# Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar

Sharline Madera, Amy Kistler, Hafaliana Ranaivoson, Vida Ahyong, Angelo

Andrianiaina, Santino Andry, Vololoniaina Raharinosy, Tsiry H.

Randriambolamanantsoa, Ny Anjara Fifi Ravelomanantsoa, Cristina M. Tato, et al.

► To cite this version:

Sharline Madera, Amy Kistler, Hafaliana Ranaivoson, Vida Ahyong, Angelo Andrianiaina, et al. Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar. Journal of Virology, 2022, pp.e00921-22. 10.1128/jvi.00921-22. hal-03765130

HAL Id: hal-03765130

https://hal.science/hal-03765130v1

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1 **Title** Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely virus, from

2 fruit bats in Madagascar.

3

4 **Running Title** Novel Henipavirus, AngV, found in Madagascar fruit bat

5

6 **Authors**

7 Sharline Madera¹, Amy Kistler², Hafaliana C. Ranaivoson³,⁴,⁵, Vida Ahyong², Angelo

8 Andrianiaina⁵, Santino Andry⁶, Vololoniaina Raharinosy⁴, Tsiry H. Randriambolamanantsoa⁴, Ny

9 Anjara Fifi Ravelomanantsoa⁵, Cristina M. Tato², Joseph L. DeRisi²,⁷, Hector C. Aguilar⁸, Vincent

10 Lacoste⁴, Philippe Dussart⁴, Jean-Michel Heraud⁴,⁹, Cara E. Brook³

11

12 **Affiliations**

13 ¹Division of Infectious Diseases, Department of Medicine, University of California, San

14 Francisco, CA, USA

15 ²Chan Zuckerberg Biohub, San Francisco, CA, USA

16 ³Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA

17 ⁴Virology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar

18 ⁵Department of Zoology and Animal Biodiversity, University of Antananarivo, Antananarivo,

19 Madagascar

20 ⁶Department of Entomology, University of Antananarivo, Antananarivo, Madagascar

21 ⁷Department of Medicine and Biochemistry & Biophysics, University of California, San

22 Francisco, CA, USA
23 <sup>8</sup> Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell

24 University, Ithaca, NY, USA

25 <sup>9</sup> Virology Department, Institut Pasteur de Dakar, Dakar, Senegal

26

27 <u>Keywords</u> emerging zoonosis, Henipavirus, novel virus, *Eidolon dupreanum*, bat-borne virus,

28 Madagascar

29

30 <u>Research area</u> Virology, Microbial genetics

31

32 <u>Word count</u>: 4,297

33 <u>Abstract word count</u>: 242 words

34 <u>Importance count</u>: 150 words
36 The genus *Henipavirus* (family *Paramyxoviridae*) is currently comprised of seven viruses, four of 37 which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 38 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit 39 bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus 40 identified in urine samples from wild, Madagascar fruit bats. We report the near-complete 41 16,740 nt genome of AngV, which encodes the six major henipavirus structural proteins 42 (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the 43 phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein 44 and a putative mRNA editing site where the insertion of one or two guanine residues encodes, 45 respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are 46 accessory proteins involved in antagonism of host immune responses during infection.

47 Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat 48 henipaviruses—HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)—but evolved more 49 recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and 50 Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to 51 bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. 52 Identification of the AngV receptor is needed to clarify the virus's potential host range. The 53 presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic 54 following zoonotic spillover.
58 Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described.

61 Here we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus which likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.

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Of these novel HNVs, at least three show evidence of zoonotic potential: serological data suggests prior human exposure to GhV or to an antigenically-related virus in West Africa (13), while MojV was first identified following a human outbreak of severe pneumonia in Chinese mine workers, all of whom died after infection (9). Additionally, LayV was first identified in part with sentinel surveillance of human febrile patients with a history of animal contact, then later found to circulate at high seroprevalence in shrews (12). Thus, in addition to their high potential for pathogenicity, HNVs possess a broad host range that spans at least seven mammalian orders, including bats (10, 14).

Cross-species viral spillover necessitates effective inter-species transmission, which first requires a virus to successfully enter the cells of diverse host species. In general, HNVs use the highly-conserved ephrin family of proteins, both type A and type B, as cell entry receptors (1, 8, 15–17). A notable exception to this pattern is MojV, which does not use ephrin proteins—or the sialic acid and CD150 receptors common to non-HNV paramyxoviruses—to gain cell entry (15, 18). Indeed, as of yet, the viral entry receptor for MojV—and the closely-related GAKV, DARV, and LayV—remain unknown. In general, viruses in the genus *Henipavirus* have broad host ranges and cause high case fatality rates following human spillover, making the characterization of new HNVs a high public health priority.

The HNV genome consists of six structural proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), glycoprotein (G), and polymerase (L). In comparison with other members of the family *Paramyxoviridae*, HNVs have relatively larger genomes (approximately 18kb vs
123 16kb). This extended length is largely due to several, long 3' untranslated regions (UTR) of the
124 N, P, F and G mRNAs (19, 20). The genome length of HNVs, like all paramyxoviruses, adheres to
125 the so-called 'Rule of Six', whereby viral genomes consistently demonstrate polyhexameric
126 length (21). The 'Rule of Six' is believed to be a requirement for efficient genome replication
127 under the unique mRNA editing features of the paramyxovirus genome (21). The paramyxovirus
128 P locus exhibits notable transcription properties that are shared across most members of the
129 Paramyxoviridae family. The P gene permits the translation of additional accessory proteins
130 from either gene editing events within the locus (prior to translation) or an overlapping ORF in
131 the P gene itself. All HNVs, with the exception of CedV, harbor a highly conserved mRNA editing
132 site at which the insertion of additional guanine residues can result in the translation of
133 accessory proteins, V and W, involved in viral antagonism and evasion of the host immune
134 system (1). The HNV P gene also contains an overlapping ORF that allows for the synthesis of a
135 third accessory protein, C, which is also involved in viral host immune evasion (1).

