar fruit bat species. 4. We uncovered serological evidence of exposure to Hendra-/Nipah-related henipaviruses in Eidolon dupreanum, Pteropus rufus and Rousettus madagascariensis, to Cedar-related henipaviruses in E. dupreanum and R. madagascariensis and to Ebola-related filoviruses in P. rufus and R. madagascariensis. We demonstrated significant seasonality in population-level seroprevalence and individual serostatus for multiple viruses across these species, linked to the female reproductive calendar. An age-structured subset of the data highlighted evidence of waning maternal antibodies in neonates, increasing seroprevalence in young and decreasing seroprevalence late in life. Comparison of mechanistic epidemiological models fit to these data offered support for transmission hypotheses permitting waning antibodies but retained immunity in adult-age bats. 5. Our findings suggest that bats may seasonally modulate mechanisms of pathogen control, with consequences for population-level transmission. Additionally, we narrow the field of candidate transmission hypotheses by which bats are presumed to host and transmit potentially zoonotic viruses globally.

Bats have received much attention in recent years for their roles as reservoirs for several virulent, emerging human pathogens, including Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher, Childs, Field, Holmes, & Schountz, 2006; Munster et al., 2016; Olival et al., 2017) . Despite their infamy, bat viruses are not well understood. Elucidation of viral transmission dynamics in bat hosts will be essential to preventing future cross-species emergence by facilitating predictions of viral shedding pulses thought to underpin spillover (Amman et al., 2012) and by highlighting intervention opportunities in enzootic disease cycles.

Serology often represents the most readily attainable empirical information for wildlife diseases; methods have been developed to infer dynamics underlying patterns of age-structured seroprevalence for immunizing infections and prevalence for persistent infections (Brook et al., 2017; Farrington, 1990; Grenfell & Anderson, 1985; Griffiths, 1974; Heisey, Joly, & Messier, 2006; Hens et al., 2010; Long et al., 2010; Muench, 1959; Pomeroy et al., 2015) . Numerous studies have reported serological evidence of bat exposure to henipa-and filoviruses across the Old World (Epstein et al., 2008 (Epstein et al., , 2013 Hayman et al., 2008 Hayman et al., , 2010 Iehlé et al., 2007; Leroy et al., 2005; Ogawa et al., 2015; Peel et al., 2012; Plowright et al., 2008; Taniguchi et al., 1999; Yuan et al., 2012) , though only a few have attempted to use mechanistic models to infer transmission dynamics from serological data for any bat virus (e.g. for rabies : Blackwood, Streicker, Altizer, & Rohani, 2013; for henipavirus: Peel et al., 2018) . The paucity of attempts to model such data may be attributable to the idiosyncratic landscape of chiropteran antibody responses. Experimental challenge trials with various bat species have demonstrated seroconversion after inoculation with Hendra (Williamson et al., 1998) and Nipah (Middleton et al., 2007) henipaviruses and with Marburg (Amman et al., 2014; Paweska et al., 2012 Paweska et al., , 2015 Schuh, Amman, Jones, et al., 2017; , Ebola and Sudan filoviruses (Jones et al., 2015; Paweska et al., 2016) , though many studies (e.g. Halpin et al., 2011) report idiosyncratic antibody dynamics of seroconversion without demonstrable viral replication. Only a few studies have followed immunized bats for longer time horizons: in Marburg-immunized Rousettus aegyptiacus, antibody titres wane after inoculation and primary seroconversion, but subsequently re-challenged seronegative bats nonetheless remain protected from reinfection and primed to remount rapid antibody responses (Paweska et al., 2015; Schuh, Amman, Jones, et al., 2017; . The underlying immunological mechanisms for these responses remain unclear, but at least two pteropodid species were recently shown to maintain a constitutively expressed interferon complex (Zhou et al., 2016) , offering an innate, non-antibody-mediated pathway for viral control.

The island of Madagascar is home to three endemic Old World Fruit Bat species, Pteropus rufus, Eidolon dupreanum and Rousettus madagascariensis, with respective Asian (Almeida, Giannini, Simmons, & Helgen, 2014) , African (Shi et al., 2014) and pan-Indian Ocean (Goodman, Chan, Nowak, & Yoder, 2010) origins. All three species are widely consumed across the island as bushmeat (Golden, Bonds, Brashares, Rodolph Rasolofoniaina, & Kremen, 2014; Jenkins & Racey, 2008; Jenkins et al., 2011) , offering abundant opportunities for zoonotic transmission. Previous work reports serological evidence of Hendra-and Nipah-related henipavirus spp. in P. rufus and E. dupreanum bats, as well as Tioman spp. virus in R. madagascariensis (Iehlé et al., 2007) . To date, no filoviruses have been investigated in any Malagasy bat, although one early serosurvey of human communities in Madagascar highlights seropositivity to Ebola-related filoviruses (but not Marburg) in several localities across the island (Mathiot, Fontenille, Georges, & Coulanges, 1989) . Recent modelling work has classed Madagascar within the "zoonotic niche" for both Ebola (Pigott et al., 2014) and Marburg virus disease (Pigott et al., 2015) . These intriguing preliminary findings, combined with the extreme virulence and heavy public health cost of known bat-tohuman henipa-and filovirus emergence events, motivated our study.

