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Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.

Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs (SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats [mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9]. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species., Highlights • 313 SARSr-CoV genomes have been sequenced (274 from human, 18 civets and 47 bats). • SARSr-CoV genomes of bats in Yunnan possess highest nt identity to those from civets. • The origin of human SARS-CoV was probably a result of multiple recombination events. • Recombination from a number of SARSr-CoV ancestors in different horseshoe bat species.

The Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a Public Health Emergency of International Concern. However, so far, there are still controversies about the source of the virus and its intermediate host. Here, we found the novel coronavirus was closely related to coronaviruses derived from five wild animals, including Paguma larvata, Paradoxurus hermaphroditus, Civet, Aselliscus stoliczkanus and Rhinolophus sinicus, and was in the same branch of the phylogenetic tree. However, genome and ORF1a homology show that the virus is not the same coronavirus as the coronavirus derived from these five animals, whereas the virus has the highest homology with Bat coronavirus isolate RaTG13.

Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoVs in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS-CoV and SARSr-CoV infection but with different binding affinities to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity for spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease., The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiencies in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS-CoV and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with that of their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that some key residues were under positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics. IMPORTANCE Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoVs in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS-CoV and SARSr-CoV infection but with different binding affinities to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity for spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.

Bats are associated with several important zoonotic viruses from different families. One example includes adeno-associated viruses (AAVs), that are extensively detected in several animals, especially primates. To understand AAVs distribution and genetic diversity in the coastal areas of Southeast China, a total of 415 intestine samples were mostly collected from two provinces of southeast China, i.e., Zhejiang and Fujian province. Intestine samples from five bat species were collected for AAVs detection. The average prevalence rate for AAV detection among these samples was 18.6% (77 positives out of 415 samples) and ranged from 11.8 to 28.9% between the five bat species. This suggests that AAVs are widely distributed in diverse bat populations in southeast coastal areas of China. Based on the genome sequence of bat adeno-associated virus-CXC1(BtAAV-CXC1) from one AAV-positive sample, the genetic diversity of the detected AAVs were assessed and analyzed. Phylogenetic analysis revealed that BtAAV-CXC1 was comparatively distant to other major AAVs from mammals and non-mammals, with only a 52.9~64.7% nucleotide identity. However, they were phylogenetically closer to Rhinolophus sinicus bat adeno-associated virus (Rs-BtAAV1), with a 74.5% nt similarity. Partial analysis of the rep and cap overlapping open reading frame (ORF) sequences from bat AAV samples revealed 48 partial rep sequences and 23 partial cap sequences from positive samples shared 86.9 to 100% and 72.3 to 98.8% nucleotide identities among themselves, respectively. This suggests that the detected AAVs had a distinctly high genetic diversity. These findings led us to conclude that diverse AAVs may be widely distributed in bat populations from the southeast regions of China.

Noroviruses (NoVs) are second only to the rotaviruses as etiologic agents of acute fulminant gastroenteritis in infants and young children worldwide. While only one species is currently recognized within the genus Norovirus, molecular epidemiological studies have demonstrated a marked genetic diversity among circulating NoVs. NoVs have been detected in a number of mammalian species, but only one sequence of bat-borne NoV, from China, has been recorded in Genbank. In this study, we performed NoV screening in 178 adult bats collected from Fujian province, southeast China, and found that 2 bats were positive for this virus. One such sample (NPIH26) was chosen to amplify the whole genome; genomic comparison showed that it possessed a genomic organization typical of noroviruses, and shared the highest nt identity (73%) with the bat NoV BtRs-CalV/YN2010 identified in Rhinolophus sinicus from Yunnan, China. Accordingly, it can be considered as comprising a new genogroup, GVII. This study may help our understanding of evolutionary origins of this virus.