136

137 Our lab has previously presented evidence of exposure to henipa-like viruses in serum collected
138 from three endemic Madagascar fruit bat species (*E. dupreanum*, *Pteropus rufus*, and *Rousettus
139 madagascariensis*) using a Luminex serological assay which identified cross reactivity to
140 CedV/NiV/HeV-G and -F proteins (22). The most significant antibody binding previously
141 detected corresponded to the NiV-G antigen for *E. dupreanum* serum and the HeV-F antigen for
142 *P. rufus* and *R. madagascariensis* serum, suggesting the potential circulation of multiple HNVs in
143 the Madagascar fruit bat system (22). Fruit bats, including *E. dupreanum*, are consumed widely
144 in Madagascar as a source of human food, presenting opportunities for cross-species zoonotic
145 emergence. This underscores the importance of further characterization of the pathogenic and
146 zoonotic potential of AngV and other potential HNVs circulating in the Madagascar fruit bat
147 system. Here, we describe and characterize a novel bat HNV genome, Angavokely virus (AngV),
148 recovered from urine samples collected from the Madagascar fruit bat, *E. dupreanum*. Our
149 work suggests AngV is part of an ancestral group of HNVs that may rely on a novel, non-ephrin-
150 mediated viral entry pathway.

151
152 Methods
153 *Ethics Statement*
154 Animal capture and handling and subsequent collection of biological samples were conducted
155 in strict accordance with the Madagascar Ministry of Forest and the Environment (permit
156 numbers 019/18, 170/18, 007/19) and guidelines posted by the American Veterinary Medical
157 Association. Field protocols were approved by the UC Berkeley Animal Care and Use Committee
158 (ACUC Protocol # AUP-2017-10-10393), as previously described (23).

159
160 *Animal capture, sample collection, and RNA extraction*
161 Fruit bats were captured and processed in part with a long-term study investigating the
162 seasonal dynamics of potentially zoonotic viruses in Madagascar, as has been previously
163 described (22–26). Animals were identified morphologically by species, sex, and age class
164 (juvenile vs. adult), and urine swabs were collected into viral transport medium from any
165 individual that urinated during handling. Urine swabs were flash-frozen in liquid nitrogen in the
166 field and delivered to -80°C freezers at Institut Pasteur de Madagascar for long-term storage.
167 Urine specimens from 206 bats were randomly selected for total RNA extraction using the Zymo

168 Quick RNA/DNA Microprep Plus kit, performed as previously described (23).

169
170 mNGS library preparation

171 Total urine RNAs were diluted with nuclease-free H₂O, and 5uL of each specimen was used as
172 input for mNGS library preparation. A 2-fold dilution series of a 25ng/uL stock of HeLa total RNA
173 (n=8 samples), along with 5 water samples were included and processed in parallel as positive
174 and negative controls, respectively. Additionally, a 25pg aliquot of External RNA Control
175 Consortium (ERCC) spike-in mix (Thermo-Fisher) was included in each sample. Dual-indexed
176 mNGS library preparations for the samples were miniaturized and performed in 384-well
177 format with NEBNext Ultra II RNAseq library preparation kit (New England Biolabs) reagents.

178 RNA samples were fragmented for 12 min at 94°C, and 16 cycles of PCR amplification were
179 performed. Per sample read yields from a small scale iSeq (Illumina) paired-end 2 x 146bp
180 sequencing run on an equivolume pool of the individual libraries were used to normalize
181 volumes of the individual mNGS libraries to generate an equimolar pool. Paired-end 2 x 146bp
182 sequencing of the resulting equimolar library pool was performed on the NovaSeq6000
183 (Illumina) to obtain approximately 50 million reads per sample.

184
185 Sequence analysis

186 Raw reads from urine sample sequencing were first uploaded to the CZBID (v6.8) platform for
187 host and quality filtering and *de novo* assembly (27). In brief, in the CZBID pipeline, adaptor
188 sequences were removed with Trimmomatic (v.0.38), and reads were quality filtered (28).
Reads then underwent host-filtration against the Malagasy fruit bat genome, *E. dupreanum*, using STAR (v 2.7.9a) (29) and a second host-removal step using Bowtie2 (30). After host filtering, reads were aligned using rapsearch2 (31) and Gsnap (32), and putative pathogen taxa were identified. Next, reads were assembled using SPADES (v.3.15.3) (33), and all contigs generated were subject to BLAST analysis against the putative taxa previously identified by rapsearch2 and Gsnap. We considered samples positive for HNV if the CZBID pipeline produced at least one contig with an average read depth of two or more, which yielded a BLAST alignment length >100 nt/aa and an e-value < 0.00001 (BLASTn v2.5.0+) or a bit score >100 (BLASTx v2.5.0+) when queried against an HNV database derived from all HNV genomes available in NCBI (Accessed July 2021).

## Genome Annotation and Comparison

One urine sample, collected from an adult female *E. dupreanum* fruit bat in March 2019, yielded a near full-length HNV genome, which we analyzed in greater depth in subsequent analyses and annotated as the novel HNV, AngV (Genbank Accession #: ON613535). Nucleotide BLAST of the AngV genome identified NiV (GenBank Accession #: AF212302) as the top hit for this novel virus and was subsequently chosen as the reference genome for further analysis. We aligned AngV to NiV (GenBank Accession #: AF212302) in the program Geneious Prime (v2020.2.4) and annotated all six major HNV structural genes, and the accessory C ORF, within the P gene. We identified the putative mRNA editing site within the P gene sequence (spanning nucleotides 1,225-1,232 of the P gene) and manually added one or two guanine (G) residues to the 3' end of the conserved HNV mRNA editing site to generate V and W ORFs, respectively,
211 and their corresponding proteins. We furthered queried all identified transcriptional elements
212 against publicly available sequences using NCBI BLAST and BLASTx (34). Resulting BLAST and
213 BLASTx hits were used in phylogenetic analyses as described below.