We aimed to (a) document the extent of henipa-and filovirus spp. exposure among endemic Malagasy fruit bats, (b) explore patterns of seasonality in seroprevalence and serostatus in these populations and (c) compare mechanistic hypotheses for possible transmission dynamics underlying these data. as serum and pelleted blood cell. A subset of adult bats (85 P. rufus and 90 E. dupreanum) were processed under anaesthesia using a halothane vaporizer (4% halothane in oxygen at 0.7 L/min), and a lower left premolar tooth was extracted from these individuals for ageing purposes. R. madagascariensis bats were deemed too small for tooth extraction and therefore not subject to anaesthesia or ageing.

Additionally, researchers at the Institut Pasteur of Madagascar (IPM) captured, sexed, weighed, measured and serum-sampled 440 E. dupreanum bats between November 2005 and July 2007 (Iehlé et al., 2007) . We included measurement and serostatus data from these capture events in our Aim 1 and 2 analyses. 

Tooth samples were exported and processed histologically at Matson's Laboratory (Missoula, Montana), following previously published protocols (Cool, Bennet, & Romaniuk, 1994; Divljan, Parry-Jones, & Wardle, 2006) , to yield integer estimates of age via cementum annuli counts. Because fruit bats birth in annual pulses , we obtained more precise estimates of age by assuming a standard birth date for captured bats of a given species and adding the duration of time between capture and birth date to the integer estimate of age via cementum annuli. We computed ages for pups <1 year in the same way. In Madagascar, births are staggered among the three species, with the largest, P. rufus, birthing first, followed by E. duprenaum and R. madagascariensis (Andrianaivoarivelo, 2015) , though the latter were not aged in our study. Assuming respective birth dates of October 1 and November 1, we computed age to the nearest day for 142 P. rufus and 109 E. dupreanum.

Serum samples were screened for antibodies against henipavirus and filovirus soluble glycoproteins (Hendra: HeV sG, HeV sF; Nipah:

NiV sG, NiV sF; Cedar: CedPV sG, CedV sF; Ebola: EBOV sGp; and Marburg: MARV sGp) using a Luminex-based, Bio-Plex® (Bio-Rad, Inc.) assay that has been previously described (Bossart et al., 2007; Chowdhury et al., 2014; Hayman et al., 2008; Peel et al., 2012 Peel et al., , 2013 (Supporting Information Text S1). (Burroughs et al., 2016; Peel et al., 2013; Trang et al., 2015) , we fit finite mixture models to the natural log of the mean fluorescence intensity (MFI) data to approximate a cut-off MFI value (and corresponding upper and lower confidence interval) for seropositivity for each species/antigen combination (Supporting Information Text S2; Tables S1 and S2; Figure S1 ).

Because our antigens were not originally obtained from Madagascar fruit bats, we required that each species/antigen data subset meet several additional criteria before further statistical analysis. For each data subset, we required that MFI values either (a) show correlation with an R 2 > 40% for associated soluble glycoproteins within the same viral genus (an indicator of reliable cross-reactivity among antibodies to related viruses; Supporting Information Figure S2 ), (b) have values >1,000 MFI for some individual(s) assayed (Gombos et al., 2013) or (c) result in >10% seroprevalence based on the mixture model cut-off (Supporting Information Text S3). We summarize all serological data, in conjunction with age and sampling data in Supporting Information Table S3 .

We next aimed to identify any seasonal trends in population-level seroprevalence or individual serostatus for antigens which met the criteria outlined under Aim 1. We restricted these analyses to adult-sized bats over 1 year in age from our own data, combined with E. dupreanum data from IPM (Iehlé et al., 2007) . We analysed each species/antigen subset of our data separately for a total of seven independent analyses (see Results, Table 1 ). For each data subset, we fit a separate generalized additive model (GAM) in the binomial family, using a matrix of seropositive/seronegative counts by sampling event as the response variable and mid-date of sampling event as the smoothing predictor, with a random effect of site and year. All GAMs were fit via REML estimation, and we fixed the number of smoothing knots (k) at seven, as recommended by the package author (Wood, 2001) . R. madagascariensis data were too sparse to permit model convergence at k = 7; in these cases, we fixed k at 6.