93. Chinese Rufous Horseshoe Bat Rhinolophus sinicus French: Rhinolophe de Chine I German: Rotbraune. China-Hufeisennase / Spanish: Herradura rufo de China Other common names: Chinese Horseshoe Bat, Little Nepalese Horseshoe Bat Taxonomy. Rhinolophus rouxi [sic] sinicus K. Andersen, 1905, “ Chin Tah, Anhwei [= Anhui Province], Lower Yangtse [River],” China. Rhinolophus sinicus is included in the rouxii species group and appears to be sister to R. thomasi. True R. sinicus is composed of three genetic lineages in the eastern, central, and Hainan portion ofits distribution while R.s. septentrionalis is sister to. thomasi, suggesting that the former is distinct at the species level. Rhinolophus sinicus was previously included in R rouxii, but shows substantial morphological, genetic, and karyological differences. There appears to be some ambiguity regarding the distribution of this species and R rouxii in published records: north Indian records previously attributed to. rouxii are now considered to refer to R.sinicus. There may also be some ambiguity regarding the distribution of this species in South-east Asia, as it is often confused with R. thomasi. Validity of races needs to be tested. Two subspecies are currently recognized, although they likely represent two distinct species, pending further revision. Subspecies and Distribution. R. s. sinicus K.Andersen, 1905 - N India (Himachal Pradesh, Uttarakhand, Sikkim, West Bengal, Meghalaya, Arunachal Pradesh, and Nagaland), Nepal, N Myanmar,, S & SE China (Xizang, Sichuan, Guizhou, Hubei, Jiangsu, Anhui, Zhejiang, Fujian, Guangdong, and Hainan I), and N & C Vietnam. On following pages: 94. Thomas's Horseshoe Bat ( Rhinolophus thomasi ); 95. Lesser Horseshoe Bat ( Rhinolophus hipposideros ); 96. rancis's Woolly Horseshoe Bat ( Rhinolophus trancisi); 97. Lesser Woolly Horseshoe Bat ( Rhinolophus sedulus ); 98. Trefoil Horseshoe Bat ( Rhinolophus trifoliatus ); 99. Northern Woolly Horseshoe Bat ( Rhinolophus perniger ); 100. Selangor Woolly Horseshoe Bat ( Rhinolophus luctoides ); 101. Malaysian Woolly Horseshoe Bat ( Rhinolophus mono); 102. Great Woolly Horseshoe Bat ( Rhinolophus luctus ); 1.03. Beddome's Woolly Horseshoe Bat ( Rhinolophus beddomei ); 104. Formosan Woolly Horseshoe Bat (Rhinolophus formosaë); 105. Thailand Horseshoe Bat ( Rhinolophus thailandensis ); 106. Dobson's Horseshoe Bat ( Rhinolophus yunanensis ); 107. Chiew Kwee's Horseshoe Bat ( Rhinolophus chiewkweeae ); 108. Pearson's Horseshoe Bat ( Rhinolophus pearsonii ); 109. Mitred Horseshoe Bat ( Rhinolophus mitratus ). R.s. septentrionalis Sanborn, 1939 — S China (Yunnan). Descriptive notes. Head-body 43-53' 5 mm, tail 21- 5—30 mm, ear 15- 8-20 mm, hindfoot 7- 5-10 mm, forearm 43-56 mm; weight 8-9-10- 9 g. Dorsal pelage is wood brown, occasionally with reddish tint; ventral pelage is slightly lighter. Ears are small. Noseleaf has hastate lancet that constricts before variably long to short tip; connecting process is rounded, as in the Indian Rufous Horseshoe Bat (. rouxii); sella is virtually parallelsided, and widely rounded off at tip; horseshoe is relatively wide (8-1-8- 2 mm) but does not completely cover muzzle, and there are usually clearly visible and well developed lateral leaflets. Lower lip has three mental grooves. Skull is massive and robust (zygomatic width is always much larger than mastoid width); anterior median swellings are relatively small; posterior swellings are moderately developed; rostral profile is concave; sagittal crest is moderate to high; frontal depression is shallow; supraorbital crests are low but visible. C1 is well developed and long; P2 is moderate in size and within tooth row or slightly extruded from it; P3 is medium-sized to small and partly to fully extruded from tooth row; P2 and P4 are touching or almost touching. Chromosomal complement has 2n = 36 and FNa = 60 (mainland China and Hainan). Habitat. The Chinese Rufous Horseshoe Bat is typically a forest species, occurring in montane forests with heavy rainfall. It has been reported from disturbed forests in Nepal and from thick moist tropical forest with some bamboo in Myanmar. Recorded at elevations of 500-2769 m, being commonest at higher altitudes. Food and Feeding. The Chinese Rufous Horseshoe Bat forages for insects by aerialhawking and occasionally perch-hunting. Breeding. No information. Activity patterns. Chinese Rufous Horseshoe Bats roost in caves, old disused tunnels, temples, houses, wells, and hollows in trees. They leave their roosts just after dusk to forage through the night. In colder portions of their range, they hibernate through the winter. Calls are FM/CF/FM shaped with a peak GF recorded at c.79-87 kHz across China and Hainan, and at 80 kHz in Vietnam. Movements, Home range and Social organization. The Chinese Rufous Horseshoe Bat roosts singly (usually males) or in colonies varying in size from a few individuals to several hundred. During the breeding season, females form large maternity colonies that can consist of several hundred bats. Status and Conservation. Classified as Least Concern on The IUCN Red List. The Chinese Rufous Horseshoe Bat is widespread and relatively common throughout its distribution, and does not seem to be facing any major threats currently. It may be locally threatened by roost disturbance and general habitat loss. Bibliography. Ao Lei et al. (2007), Bates & Harrison (1997), Bates, Csorba, Molur & Srinivasulu (2008a), Bates, Thi Mar-Mar et al. (2004), Csorba et al. (2003), Francis (2008a), Kruskop (2013a), Mao Xiuguang, Dong Ji et al. (2014), Mao Xiuguang, He Guimei et al. (2013), Mao Xiuguang, Tsagkogeorga et al. (2019), Molur et al. (2002), Smith &XieYan (2008), Stoffberg et al. (2010), Wu Yi & Harada (2005), Wu Yi, Harada & LiYanhong (2004), Wu Yi, Motokawa et al. (2009), Xie Lifen et al. (2017), Zhang Lin et al. (2018), Zhang Weidao (1985)., Published as part of Burgin, Connor, 2019, Rhinolophidae, pp. 280-332 in Handbook of the Mammals of the World – Volume 9 Bats, Barcelona :Lynx Edicions on pages 325-326, DOI: 10.5281/zenodo.3748525

