key: cord-0291327-ofn6aif9
authors: Simsek, Ceren; Corman, Victor Max; Everling, Hermann Ulrich; Lukashev, Alexander N.; Rasche, Andrea; Maganga, Gael Darren; Binger, Tabea; Jansen, Daan; Beller, Leen; Deboutte, Ward; Gloza-Rausch, Florian; Seebens-Hoyer, Antje; Yordanov, Stoian; Sylverken, Augustina; Oppong, Samuel; Sarkodie, Yaw Adu; Vallo, Peter; Leroy, Eric M.; Bourgarel, Mathieu; Yinda, Kwe Claude; Van Ranst, Marc; Drosten, Christian; Drexler, Jan Felix; Matthijnssens, Jelle
title: At least seven distinct rotavirus genotype constellations in bats with evidence of reassortment and zoonotic transmissions
date: 2020-10-26
journal: bioRxiv
DOI: 10.1101/2020.08.13.250464
sha: fd7b1aa9d86c83102e9cca786f5179c6c6d86d30
doc_id: 291327
cord_uid: ofn6aif9

Bats host many viruses pathogenic to humans, and increasing evidence suggests that Rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America and Africa were PCR-positive for RVA and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, including evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite varying levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses. Importance The increased research on bat coronaviruses after SARS-CoV and MERS-CoVallowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents shed light on the vast genetic diversity of rotaviruses and also hinted at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

13 rhinolophid bats, whereas the Chinese MSLH14-like strains were found in bats from the 283 Rhinolophidae, Hipposideridae and Emballonuridae families (Table S4a) . 284

In addition to RVAs potentially being able to infect multiple bat families, individual bat 285 families could also harbor more than one GC, as is shown in Table S4c . Pteropodid bats 286 harbor completely unique GCs (green and yellow), suggesting that the associated RVA 287 strains have a high epidemiologic fitness in these populations. This further indicates that 288 the Pteropodidae, which includes the straw-colored fruit bats, has been a substantial 289 virus reservoir for a long time already, as also shown for Marburg virus, Hendra and 290

Nipah viruses (12) (13) (14) . 291 292

The global distribution of the bat RVA GCs revealed several patterns regarding RVA 294 circulation in bats, as shown in Figure 1 . Bat RVAs belonging to the brown, purple, blue 295 and dark grey GCs have so far only been identified in Costa Rica (and perhaps Brazil), 296

Gabon, Kenya and China, respectively. On the other hand, the green and yellow GCs 297 were confirmed to be further dispersed, from Cameroon to Saudi Arabia (G25P[43]), and 298 from Ghana and Cameroon to Zambia, respectively, as was previously suggested by 299 Sasaki et al. (51) . However, highly similar RVA strains belonging to the orange MSLH14-300

like GCs span at least 3 different continents and subcontinents, e.g. Asia, Europe and 301 possibly Central America. Furthermore, it was also shown that RVA strains with distinct 302

GCs could co-circulate in the same region, as is the case in Cameroon (green, yellow 303 and purple GCs) and China (orange and dark grey GCs) (Figure 1) . 304

With powered flight, migratory bats can travel long distances between summer and 305 winter roosts, for foraging and searching for a mate (52). Among long-distance migratory 306 14 bats, E. helvum can cover a range of 270 to 2,500 km (53), vespertilionid 'tree bats' and 307 the subtropical/tropical molossid bats can fly over 1,000 km (54, 55) . Global distribution 308 and intercontinental bat virus transfers are also typical to other bat viruses (56) . In 309 addition to migration across vast distances, the fact that some distinct GCs seem to 310 have overlapping geographical ranges (such as in China and West Africa in Figure 1 ) 311 suggest a fitness advantage for these particular genotypes occurring together. However, 312

there is also ample evidence of gene reassortment events among established GCs (e.g. 313 P[47] in green and brown GC; or I8 in purple and orange GC), or with RVA strains of 314 currently unknown origin (e.g. A29, A15, E27). 315

It is clear that more bats should be sampled in order to have a comprehensive 316 understanding of the driving and restricting forces of bat RVA genetic diversity, or the 317 lack thereof. The detection of P[47] reassortment between Ghanaian and Costa Rican 318 bat RVAs, which are located more than 9,000 km's apart, cannot only be explained by 319 the flight ability of bats, but rather the lack of sampling between these 2 locations. We 320 hypothesize that with the increasing bat RVA sequencing efforts, the geographical and 321 host range of most GCs (such as the blue, dark grey, yellow and brown) will be 322 significantly expanded. 323 324

