Filoviruses are causative agents of viral haemorrhagic disease in humans and non-human primates although virus spillover is rare [1]. There are ten distinct filoviruses classified into four genera, Ebolavirus: Ebola virus, Bundibugyo virus, Taï forest virus, Sudan virus, Reston virus and Bombali virus; Marburgvirus: Marburg virus and Ravn virus; Cuevavirus: Lloviu virus; and Dianlovirus: Měnglà virus [2–4].

Bats are the proposed natural reservoir of filoviruses, involved in enzootic virus maintenance and zoonotic virus transmission to susceptible hosts [4]. The majority of described filoviruses are endemic in the African continent, although filovirus-specific antibodies were detected in bats from Bangladesh [5], the Philippines [6], and Singapore [7]. The genome of a novel filovirus, Měnglà virus, was detected in bats from China [8] and is the second Asiatic filovirus described after Reston virus [9]. Lloviu virus was discovered in Spain in 2011 and detected in Hungary in 2016 [10, 11]. Bats are hunted by humans across Africa and Asia, and at least 167 bat species are consumed [12]. High-risk activities, such as bat hunting and mining in bat-dwelling caves, pose a threat of cross-species filovirus transmission [13].

In the Northeast Indian state of Nagaland, local ethnic groups have conducted bat harvests for at least seven generations as a source of food and traditional medicine. These bat hunters are exposed to saliva, blood, and excreta from the bat species Rousettus leschenaultii and Eonycteris spelaea. We conducted a serological survey of both hunted bat species and human hunters to study if humans have been exposed to filoviruses potentially originating from bats.

The majority of bat hunters were between 18 and 50 years of age, male, and participated at least eleven times in the harvest (Table 2). All bat tissues tested were PCR-negative for filovirus-specific nucleic acid. There were a total of 13,993,300 reads from the R. leschenaultii NGS dataset and 7,975,905 reads from the E. spelaea NGS dataset and no filovirus sequences were identified. In our 2019 serum screen (that included MLAV), we detected filovirus-reactive sera in 5.9% (5/85) of human samples, 6.2% (1/16) of E. spelaea samples, and 13.3% (4/30) of R. leschenaultii samples. The highest MFI values, corresponding to levels of filovirus-specific serum IgG, were detected for EBOV-GPe in human and E. spelaea sera, and for MLAV- and RAVV-GPe in R. leschenaultii sera (Fig 2). Our results suggest human exposure to two antigenically distinct filoviruses, the first group of sera (H10, H27, H30, H40) being reactive to EBOV-, BDBV-, and SUDV-GPe, and one individual serum (H45) singly reactive to MARV-GPe. An individual E. spelaea serum sample (E34) reacted to the EBOV-, SUDV- and TAFV-GPe, displaying a similar cross-reactivity pattern as seen for the first group of human sera and was previously reported for filovirus-positive E. spelaea samples from Singapore [7]. The 2019 screening results corroborated positive samples that were detected when screened in 2018 using a Bio-Plex 200 system (S4 Fig; H27, H30, H40, E34, R39), strengthening interpretation of positive sera samples screened in two different years with two different Luminex xMAP-based machines. There was minimal intra- and inter-individual variation in the negative human sera samples and these were all well below the MFI cutoff values for each GPe (S5 Fig).

Despite the growing evidence that filoviruses are present in South and Southeast Asia, there has been a historical absence of outbreaks of filovirus haemorrhagic fever in this region. Reasons why clusters of human filovirus infections have not yet been detected in Asia, include (a) ecological barriers prevent zoonotic transmission, (b) viruses are unable to sustain transmission between humans or (c) an uncharacterized diversity of non-pathogenic, antigenically-related filoviruses exist and cause asymptomatic infection in humans. Human populations with wildlife contact and no history of Ebola virus disease in Uganda [25] and the Democratic Republic of Congo [26] were reportedly ebolavirus seropositive. Similarly, here we report the presence of filovirus (e.g. ebolavirus, marburgvirus and dianlovirus) reactive antibodies in both human (e.g. bat hunters) and bat populations in Northeast India, a region with no historical record of Ebola virus disease.

Cross-reactivity between EBOV, BDBV and SUDV of the tested samples is in agreement with a previous report [23]. The mammalian cell-culture expression system to produce oligomeric, native-like GPe capture antigens in this multiplex assay provides several benefits compared to peptide-based antigen assays, including the retention of post-translational modifications (i.e. glycosylation) and native quaternary structures allowing capture of conformational-dependent antibodies. The use of GPe from all presently described filoviruses—with the exception of the recently described Bombali virus—allows for simultaneous detection and antigenic differentiation of virus species-specific IgGs and the identification of cross-reactive IgG responses. Most ebolavirus serology surveillance studies are unable to address the complex biology of known and unknown filoviruses in terms of cross-reactivity of specific antibodies. This filovirus serological assay addresses many limitations of previously employed assays by using oligomeric, native-like virus antigens in a multiplex manner and represents an improved biosurveillance tool. Establishing thresholds for low sample number serum sets that lack both positive and negative controls is challenging, but our efforts to employ two independent statistical methods and two machine platforms yielded congruent results and is in agreement with prior approaches to estimate seropositivity [27, 28].

Furthermore, this study describes serum reactivity to MLAV, incidentally in the same bat species (R. leschenaultii) mentioned in the initial report [2]. Reactivity to RESTV, which circulates endemically among bats, pigs and monkeys in the Philippines [29], and causes subclinical infection in animal care takers and slaughterhouse workers in the Philippines [9], was not detected in our study. Interestingly, MLAV and RAVV positive R. leschenaultii sera suggest circulation of two distinct filoviruses within the same species, which is serologically distinct from reactivity with E. spelaea and human samples. These results are concordant with previous findings of exposure to filoviruses antigenically closely related to EBOV, BDBV, and SUDV in E. spelaea in Singapore [17].

Two proposed mechanisms for sustained virus infection in the studied bat species are frequent co-roosting with other bats and the introduction of large numbers of susceptible juveniles into the population [30]. The two bat species sampled in this study, R. leschenaultii and E. spelaea, roost in large colonies in caves with rolling parturition patterns [31]. Though we have serological evidence of filovirus exposure, there was no genomic data detected. There are several reasons why this may be, including; small sample size, low virus copy numbers, uncertain epidemiological shedding periodicity, and high filovirus genetic diversity that is not captured by the primers employed here. Our results reinforce the need to select sentinel sites for virus surveillance at the human-animal interface and highlights some of the gaps in our understanding of filovirus transmission and ecology.