Once each model was fit, we used the predict.gam() function to obtain a predicted estimate of seroprevalence by sampling event, bounded by an upper and lower 95% confidence interval. We list the basic structural forms of all GAMs considered in Supporting Information Text S4 and summarize outputs from fitted binomial GAMs in Supporting Information Table S4 (Supporting Information Figure S3 ). We next reformatted our data to examine seasonality within a calendar year, independent of year of study. We used binomial GAMs to test for seasonality in serostatus for adult bats of both sexes and all three species. We set a matrix of seropositive by seronegative counts per Julian day-of-year as our response variable, as computed from mean, lower and upper MFI thresholds for seropositivity, and modelled antigen type as the fixed predictor and day-of-year as the smoothing predictor. We used a "by" term to enable a separate smoother for each sex. All models included random effects of capture site and year. Because we investigated broad seasonal fluctuations, we restricted the number of smoothing knots (k) to four and used a cyclic cubic regression spline which forces the smoother to transition continuously from the end of 1 year to the beginning of the next (Supporting Information Text S4; Table S5 ; Figure S4 ).

Additionally, 17 unique E. dupreanum individuals (three female, 14 male) were captured twice across the duration of our study. Of the two E. dupreanum antigens that met criteria for statistical analysis (see Results, Table 1 ), only anti-NiV-G titres demonstrated substantial dynamism among recaptures. Data were too few for meaningful statistical analysis, but we nonetheless interpreted results anecdotally (Supporting Information Figure S5 ).

Finally, we used Gaussian GAMs to test for seasonality in mass:forearm residual for adult bats within a given year. The mass:forearm residual gives a crude measure of body condition by which to compare bat "health" within a given sex and species.

Bats above the mass:forearm length regression line are "heavier" and those below the line "lighter" than predicted, suggestive of over-and under-nourished conditions-though we caution that we did not validate this inference by comparing measured "mass" with quantification of body lipid content (Pearce, O'Shea, & Wunder, 2008) .

We first established a standardized mass:forearm residual for all adult bats in our dataset by (a) dividing the raw mass per individual by the mean mass of that particular species and sex, then (b) regressing standardized mass against forearm length and (c) calculating the residual from the species-specific linear model (Supporting Information Figure S6 ). We used "standard major axis" type 2 linear regression in this analysis since we anticipated variation and error in measurements for both x-and y-axes (Legendre, 2014) . We then modelled these data with standardized mass:forearm residual as the response variable and Julian day-of-year as the smoothing predictor, including random effects of site and year and a cyclic cubic regression spline (Supporting Information Text S4; interval threshold for the MFI cut-off for seropositivity. This is a more lenient threshold than the mean. c uci = upper confidence interval threshold for the MFI cut-off for seropositivity. This is a stricter threshold than the mean. d Of these antigen/species combinations shown here, two (in bold) met more restrictive criteria for age-seroprevalence analyses. We report only results for NiV-G in E. dupreanum

in the main text of the manuscript.

Finally, to recover the mechanistic underpinnings of our data, we fit Table S3 ).

All models were constructed using discrete-time, age-structured, matrix modelling techniques for epidemics (Klepac & Caswell, 2011; Klepac et al., 2009; Metcalf et al., 2012) , assuming frequencydependent transmission, homogeneous mixing and equilibrium structure across age classes (Supporting Information Text S5). We In all modelled epidemics, populations were jointly subjected to survival and epidemic transitions. Births were subsequently introduced into the population but restricted in duration to a 10-week, species-specific annual period. Births were distributed among the four or five epidemic states, according to parental effects: we assumed that S-class bats of reproductive age (≥2 years) produced susceptible offspring, while I-and R-class bats of reproductive age produced maternally immune offspring. We tested model forms both by which N-class dams produced S ("matSus")-and M-class ("matAB") offspring.

We controlled demographic rates under assumptions of stable (Hayman, 2015; Paweska et al., 2012; Swanepoel et al., 1996) and optimized all other epidemic parameters, depending on the chosen model structure, by minimizing the negative loglikelihood of data of a specific age and biweek, given the model's output at that same age and time. For all models, we fit rates for waning of maternally inherited antibodies (ω) and transmission (β) held constant across age and time (Supporting Information Table   S7 ). For MSIRS, MSIRN and MSIRNR models, we additionally fit a waning antibody rate for individuals exiting the R class (σ); for MSRIR models, a rate of direct seroconversion from S to R (ρ); and

for MSIRNR models, a rate of antibody boosting (γ), by which bats returned to R from N. For MSIRN/R models, we explored variations in model structure under which N-class dams produced either maternally immune (-matAB) or susceptible young (-matSus). All seven models were re-fit six different times: to NiV-G/E. dupreanum and EBOV-Gp/P. rufus data at all three MFI thresholds for seropositivity, to yield 42 distinct sets of parameter estimations.