The gut microbiota is now known as a key factor in mammalian physiology and health. Our understanding of the gut microbial communities and their effects on ecology and evolution of their hosts is extremely limited in bats which represent the second largest mammalian order. In the current study, gut microbiota of three sampling sources (small intestine, large intestine, and feces) were characterized in two sympatric and insectivorous bats (Rhinolophus sinicus and Myotis altarium) by high‐throughput sequencing of the V3‐V4 region of the 16S rRNA gene. Combining with published studies, this work reveals that Gammaproteobacteria may be a dominant class in the whole Chiroptera and Fusobacteria is less observed in bats although it has been proven to be dominant in other mammals. Our results reveal that the sampling source influences alpha diversity of the microbial community in both studied species although no significant variations of beta diversity were observed, which support that fecal samples cannot be used as a proxy of the microbiota in other gut regions in wild animals.

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa.

Habitat selection by the Chinese horseshoe bat (Rhinolophus sinicus) in the Wuling Mountains was studied in this paper. Global positioning system (GPS), remote sensing (RS) and geographic information system (GIS) technologies were used to obtain ground survey data and analyse the habitat factors driving the distribution of R. sinicus. Based on these basic data, a binary logistic regression method was used to establish habitat selection models of R. sinicus. Then, the corrected Akaike information criterion (AICC) was used to screen an optimal model, and the Hosmer-Lemeshow test indicated that the optimal model has suitable goodness of fit. Finally, the optimal model was used to predict the spatial distribution of R. sinicus in the Wuling Mountains. Verification analysis showed that the overall accuracy of the model was 72.7% and that the area under the curve (AUC) value was 0.947, which indicated that the model was effective for predicting suitable habitat for R. sinicus. The model results also showed that the main factors that influenced habitat selection were slope, annual mean temperature and distances from roads, rivers and residential land. R. sinicus preferred areas far from roads and residential land and areas near rivers. Generally, higher values of slope and annual mean temperature were associated with a greater likelihood of R. sinicus presence. Therefore, the protection of the water bodies surrounding R. sinicus habitats and fully addressing the impacts of human activities on R. sinicus habitats are recommended to protect the survival and reproduction of the population.

Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs.

Previous findings of Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses in bats, and the ability ofTylonycteris-BatCoV HKU4 spike protein to utilize MERS-CoV receptor, human dipeptidyl peptidase 4 hDPP4, suggest a bat ancestral origin of MERS-CoV. We developed 12 primary bat cell lines from seven bat species, includingTylonycteris pachypus,Pipistrellus abramusandRhinolophus sinicus(hosts ofTylonycteris-BatCoV HKU4,Pipistrellus-BatCoV HKU5 and SARS-related-CoV respectively), and tested their susceptibilities to MERS-CoVs, SARS-CoV and human coronavirus 229E (HCoV-229E). Five cell lines, includingP. abramusandR. sinicusbut notT. pachypuscells, were susceptible to human MERS-CoV EMC/2012. However, three tested camel MERS-CoV strains showed different infectivities, with only two strains capable of infecting three and one cell lines respectively. SARS-CoV can only replicate inR. sinicuscells, while HCoV-229E cannot replicate in any bat cells. Bat dipeptidyl peptidase 4 (DPP4) sequences were closely related to those of human and non-human primates but distinct from dromedary DPP4 sequence. Critical residues for binding to MERS-CoV spike protein were mostly conserved in bat DPP4. DPP4 was expressed in the five bat cells susceptible to MERS-CoV, with significantly higher mRNA expression levels than those in non-susceptible cells (P=0.0174), supporting that DPP4 expression is critical for MERS-CoV infection in bats. However, overexpression ofT. pachypusDPP4 failed to confer MERS-CoV susceptibility inT. pachypuscells, suggesting other cellular factors in determining viral replication. The broad cellular tropism of MERS-CoV should prompt further exploration of host diversity of related viruses to identify its ancestral origin.

Background In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China. Methods A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses. Results Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels. Conclusions This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae.

We report the discovery of a novel bocaparvovirus, bat bocaparvovirus (BtBoV), in one spleen, four respiratory and 61 alimentary samples from bats of six different species belonging to three families, Hipposideridae, Rhinolophidae and Vespertilionidae. BtBoV showed a higher detection rate in alimentary samples of Rhinolophus sinicus (5.7 %) than those of other bat species (0.43–1.59 %), supporting R. sinicus as the primary reservoir and virus spillover to accidental bat species. BtBoV peaked during the lactating season of R. sinicus, and it was more frequently detected among female than male adult bats (P

The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

Since the 2002–2003 severe acute respiratory syndrome (SARS) outbreak prompted a search for the natural reservoir of the SARS coronavirus, numerous alpha- and betacoronaviruses have been discovered in bats around the world. Bats are likely the natural reservoir of alpha- and betacoronaviruses, and due to the rich diversity and global distribution of bats, the number of bat coronaviruses will likely increase. We conducted a surveillance of coronaviruses in bats in an abandoned mineshaft in Mojiang County, Yunnan Province, China, from 2012–2013. Six bat species were frequently detected in the cave: Rhinolophus sinicus, Rhinolophus affinis, Hipposideros pomona, Miniopterus schreibersii, Miniopterus fuliginosus, and Miniopterus fuscus. By sequencing PCR products of the coronavirus RNA-dependent RNA polymerase gene (RdRp), we found a high frequency of infection by a diverse group of coronaviruses in different bat species in the mineshaft. Sequenced partial RdRp fragments had 80%–99% nucleic acid sequence identity with well-characterized Alphacoronavirus species, including BtCoV HKU2, BtCoV HKU8, and BtCoV1, and unassigned species BtCoV HKU7 and BtCoV HKU10. Additionally, the surveillance identified two unclassified betacoronaviruses, one new strain of SARS-like coronavirus, and one potentially new betacoronavirus species. Furthermore, coronavirus co-infection was detected in all six bat species, a phenomenon that fosters recombination and promotes the emergence of novel virus strains. Our findings highlight the importance of bats as natural reservoirs of coronaviruses and the potentially zoonotic source of viral pathogens. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s12250-016-3713-9 and is accessible for authorized users.

