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

Abstract

Isolated islands provide valuable opportunities to study the persistence of viruses in wildlife populations, including population size thresholds such as the critical community size. The straw-coloured fruit bat, Eidolon helvum, has been identified as a reservoir for henipaviruses (serological evidence) and Lagos bat virus (LBV; virus isolation and serological evidence) in continental Africa. Here, we sampled from a remote population of E. helvum annobonensis fruit bats on Annobón island in the Gulf of Guinea to investigate whether antibodies to these viruses also exist in this isolated subspecies. Henipavirus serological analyses (Luminex multiplexed binding and inhibition assays, virus neutralisation tests and western blots) and lyssavirus serological analyses (LBV: modified Fluorescent Antibody Virus Neutralisation test, LBV and Mokola virus: lentivirus pseudovirus neutralisation assay) were undertaken on 73 and 70 samples respectively. Given the isolation of fruit bats on Annobón and their lack of connectivity with other populations, it was expected that the population size on the island would be too small to allow persistence of viruses that are thought to cause acute and immunising infections. However, the presence of antibodies against henipaviruses was detected using the Luminex binding assay and confirmed using alternative assays. Neutralising antibodies to LBV were detected in one bat using both assays. We demonstrate clear evidence for exposure of multiple individuals to henipaviruses in this remote population of E. helvum annobonensis fruit bats on Annobón island. The situation is less clear for LBV. Seroprevalences to henipaviruses and LBV in Annobón are notably different to those in E. helvum in continental locations studied using the same sampling techniques and assays. Whilst cross-sectional serological studies in wildlife populations cannot provide details on viral dynamics within populations, valuable information on the presence or absence of viruses may be obtained and utilised for informing future studies.

Figure 1. Map of the Gulf of Guinea islands indicating the location of Annobón.

Figure 2. Map of Annobón indicating Eidolon helvum colonies and sampling sites.

Key: Circles indicate colony locations (open circle: colonies were reported or used to exist in the past, but no bats were found; partially filled circle: colony observed, but not sampled; filled circle: colony observed and sampled). Squares indicate sites where Eidolon bats have been observed feeding at different times of the year.

Figure 3. Forearm length of adult Eidolon helvum bats.

Values are compared among populations in the four Gulf of Guinea islands and continental Africa. Graphs are of box and whisker plots showing median (black line), 25^th and 75^th percentile (box) and 1.5x the interquartile range (dotted line) values.

Figure 4. Frequency distribution of Luminex Median Fluorescent Intensity (MFI) values against NiV in Eidolon helvum annobonensis.

The dotted line represents the where the MFI of the neutralising sample (Bat # 331) lies within this distribution.

Figure 5. Frequency distribution of Luminex MFI values against NiV in Eidolon helvum annobonensis, separated by age.

Figure 6. Luminex MFI values against NiV in Eidolon helvum annobonensis, separated by age and gender.

Age is divided into bats of approx. 8 months of age, 20 months of age (including primiparous females) and adults. Graphs are of box and whisker plots showing median (black line), 25th and 75th percentile (box) and 1.5x the interquartile range (dotted line) values.

Figure 7. Results of western blot analysis.

Samples with Luminex binding MFIs over 750 were tested using a recombinant, purified Nipah virus nucleocapsid protein. The marker is BenchMark Pre-stained Protein Ladder (Invitrogen); the positive control sera are NiV-neutralising Pteropus alecto and polyclonal Rabbit sera raised to recombinant NiV protein.