We further investigated whether there is potential for unusual RVA strains detected in 326 other mammals (including humans) to be a result of an interspecies transmission from 327 bat strains identified in the current and other studies (Table 3) . In 2013, Miño and colleagues reported an unusual Argentinian equine G3P [3] RVA 331 strain RVA/Horse-wt/ARG/E3198/2008/G3P [3] . Based on the GC, it was speculated to 332 have a common ancestor with both feline/canine RVA strains, as well as the unusual 333 rhesus RVA strain RRV. However, the nucleotide identities were below the 90% for most 334 of the genome segments, suggesting that the original host may not be identified yet (35). 335

When more bat RVA genomes became available in subsequent years, Xia and 336 colleagues, and later also Biao He and colleagues, suggested that E3198 might be of 337 bat origin, based on the GCs and nucleotide similarities (25, 26 , nearly identical to SA11-H96 ( Figure S2b ) was deposited in GenBank 353 as a potential vaccine candidate. However, the controversy about the origin of these 354 SA11-like strains (SA11-H96, B10 and ZTR-5) remained. To our surprise, the purple GC 355 described in this paper, containing only the bat RVA strains from Gabon, showed up to 356 seven genotypes in common with these SA11-like strains (Table 3) , with varying 357 degrees of nucleotide similarities (Figures S2b, c) . According to phylogenetic analyses 358 the bat RVAs from Gabon and Kenya clustered with B10 for the VP1, VP6, NSP4 gene 359 segments, and with all 3 strains (B10, SA11-H96 and ZTR-5) for VP2-4, NSP1, NSP3 360 and NSP5 (Figures 2-4) . 361

Not only for SA11-H96, but also for RVA/Simian-tc/USA/RRV/1975/G3P [3] The finding that the purple SA11-like GC was found in multiple bats in Gabon, and only 368 on a single occasion in vervet monkeys and in 2 unrelated human cases, makes bats 369 the prime suspect of being the major hosts of these viruses, making the monkey and 370 humans strains putative examples of interspecies transmissions. It should however be 371 noted that the phylogenetic clustering between these bat, simian and human strains is 372 still rather variable, and the nucleotide similarities are not as high as between bat RVA 373 strains and RVA/Horse-wt/ARG/E3198/2008/G3P [3] ( Figure S2a, Figures 2-4) , 374

suggesting that more RVAs from currently unsampled animal species will likely cluster in 375 between. However, 2 other bat strains are of further interest: 1) the bat RVA strain 376 [3] , detected in a 2 year-old severely diarrheic patient in 394

Thailand (41). It was reported to have a VP7 gene closely related to RVA/Simian-395 tc/USA/RRV/1975/G3P [3] and a VP4 gene that was caprine-like. Subsequently, Xia and 396 colleagues speculated that this strain is distinct from typical human RVA GCs and very 397 likely shared a common ancestor with Asian bat RVAs (33). Our study provides further 398 evidence for the bat origin of CMH222, as the VP6 I8 genotype of CMH222 is closely 399 related to RVA/Bat-wt/GAB/GKS-897/2009/G3P [3] (Figure 3) . 400

Later on, Wang Despite the limited number of bat species that have been screened for rotaviruses, a 429 surprisingly large genetic diversity of RVA strains is presented in this study, including 6 430 novel genotypes. With increasing screening efforts, it is without a doubt that this diversity 431 will expand both genetically and geographically. We also presented multiple examples of 432 close genetic relatedness of several mammalian and bat rotaviruses. The indicated 433 zoonoses has -to the best of our knowledge -always been restricted to sporadic cases 434 so far and has never resulted in major outbreaks in humans. However, it is believed that 435 the rotavirus genotype constellations currently circulating in humans also have a 436 common ancestor with animal rotaviruses, highlighting that interspecies transmissions 437 followed establishment in the human population could happen again (3). 438

Another notable finding is that several gene segments of bat RVA strains and the simian 439 SA11 RVA strain (the latter being used in global rotavirus research for decades), have a 440 common origin. Furthermore, SA11 strain has been recently used as the backbone of a 441 RVA reverse genetics system, and is therefore likely to be used even more in the future. 442

It would be intriguing to test whether or not SA11 grows well in bat cell lines, or in in vivo 443 infection experiments. 444

Fecal samples were collected from 2,142 bats from 10 bat families, representing 46 bat 448 species (Table S2) bats, such as coronavirus, astrovirus, and picornavirus, as described previously (56, 63-451