Several lineage B betacoronaviruses termed severe acute respiratory syndrome (SARS)–like CoVs (SL-CoVs) were identified from Rhinolophus bats in China. These viruses are characterized by a set of unique accessory open reading frames (ORFs) that are located between the M and N genes. Among unique accessory ORFs, ORF8 is most hypervariable. In this study, the ORF8s of all SL-CoVs were classified into 3 types, and, for the first time, it was found that very few SL-CoVs from Rhinolophus sinicus have ORF8s that are identical to that of human SARS-CoV. This finding provides new genetic evidence for Chinese horseshoe bats as the source of human SARS-CoV.

The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of Myotis daubentoni and Rhinolophus sinicus support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that M. daubentoni and R. sinicus are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV. Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0729-6) contains supplementary material, which is available to authorized users.

Apart from bat-SARS-CoV, we have identified a novel group 1 coronavirus, bat-CoV HKU2, in Rhinolophus sinicus (Chinese horseshoe bats). Since it has been suggested that the receptor-binding motif (RBM) of SARS-CoV may have been acquired from a group 1 coronavirus, we conducted a surveillance study and identified bat-SARS-CoV and bat-CoV HKU2 in 8.7% and 7.5% respectively of R. sinicus in Hong Kong and Guangdong. Complete genome sequencing of four strains of bat-CoV HKU2 revealed the smallest coronavirus genome (27164 nucleotides) and a unique spike protein evolutionarily distinct from the rest of the genome. This spike protein, sharing similar deletions with other group 2 coronaviruses in its C-terminus, also contained a 15-amino acid peptide homologous to a corresponding peptide within the RBM of spike protein of SARS-CoV, which was absent in other coronaviruses except bat-SARS-CoV. These suggest a common evolutionary origin in the spike protein of bat-CoV HKU2, bat-SARS-CoV, and SARS-CoV.

Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats ( Rhinolophus sinicus ) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats ( Rhinolophus ferrumequinum ), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high K a / K s ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission. IMPORTANCE Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats ( Rhinolophus sinicus ) exhibited

Wild Chinese horseshoe bats have been proven to be natural reservoirs of SARS-like coronaviruses. However, the molecular characterization of key proteins in bats still needs to be explored further. In this study, we used cloning and bioinformatics to analyze the sequence of RIG-I, STAT-1 and IFN-β in the immortalized cell lines from Rhinolophus affinis and Rhinolophus sinicus. Then, we treated different bat cells, mouse embryonic fibroblasts (MEF) and splenocytes with polyinosinic–polycytidylic acid (polyI:C) and vesicular stomatitis virus (VSV) to assess and compare antiviral immune responses between bats and mice. Our results demonstrated that bat RIG-I, STAT-1 and IFN-β showed close homology with human, mouse, pig and rhesus monkey. RIG-I and STAT-1 were both highly expressed in bat spleen. Furthermore, IFN-β was induced by polyI:C and VSV in both bat and mouse cells. These findings have provided new insight into the potential characteristics of the bat innate immune system against viral infection., Highlights • The genes of RIG-I, STAT-1 and IFN-β in Chinese horseshoe bats were sequenced and analyzed. • The expression patterns of RIG-I, STAT-1 and IFN-β were appraised in bat tissues. • The virus induced IFN-β expression is higher in bat cells than in mouse cells. • The replication of invaded VSV is more in mouse cells than in bat cells.