214
215 We used the program pySimPlot to scan the whole genome sequence of AngV for nucleotide
216 sequence identity to the NiV genome (GenBank Accession #: AF212302) and the nucleotide and
217 amino acid sequences of individual Open Reading Frames (ORFs) contained therein.

218 Respectively, window size and scanning were specified as 50 and one for nucleotide pairwise
219 identity and 50 and five for amino acid pairwise identity. Results were visualized using Prism
220 (9.2.0).

221
222 Phylogenetic analyses

223 We constructed 10 Maximum Likelihood (ML) amino acid phylogenies to analyze the
224 evolutionary relatedness of our putative HNV to previously described paramyxoviruses. We
225 note that the sequence for the newly-described LayV was not yet available at the time of this
226 writing and is, therefore, absent from all phylogenies. Our (a) first ML phylogeny compared the
227 translated L protein of AngV to all translated reference L protein paramyxovirus sequences in
228 NCBI, in addition to those of the newly-described shrew HNVs, GAKV and DARV (accessed
229 November 2021). The (b) nine subsequent ML phylogenies compared the translation of each
230 individual protein annotated for AngV (N, P, C, V, W, M, F, G, L) against the top 50 BLASTx
231 sequence hits for each protein collapsed on 98% similarity. Distinct outgroups were applied: (a)
232 Sunshine Coast Virus (GenBank Accession #: YP_009094051.1) for the first L protein phylogeny,
233 (b) Human orthopneumovirus (HRSV, GenBank Accession #: NC_001781) for N, P, M, F, G, L
234 proteins, and (c) Sendai virus (GenBank Accession #: NP_056872) for gene C.

235 For each phylogenetic tree, we aligned translated amino acid sequences via the MUSCLE
236 algorithm (v3.8.1551) (35) and determined the best fit amino acid substitution model using
237 ModelTest-NG (36). Phylogenies were then constructed in RAxML-NG (37), using the
238 corresponding best fit model: JTT (complete L-protein sequence) or LG+G4+F (individual
239 proteins). In accordance with best practices outlined in the RAxML-NG manual, twenty ML
240 inferences were made on each original alignment. Bootstrap replicate trees were inferred using
241 Felsenstein's method (38). MRE-based bootstopping test was applied after every 200 replicates
242 (39), and bootstrapping was terminated once diagnostic statistics dropped below the threshold
243 value. Bootstrap support values were drawn on the best-scoring tree.

244 We additionally computed one Bayesian time-resolved phylogeny, using all 77 full-length HNV
245 nucleotide sequences available in NCBI, including our newly contributed AngV (GenBank
246 Accession #: ON613535). As with ML trees, sequences were first aligned in MUSCLE (v3.8.1551)
247 (35), and the best fit nucleotide substitution model was subsequently queried in ModelTest-NG
248 (36). We then constructed a Bayesian timetree in the program BEAST2 (40, 41), using the best
249 fit GTR+I+G4 model inferred for the whole genome alignment from ModelTest-NG and
250 assuming a constant population prior. Sampling dates corresponded to collection data as
251 reported in NCBI Virus; we assumed a collection date of 31-July in cases where only year of
252 collection was reported. We computed trees using both an uncorrelated exponentially
253 distributed relaxed molecular clock (UCED) and a strict clock but here report results from the
strict clock only as similar results were inferred from both. We ran Markov Chain Monte Carlo (MCMC) sample chains for 1 billion iterations, checked convergence using TRACER v1.7 (42) and averaged trees after 10% burn-in using TreeAnnotator v2.6.3 (43) to visualize mean posterior densities at each node. The resulting phylogeny was visualized in R v.4.0.3 for MacIntosh in the 'ggtree' package (44).

We used the Artificial Intelligence system, AlphaFold, to predict the 3D structure of the AngV glycoprotein (G) (45). Molecular graphics and analyses of the AngV glycoprotein structure were performed with UCSF ChimeraX (46). HNV glycoprotein ephrin binding residues were aligned using the program Geneious Prime (v2020.2.4).