Rican studies, bats were caught with mist nets, put into cotton bags and fecal pellets are 453 collected. Ghanaian fecal droppings were collected with plastic foil from the trees in 454 which E. helvum bats were roosting. The pellets were kept in RNAlater RNA stabilization 455 solution (QIAGEN, Hilden, Germany). Gabonese bats were also captured with mist nets 456 just before twilight and were individually euthanized. Bat feces were collected with the 457 corresponding permissions of the host countries in all of the studies. 458

Viral RNA was isolated from the fecal specimens as described previously (65). To 461 screen the RVA presence in bats, conserved RVA-specific primer pairs targeting the 462 VP1 gene were used (277 nucleotide long PCR product) in a hemi-nested and single 463 round reverse transcription (RT-PCR) assay (Table S1 ). Among the 18 positive 464 specimens (Tables S2-S3) , 16 fecal samples, of which sufficient material was left, were 465

shipped to the Laboratory of Clinical and Epidemiological Virology, Leuven, Belgium on 466 dry ice, for further complete genome analyses (Table 1) . 467

The NetoVIR protocol was used for viral enrichment of the fecal suspensions as 468 described before (67). Briefly, the fecal samples were suspended in dPBS and 469 homogenized with a MINILYS homogenizer (Bertin Technologies) for 20s at 3,000 rpm. The genotypes were assigned using RotaC tool (http://rotac.regatools.be). The 498 sequences whose genotypes could not be determined were sent to the RCWG for 499 assignment of novel genotypes. 500

Reference strains were downloaded from Genbank in order to represent all the relevant 501 genotypes per gene segment. Codon-based nucleotide level multiple sequence 502 alignments were done using MUSCLE (76) Table 3 . 852 

strain with multiple genes related to bat rotaviruses

Bats host major mammalian 713 paramyxoviruses

Complete Genome Analysis of Three Novel 716

Picornaviruses from Diverse Bat Species

Pulau virus; a new member of the Nelson Bay orthoreovirus species isolated 719 from fruit bats in Malaysia

Novel Astroviruses in 721

Some infectious causes of diarrhea in young farm animals

Virus infections of the gastrointestinal tract of poultry

Rotavirus diarrhea in 727 bovines and other domestic animals

The zoonotic 729 potential of rotavirus

Predominance of

rotavirus G9 genotype in children hospitalized for rotavirus gastroenteritis in 733

Belgium during 1999-2003

Global Spread of the Emerging G12 Human Rotaviruses

Uniformity of rotavirus strain nomenclature proposed by the Rotavirus 742

Classification Working Group (RCWG)

Recommendations for the classification of group A rotaviruses 748 using all 11 genomic RNA segments

Identification of group A rotaviruses from Zambian fruit bats provides evidence for 752 long-distance dispersal events in Africa

Principles and Patterns of Bat Movements: From 756 Aerodynamics to Ecology

First application of satellite telemetry to track 758 African straw-coloured fruit bat migration

Continental-scale, seasonal movements 760 of a heterothermic migratory tree bat

Ecology of bat migration

Bat ecology

Close genetic relatedness of picornaviruses from 765

European and Asian bats

The cytopathic effects of vervet monkey viruses

Rotavirus Due to Reassortment with

Reverse genetics system for introduction of 771 site-specific mutations into the double-stranded RNA genome of infectious 772 rotavirus

Full 774 genomic analysis of a simian SA11-like G3P[2] rotavirus strain isolated from an 775 asymptomatic infant: Identification of novel VP1, VP6 and NSP4 genotypes

Review of global rotavirus strain prevalence data from six years post vaccine 779 licensure surveillance: Is there evidence of strain selection from vaccine pressure? 780

Complex evolutionary patterns of two rare human G3P

rotavirus strains possessing a feline/canine-like H6 genotype on an AU-1-like 784 genotype constellation

Highly diversified coronaviruses in 789 neotropical bats

Relatives of Severe Acute Respiratory Syndrome Coronavirus and Close Relatives 793 of Human Coronavirus 229E in Bats

Genomic 797 Characterization of Severe Acute Respiratory Syndrome-Related Coronavirus in 798

European Bats and Classification of Coronaviruses Based on Partial RNA-799

Dependent RNA Polymerase Gene Sequences

Characterization and phylogenetic analysis of new bat astroviruses detected 803 in Gabon, Central Africa

Modular 806 approach to customise sample preparation procedures for viral metagenomics: A 807 reproducible protocol for virome analysis

Human and other mammal RVAs Avian RVAs R19_RVA/Bat-wt/CHN

Human (incl. E2451 and L621) and camelid RVAs

Murine RVAs

Human and other mammal RVAs RVA/Bat-wt/GHA