In all, seven species/antigen combinations met criteria for further analysis, indicating the presence of reliable reactive antibodies to tested antigens in serum from species in question: NiV-G and CedPV-G in E. dupreanum, HeV-F and EBOV-Gp in P. rufus, and

HeV-F, CedPV-G and EBOV-Gp in R. madagascariensis (Table 1 and Supporting Information Table S2 ). These Luminex results indicate that all three Madagascar fruit bat species demonstrated antibody reactivity to Hendra and/or Nipah-related henipaviruses; the inclusion of R. madagascariensis represents an expansion on previous findings (Iehlé et al., 2007 ; Table 1 ). MFI values from the NiV-G/HeV-G and NiV-F/HeV-F Luminex assays were highly correlated (Supporting Information Figure S2 ), suggesting cross-reactivity against related Nipah/Hendra-like henipavirus antigens. For each species, we selected the Nipah/Hendra-like antigen that yielded the highest MFI per species for further ecological analysis: NiV-G for E. dupreanum

and HeV-F for P. rufus and R. madagascariensis (Table 1) .

Additionally, we document the first serological evidence of cross-reactivity with a third henipavirus, Cedar virus, in E. dupreanum and R. madagascariensis, although seroprevalences were low (anti-CedPV-G and anti-CedV-F seroprevalence = 1.27%

and 9.33%, respectively). We also report the first serological ev- Information Table S3 ).

Because ages were unavailable for R. madagascariensis, and seroprevalences were low for HeV-F in P. rufus and CedPV-G in E. dupreanum (6.97% and 1.27%, respectively), we restricted mechanistic modelling of age-seroprevalence trends (Aim 3) to NiV-G in E. dupreanum and EBOV-Gp in P. rufus data only. Due to concerns over the lack of specificity and validation in our assay for EBOV-Gp in P. rufus (which met only one of our three criteria for analysis),

we ultimately reported results for these fits in the Supporting Information only and reserved the main text of our manuscript for modelling of anti-NiV-G in E. dupreanum data, which met all three of criteria for analysis. This Luminex has been previously validated on samples from the sister species E. helvum (Hayman et al., 2008; Peel et al., 2018) . Male bats did not exhibit significant seasonality in seroprevalence at the population level (Supporting Information Figure S4 ), though three of fourteen recaptured male and one of three recaptured female E. dupreanum demonstrated dynamic anti-NiV-G titres (Supporting Information Figure S5 ). 

Teeth were processed histologically to yield integer estimates of fruit bat age (see Materials and Methods), producing species-specific age-frequency distributions for E. dupreanum and P. rufus (Figure 3 ).

Adult mortality rates derived from exponential models fit to E. dupreanum data are compatible with assumptions of stable population structure, but age-frequencies recovered for P. rufus indicate that the species is likely in serious population decline. As such, we adopted juvenile mortality rates from E. dupreanum for epidemiological modelling of P. rufus data (Supporting Information Text S5).

We combined age data with serological data amassed under Aim 1 to develop age-seroprevalence curves for NiV-G in E. dupreanum and EBOV-Gp in P. rufus (Supporting Information Text S5).

Composite age-seroprevalence data for E. dupreanum NiV-G demonstrated high seroprevalence in neonates, suggestive of inherited maternal antibodies (Supporting Information Text S5; Figure S7 ). This neonatal seroprevalence peak decreased rapidly Figures S8 and S9 ). The neonatal decline and early age increase in seroprevalence in our data replicates patterns previously reported for NiV-G exposure in African E. helvum (Peel et al., 2018) , but our observed late-age seroprevalence decline contrasts with the late-age plateau of anti-NiV-G seroprevalence in the African system. We recovered similar age-seroprevalence patterns of EBOV-Gp exposure in P. rufus (Supporting Information Text S5; Figures S10-S12).

We report composite age-seroprevalence data for E. dupreanum NiV-G, combined with model outputs summarized across one age-structured equilibrium year, in Figure 4 (Supporting Information Figure S10 ). The right-hand panel in each subplot shows relative AIC within a given data subset; raw AIC scores are listed in Supporting Information Figure S4 MSIRNR performing too poorly in AIC comparison for true consideration as a best fit model), while MSIRS predicted a late-age seroprevalence plateau.

Parameter estimates varied between the two best fit mod- (3) we use mechanistic models to reveal the critical role of waning humoral immunity and the potential for alternative immune processes in governing serological patterns witnessed in our data.