To the Editor: The severe acute respiratory syndrome (SARS) outbreak of 2002–03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (CoV), prompted numerous studies of bats and the viruses they harbor. A novel clade 2c betacoronavirus, termed Middle East respiratory syndrome (MERS)–CoV, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the Arabian Peninsula (1). Extending on previous work (2), we described European Pipistrellus bat–derived CoVs that are closely related to MERS-CoV (3). We now report the identification of a South Africa bat derived CoV that has an even closer phylogenetic relationship with MERS-CoV. During 2011–2012, fecal pellets were collected from 62 bats representing 13 different species in the KwaZulu-Natal and Western Cape Provinces of South Africa and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA). Details about the bat sample are available in the Technical Appendix. RNA was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Screening for CoVs was done by nested reverse transcription PCR using broadly reactive oligonucleotide primers targeting a conserved region in the RNA-dependent RNA polymerase (RdRp) gene (online Technical Appendix). PCR results were positive for 5 (8%) of the 62 specimens. PCR amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from South Africa (4). The other positive specimen, termed PML/2011, was from an adult female Neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC869678","term_id":"666386896"}}KC869678). Technical Appendix Figure 1 shows the distribution of this bat species. To obtain better phylogenetic resolution, we extended the 398-nt RdRp fragment generated by the screening PCR to 816 nt, as described (5). PML/2011 differed from MERS-CoV by only 1 aa exchange (0.3%) in the translated 816-nt RdRp gene fragment. Thus, PML/2011 was much more related to MERS-CoV than any other known virus. The amino acid sequence of the next closest known relatives of MERS-CoV, from European Pipistrellus bats (3), differed from MERS-CoV by 1.8%. The amino acid sequences of viruses from Nycteris bats in Ghana (3) and the 2c prototype bat CoVs, HKU4 and HKU5, from China (6) differed by 5.5%–7.7% from MERS-CoV. The smaller 152- to 396-nt RdRp fragments of 2c bat CoVs from a Hypsugo savii bat in Spain (7), bat guano in Thailand (8), and a Nyctinomops bat in Mexico (9) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. However, in their respective RdRp fragments, these CoVs yielded amino acid sequence distances of 3.5%–8.0% and were thus probably more distant from MERS-CoV than the virus described here. A Bayesian phylogenetic analysis of the 816-nt RdRp sequence confirmed the close relationship between PML/2011 and MERS-CoV (Figure). Their phylogenetic relatedness was as close as that of SARS-CoV and the most closely related bat coronavirus known, Rs672 from a Rhinolophus sinicus bat (Figure). Like PML/2011 and MERS-CoV, Rs672 and SARS-CoV showed only 1 aa exchange in the translated 816-nt RdRp fragment. To confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for PML/2011 (oligonucleotide primers available upon request from the authors). A partial spike gene–based phylogeny using this sequence yielded the same topology as that using the partial RdRp sequence (Technical Appendix Figure 2). Again, PML/2011 was most closely related to MERS-CoV, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for CoV attachment and cellular entry. This distance was less than the 13.3% aa sequence distance between MERS-CoV and the European Pipistrellus CoVs (3) and less than the 20.5%–27.3% aa sequence distance between MERS-CoV and HKU5 and between MERS-CoV and HKU4 (6) in the same sequence fragment. Figure Partial RNA-dependent RNA polymerase (RdRp) gene phylogeny, including the novel betacoronavirus from a Neoromicia zuluensis bat in South Africa (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC869678","term_id":"666386896"}}KC869678 ... Our results further support the hypothesis that, like human CoV-229E and SARS-CoV, ancestors of MERS-CoV might exist in Old World insectivorous bats belonging to the family Vespertilionidae, to which the genera Neoromicia and Pipistrellus belong (3). Knowledge of the close relatedness of PML/2011 and MERS-CoV, which contrasts with the more distant relatedness of CoVs in bats from the Americas and Asia, enables speculations of an African origin for bat reservoir hosts of MERS-CoV ancestors. This hypothesis is limited by a global sampling bias, the small sample size, and the single clade 2c betacoronavirus detection in this study. Still, a putative transfer of MERS-CoV ancestors from Africa to the Arabian Peninsula would parallel the transfer of other viruses (e.g., the exportation of Rift Valley fever virus from East Africa, which led to a severe outbreak in Saudi Arabia in 2000) (10). Studies of Vespertilionidae bats and potential intermediate hosts (e.g., carnivores and ungulates, such as camels) are urgently needed to elucidate the emergence of MERS-CoV. Such studies should focus on the Arabian Peninsula and Africa. Technical Appendix: Description of bat sampling, screened bat species, distribution of Neoromicia zuluensis bats, and spike gene phylogeny of the 2c betacoronavirus clade. Click here to view.(326K, pdf)