## Results

### Discovery and prevalence of HNV in Malagasy bats

Urine swab specimens from 206 bats were collected in 8 roosting sites across the island of Madagascar from 2013 to 2019 (Figure 1). Urine samples were collected during wet and dry seasons from all three Madagascar fruit bat species, *P. rufus*, *E. dupreanum*, and *R. madagascariensis* (Table 1). Isolated RNA from urine swab specimens generated an average of 19 million paired-read sequences. In total, 10/206 (4.9%) bats were positive for HNV; all positive samples were collected from *E. dupreanum* bats (10/106; 9.4%) at the Angavokely cave roosting site (Table 1). Positive samples were collected in wet and dry seasons from both male and female adults.
276
277 *Genomic characterization of AngV*
278 We recovered one near-full-length HNV contig (16,740 nt), supported by an average sequencing
depth of 14 reads (Figure 2A), from a urine sample collected from an adult, non-lactating E.
279 *dupreanum* female in the 2018-2019 wet season (capture date: 15-March 2019). We focused
280 subsequent genomic analyses on this longest sequence, which we named Angavokely virus
281 (AngV) after the site of E. *dupreanum* capture.
282
283
284 As with other members of the *Henipavirus* genus, the genome of AngV is organized into 6 open
285 reading frames (ORF) arranged in the order 3'-N-P-M-F-G-L-5'. AngV shares an average
286 nucleotide identity of 36% with the NiV reference genome (AF212302) and a varying amino acid
287 identity that is highest across the ORFs encoding for the nucleocapsid and L polymerase
288 proteins (Figures 2B, 2C).
289
290 *AngV coding regions*
291 The P gene of AngV follows an organization similar to most members of the *Henipavirus* genus.
292 AngV harbors alternative start sites which, respectively, encode the P and C proteins, as well as
293 a conserved putative mRNA editing site common to most paramyxoviruses A⁴⁻⁶G²⁻³ (Figure 3A
294 and B). AngV shares an identical putative mRNA editing site with the recently discovered HNVs,
295 MojV and GAKV. Pseudotemplated addition of one or two G residues at the conserved putative
296 mRNA editing site generates a putative V and W protein, respectively (Figure 3C; W protein in
297 Supplemental Figure 1). In congruence with members of the *Henipavirus* genus that encode the
298 conserved putative mRNA editing site, the putative V protein of AngV harbors a unique C-

299 terminal region that contains a highly conserved cysteine-rich zinc finger domain (Figure 3C).

300
301 The length of each ORF in the AngV genome resembles those from previously described HNVs,
302 with the exception of the gene encoding the glycoprotein (G)—which, at 688 aa, is 56 aa longer
303 than the longest previously-characterized HNV glycoprotein from GhV (632 aa; Table 2) (16).

304 BLAST analysis indicates that AngV ORFs for genes encoding the nucleocapsid (N), matrix (M),
305 and polymerase (L) proteins exhibit the highest nucleotide and amino acid pairwise identity
306 with other HNVs, with highest similarity shared with the NiV L protein (nt 74.7%, aa 52.2%,
307 Table 2). In contrast to many emerging viruses, AngV largely exhibits higher nucleotide vs.
308 amino acid identity with other HNVs (Table 2). The more recently discovered HNVs (MojV,
309 CedV, GAKV, DARV, GhV) mirror this pattern, showing higher nucleotide vs. amino acid identity
310 when compared to NiV and HeV (data not shown).

311
312 *AngV non-coding regions*
313 Examination of all viral intergenic regions (in cRNA orientation) reveals that AngV exhibits the
314 highly conserved CTT intergenic junction site characteristic of other HNVs, as well as gene stop
315 and gene start sites with high similarity to those of previously described HNVs (Supplemental
316 Table 1). We were unable to locate the intergenic junction site and transcriptional start or stop
317 site in the 5' region of the N ORF for AngV, suggesting that the genomic 3' untranslated regions
318 (UTR) for AngV have not yet been fully recovered. Comparison of the 5' and 3' UTRs for AngV
319 with those of other HNVs reveals UTRs of varying lengths within the *Henipavirus* genus
320 (Supplemental Table 2). Nevertheless, AngV exhibits similar lengths and a nucleotide identity of
321 roughly 30-40% for the 5' and 3' UTRs of most HNV genes; however, the P gene 3' UTR, the M
322 gene 5' and 3' UTRs, and the F gene 5' and 3' UTRs, are significantly shorter in AngV compared
323 with previously described HNVs. Correspondingly, nucleotide identity varies when comparing
324 this shorter subset of 5' and 3' UTRs for AngV against other HNVs (Supplemental Table 2).
325

326

Phylogenetic Analyses

327 Phylogenetic analysis of complete L protein amino acid sequences across the *Paramyxoviridae*
328 family places AngV within the *Henipavirus* genus at <0.82 nucleotide substitutions away from
329 the node distinguishing the family *Paramyxoviridae* from the *Sunviridae* (Figure 4A). AngV
330 clusters independently within the *Henipavirus* genus and diverges ancestral to all currently
331 known bat-borne HNVs. Our time-resolved Bayesian phylogeny further corroborates this result,
332 placing AngV ancestral to all previously described bat-borne HNVs but more recently diverged
333 than the rodent- and shrew-borne HNVs, MojV, GAKV, and DARV (Figure 4B; Supplemental
334 Figure 2). We estimate the divergence of the AngV lineage from the rest of the HNV clade at
335 9,794 years ago (95% HPD 6,519-14,024 years), and the time to the Most Recent Common
336 Ancestor (MRCA) for the entire HNV genus as 11,195 years ago (95% HPD 7,351-15,905 years).
337

338

338 In N, P, C, V, W, M and G amino acid phylogenies, the AngV proteins cluster closely with those
339 of other HNVs (Supplemental Figure 3). Interestingly, in the amino acid phylogeny, the AngV F
340 protein, like the F proteins of MojV, GAKV, and DARV, localizes ancestral to non-HNV
341 paramyxoviruses and distinct from the bat-borne HNV clade (Supplemental Figure 3). The AngV
342 L protein shows the highest amino acid identity to the L protein of rodent-borne Mount Mabu

343 Lophuromys virus 2 (MMLV-2), a putative Jeilongvirus (47), but is nonetheless nested between

344 the MojV/GAKV/DARV clade and the bat-borne HNV clade (Supplementary Figure 3).