We report many serological findings novel for the Madagascar ecosystem-including the first evidence of antibodies cross-reactive F I G U R E 3 Ageing Madagascar fruit bats via cementum annuli. (a) Age-frequency distribution generated from cementum annuli counts of extracted Eidolon dupreanum teeth. Histogram is binned by year, with 95% exact binomial confidence intervals shown as dotted lines. The red curve is the predicted age-frequency distribution generated from the fit of a simple exponential model to age distribution >6 months, incorporating an annual adult survival rate of 0.793 and a juvenile annual survival rate of 0.544 (determined using Leslie matrix techniques to maintain a stable age distribution and constant population size; Supporting Information Text S5). Translucent shading shows 95% confidence intervals of the exponential fit by standard error. (b) Age-frequency distribution from cementum annuli counts of extracted Pteropus rufus teeth, with a fitted exponential model (red line) and 95% confidence intervals (red shading), incorporating an annual adult survival rate of 0.511 and a juvenile survival rate of 0.544 (constant population size was impossible for P. rufus, so we adopted the same rate as for Marburg) antigen will interest the global public health community, as recent work classes Madagascar within the "zoonotic niche" of both Ebola (Pigott et al., 2014; Schmidt et al., 2017) and Marburg (Pigott et al., 2015) filoviruses. Ironically, Madagascar's inclusion in these risk maps has been largely derived from the species distribution of Eidolon dupreanum (Han et al., 2016; Pigott et al., 2014) , the one Malagasy fruit bat for which we found no filovirus seropositive samples. This finding is not hugely surprising if we consider the relative rarity of Ebola seropositivity in E. dupreanum's sister taxon, E. helvum (Olival & Hayman, 2014) , which possesses a receptor-level substitution that makes it refractory to Ebola infection (Ng et al., 2015) .

Given Madagascar's geographic isolation and the considerable phylogenetic distance separating its fruit bats from their nearest mainland relatives (Almeida et al., 2014; Goodman et al., 2010; Shi et al., 2014) , it seems likely that some of the seropositives recovered in this study result from cross-reactivity of Malagasy bat antibodies to related, but distinct, antigens from those assayed here. To date, no henipaviruses or filoviruses have been identified (via live virus or RNA) in Madagascar. Detection and characterization of these viruses, together with description of the specificity, avidity and neutralization capacity of their antibodies, thus represents a critical research priority. The probable cross-reactivity of Malagasy bat antibodies derived from different-and potentially novel-henipa-and filovirus antigens adds considerable uncertainty to our tabulation of MFI thresholds for seropositivity.

The greatest challenge to our dynamical inference is the possibility that seropositive samples do not signify true circulating virus within any of our three species. In laboratory trials, for example, R. aegyptiacus bats are known to seroconvert upon contact with inoculated individuals without ever becoming detectably infectious (Jones et al., 2015; Paweska et al., 2015) . While we attempted to explore these dynamics within a single bat population using our MSRIR model, it is possible that focal viruses circulate in species distinct from those studied here, resulting in seropositive samples via dead-end seroconversion from transient bat contact with an alternative reservoir. Although we cannot falsify this hypothesis, there are a few specifics of the Madagascar ecosystem that make such a scenario unlikely. In particular, all but one of the roosts surveyed in this study are largely single-species conglomerations: P. rufus is a tree-dwelling pteropodid which only roosts in single-species assemblages, while E. dupreanum predominantly inhabits cracks and crevasses with conspecifics (Goodman, 2011) .

F I G U R E 4 Model fits to age-seroprevalence data. Age-seroprevalence curves for Eidolon dupreanum NiV-G, using the mean MFI cut-off for seropositive status. Seroprevalence data (left y-axis) are shown as open circles, binned for 0-0.5 years, 0.5-1 years, 1-1.5 years, 1.5-3 years, and for 3-year increments increasing after that. Shape size corresponds to the number of bats sampled per bin (respective sample sizes, by age bin, are as follows: N = 10, 2, 20, 9, 18, 5, 7, 1). Solid purple lines indicate model outputs, and translucent shading highlights the 95% confidence interval derived from the Hessian matrix of the maximum likelihood of each model fit to the data. Panels are stratified into columns by model structure: (a) MSIR = maternally immune, susceptible, infectious, recovered; (b) MSRIR = maternally immune, susceptible, recovered via direct seroconversion, infectious, recovered; (c) MSIRS = maternally immune, susceptible, infectious, recovered, susceptible; (d) MSIRN = maternally immune, susceptible, infectious, recovered, non-antibody immune; (e) MSIRNR = maternally immune, susceptible, infectious, recovered, non-antibody immune; recovered). All MSIRN/R model outputs depicted assume that non-antibody immune dams produce maternally immune-class young. The right-hand y-axis (in navy) of each subplot shows ΔAIC for each model fit, relative to all other models in the figure (navy diamonds). The MSIRN model (d) offered the best fit to the data, corresponding to ΔAIC = 0. All parameter values, confidence intervals and raw AIC scores for each model fit are reported in Supporting Information Table S7 . Model fits including MSIRN/R fits assuming N-class mothers produce susceptible young are shown in Supporting Information Figure S10 , along with fits to seroprevalence data for P. rufus EBOV-Gp. Fits calculated using the lower and upper MFI thresholds for seropositivity are shown in Supporting Information Figures S11 and S12 In cave environments, E. dupreanum and R. madagascariensis occasionally co-roost and roost with insectivorous bats (Cardiff, Ratrimomanarivo, Rembert, & Goodman, 2009) , and all three fruit bat species contact at feeding sites. Nonetheless, given the relative rarity of these cross-species contacts, it is unlikely that the high seroprevalence recovered in our data for anti-NiV-G antibodies in E. dupreanum (24.2%) and anti-EBOV-Gp antibodies in P. rufus (10.2%) result from dead-end seroconversion alone.

