African Henipavirus Research Articles

Ecological and Reproductive Cycles Drive Henipavirus Seroprevalence in the African Straw‐Coloured Fruit Bat (Eidolon helvum)

ABSTRACTBats are known to host zoonotic viruses, including henipaviruses that cause high fatality rates in humans (Nipah virus and Hendra virus). However, the determinants of zoonotic spillover are generally unknown, as the ecological and demographic drivers of viral circulation in bats are difficult to ascertain without longitudinal data. Here we analyse serological data collected from African straw‐coloured fruit bats (Eidolon helvum) in Ghana over the course of 2 years and across four sites, comprising three wild roosts and one captive colony. We focus on antibody affinity to five henipavirus antigens: Ghanaian bat henipavirus (GhV), Nipah virus (NiV), Hendra virus (HeV), Mojiang virus (MojV) and Cedar virus (CedV). In the wild roosts, we detected seasonal variations in henipavirus antibody binding, possibly associated with bat life‐history cycles and migration patterns. In the captive colony, we identified increases in antibody affinity levels among pregnant bats, suggesting possible shifts in the immune system during pregnancy. These bats then pass maternal antibodies to their pups, which wane before antibody affinity levels rise later in life following initial infections and/or reactivation of latent infections. These results improve our understanding of the links between bat ecology and viral circulation, including for GhV, a locally‐circulating African henipavirus.

Henipaviruses at the Interface Between Bats, Livestock and Human Population in Africa.

Nipah virus (NiV) and Hendra virus (HeV) are closely related members within the genus Henipavirus, family Paramyxoviridae, for which fruit bats serve as the reservoir. The initial emergence of NiV infections in pigs and humans in Malaysia, and HeV infections in horses and humans in Australia, posed severe impacts on human and animal health, and continues threatening lives of humans and livestock within Southeast Asia and Australia. Recently, henipavirus-specific antibodies have also been detected in fruit bats in a number of sub-Saharan African countries and in Brazil, thereby considerably increasing the known geographic distribution of henipaviruses. Africa is progressively being recognized as a new high prevalence zone for henipaviruses, as deduced from serological and molecular evidence of past infections in Madagascar, Ghana, Republic of Congo, Gulf of Guinea, Zambia, Tanzania, Cameroon, and Nigeria lately. Serological data suggest henipavirus spillover from bats to livestock and human populations in Africa without reported clinical disease in any of these species. All virus isolation attempts have been abortive, highlighting the need for further investigations. The genome of the Ghanaian bat henipavirus designated Ghana virus (GhV), which was detected in a pteropid Eidolon helvum bat, is the only African henipavirus that has been completely sequenced limiting our current knowledge on the genetic diversity and pathogenesis of African henipaviruses. In this review, we summarize the available data on the circulation of henipaviruses in Africa, discuss potential sources for virus spillover, and highlight existing research gaps.

Changing resource landscapes and spillover of henipaviruses.

Old World fruit bats (Chiroptera: Pteropodidae) provide critical pollination and seed dispersal services to forest ecosystems across Africa, Asia, and Australia. In each of these regions, pteropodids have been identified as natural reservoir hosts for henipaviruses. The genus Henipavirus includes Hendra virus and Nipah virus, which regularly spill over from bats to domestic animals and humans in Australia and Asia, and a suite of largely uncharacterized African henipaviruses. Rapid change in fruit bat habitat and associated shifts in their ecology and behavior are well documented, with evidence suggesting that altered diet, roosting habitat, and movement behaviors are increasing spillover risk of bat-borne viruses. We review the ways that changing resource landscapes affect the processes that culminate in cross-species transmission of henipaviruses, from reservoir host density and distribution to within-host immunity and recipient host exposure. We evaluate existing evidence and highlight gaps in knowledge that are limiting our understanding of the ecological drivers of henipavirus spillover. When considering spillover in the context of land-use change, we emphasize that it is especially important to disentangle the effects of habitat loss and resource provisioning on these processes, and to jointly consider changes in resource abundance, quality, and composition.

Formation of high-order oligomers is required for functional bioactivity of an African bat henipavirus surface glycoprotein

Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic henipaviruses originating from fruit bats in Australia and Asia that can cause severe infections in livestock and humans. In recent years, also African bat henipaviruses were identified at the nucleic acid level. To assess their potential to replicate in non-bat species, several studies were performed to characterize the two surface glycoproteins required for virus entry and spread by cell-cell fusion. It has been shown that surface expression and fusion-helper function of the receptor-binding G protein of Kumasi virus (KV), the prototypic Ghanaian bat henipavirus, is reduced compared to other non-African henipavirus G proteins. Immunostainings and pulse-chase analysis revealed a delayed export of KV G from the ER. As defects in oligomerization of viral glycoproteins can be responsible for limited surface transport thereby restricting the bioactivity, we analyzed the oligomerization pattern of KV G. In contrast to HeV and NiV whose G proteins are known to be expressed at a dimer-tetramer ratio of 1:1, KV G almost exclusively formed stable tetramers or higher oligomers. KV G also showed less stringent requirements for defined stalk cysteines to form dimers and tetramers. Interestingly, any changes in the oligomeric forms negatively affected the fusion-helper activity although surface expression and receptor binding was unchanged. This clearly indicates that the formation of mostly higher oligomeric KV G forms is not a deficiency responsible for ER retention, but is rather a basic structural feature essential for the bioactivity of this African bat henipavirus glycoprotein.