345
346 AngV glycoprotein
347 We further examined the AngV G protein for conserved structural features and amino acid
348 residues historically associated with HNV ephrin binding. AlphaFold analysis predicted a six-
349 bladed β-propeller fold that is characteristic of *Paramyxoviridae* glycoproteins, with each blade

350 largely composed of 4 antiparallel β-strands (Figure 5A). The β-propeller fold is stabilized by
351 seven disulfide bonds that are conserved among HNVs (Supplemental Figure 4). This HNV
352 protein G structure-based alignment reveals that the elongated AngV G protein primarily
353 results from a lengthy C-terminal tail with an additional 67 aa beyond that of NiV G protein
354 (Supplemental Figure 4). Similar to the MojV G protein, the AngV G protein lacks previously
355 described ephrin binding residues (NiV W504, E505, Q530, T531, A532, E533, N557, and Y581)
356 (Figure 5B, 5C, and Supplemental Figure 4) (17, 48, 49).

357
358 Discussion
359 We describe and characterize a novel HNV, AngV, from a urine sample collected from an *E. dupreanum* Malagasy fruit bat. In this study, urine samples from 206 unique fruit bats were assessed by metagenomic sequencing, yielding an overall positive HNV detection rate of 4.9% 360
361 (10/206) for all bats studied and a HNV prevalence of 9.4% (10/106) for the *E. dupreanum* 362
363 hosts. Of all the HNV positive samples, only one sample yielded sufficient reads for assembly of
364 a complete coding sequence and subsequent genomic analysis. In a 6-year collection period
365 spanning multiple wet/dry seasons, HNV positive samples were only recovered from *E.*
366 *dupreanum* bats in the Angavokely roosting site, despite prior serological evidence of HNV
367 infection in *P. rufus* and *R. madagascariensis* bats, as well (22). HNV RNA was recovered from *E.*
368 *dupreanum* in both wet and dry seasons, though higher sampling intensity throughout the wet
369 season precludes any conclusions regarding underlying seasonal patterns in these data.
370 Previous work in this system has suggested a seasonal increase in fruit bat seroprevalence
371 across the winter low nutrient season, which also overlaps the gestation period for these
372 synchronously breeding fruit bats (22). In fruit bat systems elsewhere, HNVs are also shed in
373 urine at higher rates during the nutrient-poor dry seasons for the localities in question (50–52);
374 in the case of NiV and HeV, these seasonal viral shedding pulses have been linked to zoonotic
375 spillover.
376
377 The recovered genome of AngV exhibits a structural organization characteristic of the
378 *Henipavirus* genus and a nucleotide and amino acid identity to HeV and NiV that is comparable
379 to those shared with the more distantly related HNVs, MojV, GhV and CedV. A limited quantity
380 of available original sample precluded full genome recovery for AngV (as evidenced by the lack
381 of the 5' UTR region of the N ORF), which prevented analysis of the extent to which the full
382 AngV genome may abide by the 'Rule-of-Six', observed by all other members of the
383 *Orthoparamyxovirinae* subfamily (53). Phylogenetic analyses of AngV support classification of
384 this virus as a distinct novel bat-borne *Henipavirus* (L gene amino acid distance <0.82 distance
for the subfamily *Orthoparamyxovirinae*), in accordance to the International Committee on Taxonomy of Viruses (ICTV) criteria (20). This novel HNV is estimated to have diverged approximately 9,800 years ago, prior to the currently known African and Asian bat-borne HNV lineages but considerably more recently than the estimated mid-to late-Miocene divergence of *E. dupreanum* from its sister species, *E. helvum*, on the African continent (54). Recent characterization of *Betacoronaviruses* in Madagascar fruit bats demonstrates surprising identity to lineages circulating in West Africa (23), suggesting that, though resident only in Madagascar, Malagasy fruit bats likely experience some form of contact with the African continent. Of the 49 bat species that inhabit the island nation of Madagascar, nine species are widely distributed across Africa, Asia, and/or Europe, presenting opportunities for inter-species viral transmission via island-hopping. Intensified viral sampling of Madagascar's insectivorous bat populations for HNVs thus represents an important future research priority.

As an ancestral bat-borne HNV, AngV may provide important insight into HNV evolution and pathogenesis. Similar to other paramyxoviruses, the encoded AngV P gene is able to produce multiple immunomodulatory protein products (55). One such protein product is the V protein, thought to be involved in immune evasion and considered a significant determinant of viral pathogenicity and lethality (56, 57). AngV harbors the highly conserved mRNA editing site and a predicted ORF that encodes a V protein with a conserved cysteine-rich C-terminus, suggesting that AngV has the capacity to produce a functional V protein. With the exception of the newly discovered HNVs in shrews, GAKV and DARV, all HNVs harboring a V protein have previously demonstrated evidence of human infection, highlighting the potential for AngV to cause
407 productive infection in humans (1, 9, 13). Further studies are needed to ascertain the virulence
408 potential and host breadth of this novel virus.

409
410 Characterization of the AngV glycoprotein (G) through AlphaFold modeling and structure-based
411 alignments revealed a similar structural organization to other HNV glycoproteins. Notably, the
412 AngV glycoprotein surpasses that of GhV as the longest glycoprotein of the *Henipavirus* genus.
413 Like that of GhV, the AngV glycoprotein harbors a long C terminal extension (Supplemental
414 Figure 4). It is unclear if the C terminal extension of the AngV glycoprotein has a functional role,
415 though the C terminal extension of the glycoprotein in GhV is known to play a functional role in
416 receptor-mediated fusion (16).