Previous work has investigated paramyxovirus spp. by PCR among insectivorous bats in Madagascar (Wilkinson et al., 2012 (Wilkinson et al., , 2014 , and no henipavirus spp. have been identified, further supporting our assumptions that Malagasy fruit bats maintain their own endemic viral transmission cycles.

The lack of specificity in our serological assay also permits the possibility that a given bat population might maintain active infections with multiple serologically indistinguishable viruses of the same family, which are nonetheless epidemiologically unique; serum from Ebola-infected humans, for example, will recognize all five known species of ebolavirus (MacNeil, Reed, & Rollin, 2011 ). An analysis like ours would consider serological evidence of any ebolavirus infection equivalently and model all seropositives as one population, though, in reality, each specimen could represent a distinct virus that maintains its own transmission cycle. Again, we cannot falsify this hypothesis, but recent molecular work supports a theory of single-bat, single-filovirus species interactions that runs counter to this claim (Ng et al., 2015) . We observed vast differences in the range of MFI titres recovered for each antigen among our three bat species, recovering high MFI titres for EBOV-Gp in R. madagascariensis but only mid-range titres in P. rufus (Table 1 ). We also found that E. dupreanum serum reacted most strongly to the NiV-G antigen, while P. rufus and R. madagascariensis serum bound more tightly to the HeV-F antigen.

Such differences could be attributable to cross-species variation in the robustness of the humoral immune response or could indicate that our tested antigens more closely align with the wild antigen from which one species' antibodies were derived vs. that of another.

This species-specific variation in antibody binding to the same antigen challenge supports our decision to model each bat species-virus relationship independently, rather than allowing for significant interspecies transmission to govern viral dynamics in this system.

Female serostatus for both henipavirus and filovirus spp. varied seasonally in our data, tracking reproduction for E. dupreanum and R. madagascariensis; female bats showed elevated antibody titres during reproduction, consistent with previous work (Baker et al., 2014) . This pattern suggests that viral control is one of many costs to which resource-limited hosts must allocate energy and that male and female bats do so differently while facing distinct metabolic demands. While higher serotitres in reproductive females may seem counterintuitive if viewed as increased investment in immunity, recent research suggests that bats may control viral infections primarily via innate immune pathways (Zhou et al., 2016) , which are more metabolically costly than adaptive immunity (Raberg et al., 2002) .

It is possible then that female bats trade off innate immunity with less metabolically demanding means of viral control (i.e. antibodies) during reproductive periods or that contact rates with infectious individuals are elevated during these seasons, resulting in antibody-boosting effects (e.g. Paweska et al., 2015; Schuh, Amman, Jones, et al., 2017; . Alternatively, elevated antibody titres might be independent of both exposure and metabolic trade-offs; for example, production of the milk protein prolactin (typically elevated in late pregnancy and early lactation for mammals) is known to stimulate antibody production and facilitate maternal antibody transfer to young (Spangelo, Hall, Ross, & Goldstein, 1987) .

Males, with fewer reproductive constraints, demonstrate no clear shifts in seasonal serostatus at the population level. Nonetheless, recapture data suggest that male antibody titres subtly track seasonal peaks and troughs in body mass, increasing during the fruit-abundant wet season and declining during the dry season. Understanding seasonal trade-offs in bat immune investment will be critical to enhancing our capacity for predicting seasonal pulses in viral transmission and informing possible zoonotic risk. Paired field studies, tracking viral excretion in conjunction with individual serostatus, will be essential to elucidating these dynamics in the future.