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

Spatiotemporally-localised prediction of virus emergence from wildlife requires focused studies on the ecology and immunology of reservoir hosts in their native habitat. Reliable predictions from mathematical models remain difficult in most systems due to a dearth of appropriate empirical data. Our goal was to study the circulation and immune dynamics of zoonotic viruses in bat populations and investigate the effects of maternally-derived and acquired immunity on viral persistence. Using rare age-specific serological data from wild-caught Eidolon helvum fruit bats as a case study, we estimated viral transmission parameters for a stochastic infection model. We estimated mean durations of around 6 months for maternally-derived immunity to Lagos bat virus and African henipavirus, whereas acquired immunity was long-lasting (Lagos bat virus: mean 12 years, henipavirus: mean 4 years). In the presence of a seasonal birth pulse, the effect of maternally-derived immunity on virus persistence within modelled bat populations was highly dependent on transmission characteristics. To explain previous reports of viral persistence within small natural and captive E. helvum populations, we hypothesise that some bats must experience prolonged infectious periods or within-host latency. By further elucidating plausible mechanisms of virus persistence in bat populations, we contribute to guidance of future field studies.

Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus-receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure-function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.

Fusion activity of African henipavirus F proteins with a naturally occurring start codon directly upstream of the signal peptide

Compared to the fusion proteins of pathogenic Nipah and Hendra viruses, the F protein of prototype African henipavirus GH-M74a displays a drastically reduced surface expression and fusion activity. A probable reason for limited F expression is the unusually long sequence located between the gene start and the signal peptide (SP) not present in other henipaviruses. Such a long pre-SP extension can prevent efficient ER translocation or protein maturation and processing. As its truncation can therefore enhance surface expression, the recent identification of a second in-frame start codon directly upstream of the SP in another African henipavirus F gene (GH-UP28) raised the question if such a naturally occurring minor sequence variation can lead to the synthesis of a pre-SP truncated translation product, thereby increasing the production of mature F proteins. To test this, we analyzed surface expression and biological activity of F genes carrying the second SP-proximal start codon of GH-UP28. Though we observed minor differences in the expression levels, introduction of the additional start codon did not result in an increased fusion activity, even if combined with further mutations in the pre-SP region. Thus, limited bioactivity of African henipavirus F protein is maintained even after sequence changes that alter the gene start allowing the production of F proteins without an unusually long pre-SP.

Evidence for henipavirus spillover into human populations in Africa.

Zoonotic transmission of lethal henipaviruses (HNVs) from their natural fruit bat reservoirs to humans has only been reported in Australia and South/Southeast Asia. However, a recent study discovered numerous HNV clades in African bat samples. To determine the potential for HNV spillover events among humans in Africa, here we examine well-curated sets of bat (Eidolon helvum, n=44) and human (n=497) serum samples from Cameroon for Nipah virus (NiV) cross-neutralizing antibodies (NiV-X-Nabs). Using a vesicular stomatitis virus (VSV)-based pseudoparticle seroneutralization assay, we detect NiV-X-Nabs in 48% and 3–4% of the bat and human samples, respectively. Seropositive human samples are found almost exclusively in individuals who reported butchering bats for bushmeat. Seropositive human sera also neutralize Hendra virus and Gh-M74a (an African HNV) pseudoparticles, as well as live NiV. Butchering bat meat and living in areas undergoing deforestation are the most significant risk factors associated with seropositivity. Evidence for HNV spillover events warrants increased surveillance efforts.

Attachment protein G of an African bat henipavirus is differentially restricted in chiropteran and nonchiropteran cells.

Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1) but not in nonchiropteran BHK-21 and Vero76 cells. Here, we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon coexpression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the nonchiropteran cells was detectable only in a fraction of cells. In contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and nonchiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the endoplasmic reticulum (ER), as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type, as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems, with larger amounts of viral glycoprotein present on chiropteran cells than on nonchiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses, which is necessary to assess the risk of zoonotic virus transmission. Importance: Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a zoonotic potential as high as that of their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein expression of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in nonchiropteran cells than it is in chiropteran cells, explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.

Functional Rectification of the Newly Described African Henipavirus Fusion Glycoprotein (Gh-M74a)

Recent evidence identified multiple Henipavirus species in Africa distinct from those in Southeast Asia and Australia. The reported fusion glycoprotein (F) sequence of the African Gh-M74a strain (GhV-F) is likely incorrect: a single base pair deletion near the N terminus results in multiple aberrancies. Rectifying this by adding single nucleotide insertions results in a GhV-F that now possesses a signal peptide, is efficiently cell surface expressed, exhibits syncytium formation when coexpressed with GhV-G protein, and mediates pseudotyped viral particle entry.

Surface glycoproteins of the recently identified African Henipavirus promote viral entry and cell fusion in a range of human, simian and bat cell lines

The recent discovery of a wide range of henipavirus-like viruses circulating in Megabats in Africa raises the question as to the zoonotic potential of these pathogens given the high human mortality rates seen with their pathogenic relatives Nipah virus and Hendra virus. In the absence of cultured infectious African Henipavirus we have performed experiments with recombinant F and G glycoproteins from the representative African Henipavirus strain M74a aimed at estimating its cellular tropism and capacity to use similar receptors to its highly pathogenic counterparts. The ability of the M74a virus G surface protein to use the ubiquitous Ephrin B2 host cell receptor and its heterologous cross-compatibility with Nipah virus could be expected to impart upon this virus a reasonable potential for species spillover, although differences in fusion efficiency seen with the M74a virus F protein in certain cell lines could present a barrier for zoonotic transmission.

Characterization of African bat henipavirus GH-M74a glycoproteins

In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression.

Surface Glycoproteins of an African Henipavirus Induce Syncytium Formation in a Cell Line Derived from an African Fruit Bat, Hypsignathus monstrosus

Serological screening and detection of genomic RNA indicates that members of the genus Henipavirus are present not only in Southeast Asia but also in African fruit bats. We demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional similarities to glycoproteins of Nipah virus.