417
418 Henipavirus host tropism and virulence rely on a myriad of factors, one of which is the HNV
419 glycoprotein. The previously characterized HNV glycoproteins of NiV, HeV, CedV, and GhV,
420 utilize members of the ephrinA and ephrinB class family as host-cell receptors for viral entry
421 into human cells (16–18, 48, 58). However, like MojV, the AngV glycoprotein lacks these well-
422 conserved ephrin binding residues. Structure-based alignments can shed light on potential
423 receptor binding residues when characterizing novel viruses. For instance, sequence-based
424 comparisons of the GhV and NiV glycoproteins were used to predict GhV ephrin binding (13),
425 which was later confirmed by crystallography (16). Structure-based alignment of the AngV
426 glycoprotein shows a lack of highly conserved ephrin binding residues, including NiV E533 – a
427 seminal residue for ephrinB2 binding that is conserved across all ephrin binding HNVs. This
428 suggests that, like MojV—and probably DARV, GAKV, and LayV—the AngV glycoprotein may not
429 bind ephrins, pointing to the possible use of an ancestral viral entry pathway. The growing
430 number of novel HNVs that appear not to rely on ephrin binding for cellular entry could warrant
431 re-evaluation of the existing HNV genus to better reflect conserved function and pathobiology.

432
433 This work presents a novel bat-HNV, AngV, identified from a Malagasy fruit bat. AngV joins a
434 growing group of ancestral HNVs with unknown cell-entry receptors. Discovery of the cell
435 surface receptor for AngV represents an important future research priority that will shed light
436 on the breadth of host range for this virus, including its zoonotic potential.

437
438
439
440
441 Acknowledgements
442 The authors thank Anecia Gentles and Kimberly Rivera for help in the field and the lab and
443 acknowledge the Virology Unit at the Institut Pasteur de Madagascar and Maira Phelps of the
444 Chan Zuckerberg Biohub (CZB) for logistical support. They additionally thank Angela Detweiler,
445 Michelle Tan, and Norma Neff of the CZB genomics platform for mNGS support. Molecular
446 graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for
447 Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with
448 support from the National Institutes of Health R01-GM129325 and the Office of Cyber
449 Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

450
451 Funding

452 This work was supported by the National Institutes of Health (1R01AI129822-01 grant to JMH,

453 PD, and CEB; 5T32AI007641-19 to SM; R01AI109022 to HAC), DARPA (PREEMPT Program

454 Cooperative Agreement no. D18AC00031 to CEB), the Bill and Melinda Gates Foundation

455 (GCE/ID OPP1211841 to CEB and JMH), the Adolph C. and Mary Sprague Miller Institute for

456 Basic Research in Science (postdoctoral fellowship to CEB), AAAS/L'Oréal USA (For Women in

457 Science fellowship to CEB), the Branco Weiss Society in Science (fellowship to CEB), and the

458 Chan Zuckerberg Biohub.

459

460 Conflict of Interests

461 The authors declare no competing interests.

462

463 Data Availability

464 Raw and assembled sequencing data are deposited in NCBI Bioproject PRJNA837298. The full

465 genome of AngV is available in GenBank under Accession # ON613535. All raw data and code

466 for figures can be obtained in our open-access GitHub repository:

467 https://github.com/brooklabteam/angavokely-virus

468

469

470

471

472
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664
665
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675
676
677 **Table 1.** Prevalence of HNV infections in the urine of Malagasy bats captured during 2013-2019

678 collection period.

<table><thead><tr><th rowspan="2">Roost site</th><th rowspan="2">Species</th><th colspan="3">By Site</th><th colspan="2">By Season/Sex</th></tr><tr><th>Total sampled</th><th>Total henipavirus positive</th><th>Season</th><th>Total sampled # (M,F)</th><th>Total henipavirus positive # (M,F)</th></tr></thead><tbody><tr><td rowspan="5">Ambakoana</td><td rowspan="5">P. rufus</td><td rowspan="5">18</td><td rowspan="5">0</td><td>wet '13</td><td>2 (1,1)</td><td rowspan="5">0 (0,0)</td></tr><tr><td>wet '14/'15</td><td>3 (3,0)</td></tr><tr><td>wet '15/'16</td><td>2 (2,0)</td></tr><tr><td>wet '17/'18</td><td>2 (2,0)</td></tr><tr><td>wet '18/'19</td><td>9 (6,3)</td></tr><tr><td rowspan="2">Angavobe</td><td rowspan="2">E. dupreanum</td><td rowspan="2">8</td><td rowspan="2">0</td><td>wet '17/'18</td><td>5 (1,4)</td><td>0 (0,0)</td></tr><tr><td>dry '18</td><td>3 (1,2)</td><td>0 (0,0)</td></tr><tr><td rowspan="4">Angavokely</td><td rowspan="4">E. dupreanum</td><td rowspan="4">98</td><td rowspan="4">10</td><td>wet '15/'16</td><td>2 (1,1)</td><td>0 (0,0)</td></tr><tr><td>wet '17/'18</td><td>38 (5,33)</td><td>4 (0,4)</td></tr><tr><td>dry '18</td><td>11 (8,3)</td><td>2 (1,1)</td></tr><tr><td>wet '18/'19</td><td>47 (20,27)</td><td>4 (2,2)</td></tr><tr><td>Mahabo</td><td>P. rufus</td><td>8</td><td>0</td><td>dry '14</td><td>8 (4,4)</td><td>0 (0,0)</td></tr><tr><td>Mahialambo</td><td>P. rufus</td><td>5</td><td>0</td><td>wet '18/'19</td><td>5 (2,3)</td><td>0 (0,0)</td></tr><tr><td>Makira</td><td>P. rufus</td><td>2</td><td>0</td><td>dry '15</td><td>2 (2,0)</td><td>0 (0,0)</td></tr><tr><td rowspan="3">Marovitsika</td><td rowspan="3">P. rufus</td><td rowspan="3">62</td><td rowspan="3">0</td><td>wet '17/'18</td><td>9 (3,6)</td><td rowspan="3">0 (0,0)</td></tr><tr><td>dry '18</td><td>7 (3,4)</td></tr><tr><td>wet '18/'19</td><td>46 (20,26)</td></tr><tr><td rowspan="3">Maromizaha</td><td rowspan="3">R. madagascariensis</td><td rowspan="3">5</td><td rowspan="3">0</td><td>wet '13</td><td>2 (1,1)</td><td rowspan="3">0 (0,0)</td></tr><tr><td>dry '14</td><td>1 (0,1)</td></tr><tr><td>wet '14/'15</td><td>2 (2,0)</td></tr></tbody></table>