One of the largest questions arising from our investigation addresses the extent to which seropositive status correlates with infectiousness and immunity. Previous work highlights notable seasonality in spillover of both Hendra (Plowright et al., 2015) and

Ebola viruses (Schmidt et al., 2017) , although the mechanistic contributions of bat demography versus physiology remain unclear. In our models, the force of infection varied seasonally as a result of birth pulse-mediated cycles in the infectious population (Supporting Information Figure S13 ). Seasonal fluctuations in the magnitude of transmission-which could emerge from changes in host contact rates (Ferrari et al., 2008; , variation in within-host immunological susceptibility (Dowell, 2001) or periodicity in viral shedding (Plowright et al., 2015) -might further modulate seasonality in FOI. Several studies have highlighted the possible role that latent infections and viral recrudescence could play in bat virus transmission (Plowright et al., 2016; Rahman et al., 2011) , but data from longitudinally resampled individuals were too few to allow for evaluation of any such model in our study. If future field work is able to demonstrate a role for seasonal transmission independent of demography, then the extent to which observed seasonality in serotitre could serve as a biomarker for an individual bat's infectiousness or susceptibility will be critical to resolving predictive power from cross-sectional serological data.

We fit age-structured, epidemic models to age-seroprevalence data and recovered strong support for models incorporating waning humoral immunity (i.e. MSIRS, MSIRN). This result is consistent with previous experimental findings, which demonstrate rapidly declining antibody titres in Marburg-infected R. aegyptiacus, after inoculation and seroconversion (Paweska et al., 2015; Schuh, Amman, Jones, et al., 2017; Schuh, Amman, Jones, et al., 2017; Williamson, Hooper, Selleck, Westbury, & Slocombe, 2000; Williamson et al., 1998) . Although we chose not to explore models of this form at this time, we caution that we should remain cognizant of these possible mechanisms in the future.

Although no known bat-borne zoonoses have been documented in Madagascar, our work confirms a history of exposure to potentially zoonotic henipaviruses and filoviruses in several widespread, endemic fruit bat species. These species are widely consumed throughout Madagascar, and the majority of bat hunting-and corresponding bat-human contact-is concentrated during the resource-poor winter, overlapping with bat gestation and elevated anti-viral seroprevalence in our data (Golden et al., 2014; Jenkins & Racey, 2008; Jenkins et al., 2011) . If seasonal changes in serostatus are revealed to have any bearing on viral transmission, insights from our modelling will offer a predictive framework to safeguard public health.

We 

Raw data used in all analyses described in this manuscript are available for public access in the following Dryad Digital Repository:

https://doi.org/10.5061/dryad.61tc3hd .

Each flying fox on its own branch: A phylogenetic tree for Pteropus and related genera (Chiroptera: Pteropodidae)

Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human infection

Oral shedding of Marburg virus in experimentally infected Egyptian Fruit Bats (Rousettus aegyptiacus)

Viral antibody dynamics in a chiropteran host

Dynamics of measles epidemics: Estimating scaling of transmission rates using a times series SIR model

Resolving the roles of immunity, pathogenesis, and immigration for rabies persistence in vampire bats

Estimating time of infection using prior serological and individual information can greatly improve incidence estimation of human and wildlife infections

Neutralization assays for differential henipavirus serology using Bio-Plex protein array systems

Bartonella spp. in fruit bats and blood-feeding ectoparasites in Madagascar

Elucidating transmission dynamics and host-parasite-vector relationships for rodent-borne Bartonella spp

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Population trends for two Malagasy fruit bats

Data from: Disentangling serology to elucidate henipa-and filovirus transmission in Madagascar fruit bats

Hendra virus infection dynamics in the greyheaded flying fox (Pteropus poliocephalus) at the southern-most extent of its range: Further evidence this species does not readily transmit the virus to horses

Bats: Important reservoir hosts of emerging viruses

Hunting, disturbance and roost persistence of bats in caves at Ankarana, northern Madagascar

Serological evidence of henipavirus exposure in cattle, goats and pigs in Bangladesh

Age estimation of pteropodid bats (Megachiroptera) from hard tissue parameters

Age determination in the grey-headed flying fox

Seasonal variations in host susceptibility and cycles of certain infectious diseases

Duration of maternal antibodies against Canine Distemper Virus and Hendra virus in Pteropid bats

Henipavirus infection in fruit bats (Pteropus giganteus)

Modelling forces of infection for measles, mumps, and rubella

The dynamics of measles in sub-Saharan Africa

Economic valuation of subsistence harvest of wildlife in Madagascar

Influence of test technique on sensitization status of patients on the kidney transplant waiting list

Les chauves-souris de Madagascar

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The estimation of age-related rates of infection from case notifications and serological data