679

680

681

682

683

684

685

686
<table><caption>Table 2. Length and pairwise sequence identity of predicted open reading frames of AngV and other HNV.</caption><thead><tr><th>Gene</th><th>AngV<br>length aa</th><th>NiV<br>length aa<br>(%nt, %aa)</th><th>HeV</th><th>MojV</th><th>CedV</th><th>GAKV</th><th>DARV</th><th>GhV</th></tr></thead><tbody><tr><td>N</td><td>514</td><td>532<br>(56.4, 48.0)</td><td>532<br>(55.4, 46.7)</td><td>539<br>(55.8, 45.3)</td><td>510<br>(57.5, 48.3)</td><td>533<br>(58.0, 47.5)</td><td>574<br>(55.3, 24.0)</td><td>514<br>(56.2, 48.9)</td></tr><tr><td>P</td><td>693</td><td>709<br>(47.8, 25.1)</td><td>707<br>(48.1, 24.1)</td><td>694<br>(47.6, 23.6)</td><td>737<br>(42.3, 21.6)</td><td>586<br>(43.8, 22.5)</td><td>698<br>(47.3, 23.2)</td><td>870<br>(42.2, 23.1)</td></tr><tr><td>C</td><td>173</td><td>166<br>(51.3, 25.7)</td><td>166<br>(51.5, 25.1)</td><td>177<br>(47.6, 22.5)</td><td>177<br>(54.5, 29.8)</td><td>184<br>(47.4, 27.5)</td><td>175<br>(48.1, 24.0)</td><td>163<br>(46.1, 23.1)</td></tr><tr><td>V</td><td>461</td><td>456<br>(46.9, 22.8)</td><td>457<br>(48.5, 23.6)</td><td>464<br>(47.2, 24.5)</td><td></td><td>370<br>(43.0, 22.3)</td><td>468<br>(47.2, 22.2)</td><td>621<br>(24.7, 19.8)</td></tr><tr><td>W</td><td>412</td><td>450<br>(47.0, 21.3)</td><td>448<br>(45.0, 20.0)</td><td>435<br>(46.5, 22.5)</td><td></td><td>331<br>(43.9, 22.7)</td><td>437<br>(45.5, 21.1)</td><td>572<br>(39.3, 16.8)</td></tr><tr><td>M</td><td>354</td><td>352<br>(60.0, 54.6)</td><td>352<br>(59.5, 54.0)</td><td>340<br>(57.1, 53.4)</td><td>360<br>(56.9, 53.1)</td><td>340<br>(56.1, 50.8)</td><td>345<br>(56.5, 52.3)</td><td>343<br>(56.9, 51.4)</td></tr><tr><td>F</td><td>539</td><td>546<br>(53.7, 39.4)</td><td>546<br>(51.7, 39.4)</td><td>545<br>(52.2, 40.6)</td><td>557<br>(51.8, 36.3)</td><td>565<br>(53.3, 39.4)</td><td>545<br>(58.8, 40.1)</td><td>662<br>(45.5, 35.4)</td></tr><tr><td>G</td><td>688</td><td>602<br>(43.5, 21.4)</td><td>604<br>(43.8, 20.4)</td><td>625<br>(43.0, 21.2)</td><td>622<br>(43.3, 19.8)</td><td>635<br>(44.9, 17.8)</td><td>628<br>(44.3, 18.7)</td><td>632<br>(42.5, 19.8)</td></tr><tr><td>L</td><td>2,259</td><td>2,244<br>(74.7, 52.2)</td><td>2,244<br>(57.6, 51.9)</td><td>2,277<br>(57.5, 51.8)</td><td>2,501<br>(52.4, 44.8)</td><td>2,291<br>(57.7, 51.0)</td><td>2,271<br>(58.8, 51.3)</td><td>2,250<br>(57.0, 49.1)</td></tr></tbody></table>
702 **Figure Legends**

703 **Figure 1.** Geographic location of sampling sites used in this study. Sampling sites grouped by bat

704 species found depicted as follows *P. rufus* (pink circles) Ambakoana (–18.51 S, 48.17 E) /

705 Mahabo (-20.46 S, 44.68 E) / Mahialambo (-18.11 S, 48.21 E) / Makira (-15.11 S, 49.59 E) /

706 Marovitsika (-18.84 S, 48.06 E) roosts; *E. dupreanum* (green triangles) Angavobe (–18.94 S, 47.95 E) /Angavokely (–18.93 S, 47.76 E) caves; *R. madagascariensis* (orange squares)

708 Maromizaha cave (–18.96 S, 48.45). Dashed lines link each sampling site to corresponding pie

709 charts, which show the percentage of HNV positive specimens for all sampled species and sites.

710 Dashed lines are included for visualization purposes only, and variation in length is not
711 significant. Pies are size-weighted by the total bat population sampled at each site,
712 corresponding to the legend. HNV positive samples were only recovered from the *E.*
713 *dupreanum* Angavokely site.

714
715 **Figure 2.** AngV genome organization. A. Coding regions for each gene are shown and depicted
716 in color, non-coding intergenic and terminal regions are highlighted in gray. Depicted genes
717 represented as follows: nucleocapsid (N), RNA polymerase (P and L), matrix (M), fusion (F), and
718 glycoprotein (G). Sequencing read depth supporting each position of the recovered genome
719 sequence is plotted below the genomic schematic. Scanning nucleotide (B) and amino acid (C)
720 pairwise identity to Nipah virus (GenBank Accession #: AF212302). Dotted horizontal lines
721 represent average read depth (14.29) or average nucleotide pairwise identity (36%).

722
**Figure 3.** Organization of the P gene of AngV. A. Alternative transcriptional start sites (pink triangle) generate the P and C protein. Pseudotemplated addition of one or two guanine nucleotides at the putative mRNA editing site generates a V and W protein, respectively. B. Sequence alignment of the putative mRNA editing site across members of the *Henipavirus* genus (cRNA depicted). C. Amino acid alignment of the unique C terminal region of the V protein following the addition of one guanine nucleotide to the putative mRNA editing site.

Gray boxes denote conserved cysteine and histidine residues suggested to directly coordinate bound zinc ions (55). Individual nucleotides or amino acids are color coordinated if at least 75% conserved at the alignment position. Nucleotide or amino acid position numbers displayed represent the position within the AngV gene or protein. Virus name (abbreviation), followed by GenBank Accession #: Angavokely virus (AngV) ON613535; Nipah virus (NiV) AF212302; Hendra virus (HeV) AF017149; Mojiang virus (MojV) KF278639; Ghanaian bat Henipavirus (GhV) HQ660129; Daeryong virus (DARV) MZ574409; Gamak virus (GAKV) MZ574407, Cedar virus (CedV) JQ001776. CedV is shown here only for comparison, as the CedV P protein is not believed to undergo RNA editing or to generate a functional V protein (8, 55).

**Figure 4.** A. Phylogenetic analysis of the complete L protein sequences of members of the family *Paramyxoviridae*. We note that the sequence for the newly-described Langya henipavirus (LayV) (12) was not yet available at the time of this writing and is, therefore, not included in the phylogenies. Tree is rooted with Sunshine Coast Virus (GenBank Accession #: YP_009094051.1) as an outgroup, with outgroup branch length shrunk for ease of viewing.

Novel HNV, AngV, is depicted in green. Subfamilies and genera are demarcated, excluding those
745 unassigned to subfamily (genera *Scolidonvirus*, *Cynoglossusvirus*, *Hoplichthysvirus*). Bootstrap
746 support is depicted and GenBank Accession numbers displayed next to virus names. Scale bar
747 represents substitutions per site. B. Time-resolved Bayesian phylogeny computed in BEAST2
748 incorporating all available *Henipavirus* whole genome nucleotide sequences, with the addition
749 of newly discovered GAKV, DARV, and AngV. Closely-related sequences are collapsed at triangle
750 nodes for NiV and HeV (phylogeny with un-collapsed branches available in Supplemental Figure
751 2). 95% HPD intervals around the timing of each branching node are visualized as red horizontal
752 bars. Posterior support >.9 is indicated by black coloring of the corresponding node, and distinct
753 *Henipavirus* species are indicated by colored tip points, with AngV highlighted in yellow for
754 further emphasis. The estimated time to MRCA for Angavokely virus and the previously-
755 described bat-borne HNVs is 9,794 (95% HPD 6,519 – 14,025) years ago.

756
757 **Figure 5.** AlphaFold-predicted AngV glycoprotein 3D structure and ephrin binding residue
758 sequence alignment. A. AlphaFold-predicted 3D structure of AngV glycoprotein. N and C
759 termini are indicated in white text, and residues corresponding to ephrin binding sites in other
760 HNVs that are not conserved in AngV are colored green. B. Alignment of HNV ephrin binding
761 residues. The position of previously-described HNV ephrin binding residues are noted by a star,
762 and residues conserved across most HNVs are highlighted yellow. Amino acid position numbers
763 displayed represent the position within the AngV or NiV glycoproteins. Virus name
764 (abbreviations) followed by GenBank Accession #: Angavokely virus (AngV) ON613535; Nipah
765 virus (NiV) AF212302; Hendra virus (HeV) AF017149; Mojiang virus (MojV) KF278639; Cedar
766 virus (CedV) JQ001776; Ghanaian bat Henipavirus (GhV) HQ660129; Gamak virus (GAKV)
767 MZ574407; Daeryong virus (DARV) MZ574409. C. AlphaFold detail from full structure prediction

768 for AngV. Localization of ephrin binding sites conserved across most HNVs is colored green and

769 labeled corresponding to position in the NiV and AngV genomes.
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henipavirus pos

henipavirus neg

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Pteropus rufus

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Eidolon dupreanum

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Rousettus madagascariensis

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60 km
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AUG

mRNA editing site

V

W

C

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mRNA editing site

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AngV KKGHKRNYNIQWDGKFVTVYRWCTPNCREDRPTTTADINCICGRCPIKCPKCF--E

NiV KKGHRREISICWDGKRAWVEEWCNPACSRITPLPRRQECCQCGECPTECFH-C--G

HeV KKGHRREVSIWCDDRRRAWVEEWCNPVCSCRITPQPRKQECYCGECPTECSQCCHE--E

MojV KKGHRREYSLCWDGSEIKVEEWCNPICSVKNSEPSREKCTCKQCPIMCQDEHCTKIEY--E

GhV KRGHRREFLMYWDKGRLKTIEWCNPICTKIQIEPETGECICGECPTWCDQCTI--

GAKV KKGHRKEYQLCWDGERIDINEWCNPCTCTSIKPDEGQQTCICGQCPLYCEDPNCNGIIK--G

DARV KKGHRREYSLSWDGSEIRVEEWCNPSCARIKSCATRERCKCRTCPRYCQQEDCTGIEYDTK

CedV KMRYKEIDICWDGNGVWTNEYWVRRSTRIKG-WDF
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A

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B

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C

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