Dynamics of measles epidemics: Scaling noise, determinism, and predictability with the TSIR model

A catalytic model of infection for measles

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Biannual birth pulses allow filoviruses to persist in bat populations

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Demography of straw-colored fruit bats in Ghana

Evidence of henipavirus infection in West African fruit bats

The fitting of general force-of-infection models to wildlife disease prevalence data

75 years of estimating the force of infection

Henipavirus and Tioman virus antibodies in Pteropodid bats

Analysis of patterns of bushmeat consumption reveals extensive exploitation of protected species in eastern Madagascar

Bats as bushmeat in Madagascar

Experimental inoculation of Egyptian rousette bats (Rousettus aegyptiacus) with viruses of the Ebolavirus and Marburgvirus genera

The stage-structured epidemic: Linking disease and demography with a multi-state matrix approach model

Stage-structured transmission of phocine distemper virus in the Dutch 2002 outbreak

lmodel2: Model II Regression

Fruit bats as reservoirs of Ebola virus

Identifying the age cohort responsible for transmission in a natural outbreak of Bordetella bronchiseptica

Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus

Antibodies populations to haemorrhagic fever viruses in Madagascar

The epidemiology of rubella in Mexico: Seasonality, stochasticity and regional variation

Seasonality and comparative dynamics of six childhood infections in pre-vaccination Copenhagen

Structured models of infectious disease: Inference with discrete data

Experimental Nipah virus infection in pteropid bats (Pteropus poliocephalus)

Catalytic models in epidemiology

Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis)

NPC1 contributes to species-specific patterns of Ebola virus infection in bats. ELife, 4, e11785

Seroepidemiological prevalence of multiple species of filoviruses in fruit bats (Eidolon helvum) migrating in Africa

Filoviruses in bats: Current knowledge and future directions

Host and viral traits predict zoonotic spillover from mammals

Lack of Marburg virus transmission from experimentally infected to susceptible in-contact Egyptian fruit bats

Virological and serological findings in Rousettus aegyptiacus experimentally inoculated with Vero cells-adapted Hogan strain of Marburg virus

Experimental inoculation of Egyptian fruit bats (Rousettus aegyptiacus) with Ebola Virus

Evaluation of morphological indices and total body electrical conductivity to assess body composition in big brown bats

Henipavirus neutralising antibodies in an isolated island population of African fruit bats

Support for viral persistence in bats from age-specific serology and models of maternal immunity

Use of cross-reactive serological assays for detecting novel pathogens in wildlife: Assessing an appropriate cutoff for henipavirus assays in African bats

The effect of seasonal birth pulses on pathogen persistence in wild mammal populations

Inferring infection hazard in wildlife populations by linking data across individual and population scales

Mapping the zoonotic niche of Ebola virus disease in Africa

Mapping the zoonotic niche of Marburg virus disease in Africa

Ecological dynamics of emerging bat virus spillover

Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)

Urban habituation, ecological connectivity and epidemic dampening: The emergence of Hendra virus from flying foxes (Pteropus spp

Transmission or within-host dynamics driving pulses of zoonotic viruses in reservoir-host populations

Serotype-specific transmission and waning immunity of endemic foot-and-mouth disease virus in Cameroon

Basal metabolic rate and the evolution of the adaptive immune system

Evidence for Nipah virus recrudescence and serological patterns of captive Pteropus vampyrus

Spatiotemporal fluctuations and triggers of Ebola virus spillover

Modelling filovirus maintenance in nature by experimental transmission of Marburg virus between Egyptian rousette bats

Egyptian rousette bats maintain long-term protective immunity against Marburg virus infection despite diminished antibody levels

A deep divergence time between sister species of Eidolon (Pteropodidae) with evidence for widespread panmixia

Stimulation of in vivo antibody production and concanavalin-A-induced mouse spleen cell mitogenesis by prolactin

Experimental inoculation of plants and animals with Ebola virus

Reston ebolavirus antibodies in bats, the Philippines. Emerging Infectious Diseases

Determination of cut-off cycle threshold values in routine RT-PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis

Introduction of rubellacontaining-vaccine to Madagascar: Implications for roll-out and local elimination

Highly diverse Morbillivirusrelated paramyxoviruses in the wild fauna of southwestern Indian Ocean islands: Evidence of exchange between introduced and endemic small mammals

Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean

Estimating HIV incidence rates from age prevalence data in epidemic situations

Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats

Experimental Hendra virus infection in pregnant guinea-pigs and fruit bats (Pteropus poliocephalus)

mgcv: GAMs and generalized ridge regression for

Serological evidence of ebolavirus infection in bats

Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats

Disentangling serology to elucidate henipa-and filovirus transmission in Madagascar fruit bats