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.

We present a complete mitochondrial genome sequence of Rhinolophus sinicus sinicus from Central China and provide its annotation, as well as showed the phylogenetic relationship and mitogenomic variation with other published mitochondrial genomes of congeneric bat species. Our results revealed a relatively high mitogenomic variation between two R. s. sinucus from Central and East China, which is similar to interspecific divergence level.

There are two subspecies of Rhinolophus sinicus currently recognized in China. In this study, using next generation sequencing approaches, the complete mitochondrial genome of one subspecies R. s. sinicus was obtained. The total length of the genome sequence is 16,898 bp. The arrangement and contents of R. s. sinicus mitochondrial genes exhibit high similarity with other bats of family Rhinolophida. Phylogenetic reconstructions support the sister relationship of the two subspecies and confirm the subspecies status of our specimen.

The present study was conducted to investigate the prevalence and genotypes of Cryptosporidium in bats. A total of 247 bats, belonging to Rhinolophus sinicus , Rousettus leschenaultia, Aselliscus stoliczkanus , and Hipposideros fulvus , were collected in Yunnan Province, Southwestern China, and the intestinal tissues were examined for Cryptosporidium infection by PCR amplification of the small subunit ribosomal RNA (SSU rRNA). The overall infection rate was 7.7% (95% CI, 4.5 to 11.0%), with R. sinicus having the highest level at 9.5% (95% CI, 2.8 to 16.1%) followed by A. stoliczkanus at 7.8% (95% CI, 2.2 to 18.9%), H. fulvus at 7.2% (95% CI, 1.1 to 13.4%), and R. leschenaultia at 5.7% (95% CI, 1.2 to 15.7%). DNA sequence and phylogenetic analyses based on SSU rRNA revealed the presence of 2 novel genotypes, designated as Cryptosporidium bat genotype I in A. stoliczkanus and R. sinicus and Cryptosporidium bat genotype II in R. leschenaultia, R. sinicus , and H. fulvus . This is the first report of Cryptosporidium genotypes in bats. Further biological and genetic characterization is needed to determine the relationship of the 2 novel genotypes to established Cryptosporidium species-genotypes.

Closely related taxa living in sympatry provide good opportunities to investigate the origin of barriers to gene flow as well as the extent of reproductive isolation. The only two recognized subspecies of the Chinese rufous horseshoe bat Rhinolophus sinicus are characterized by unusual relative distributions in which R. s. septentrionalis is restricted to a small area within the much wider range of its sister taxon R. s. sinicus. To determine the history of lineage divergence and gene flow between these taxa, we applied phylogenetic, demographic and coalescent analyses to multi-locus datasets. MtDNA gene genealogies and microsatellite-based clustering together revealed three divergent lineages of sinicus, corresponding to Central China, East China and the offshore Hainan Island. However, the central lineage of sinicus showed a closer relationship with septentrionalis than with other lineages of R. s. sinicus, in contrary to morphological data. Paraphyly of sinicus could result from either past asymmetric mtDNA introgression between these two taxa, or could suggest septentrionalis evolved in situ from its more widespread sister subspecies. To test between these hypotheses, we applied coalescent-based phylogenetic reconstruction and Approximate Bayesian Computation (ABC). We found that septentrionalis is likely to be the ancestral taxon and therefore a recent origin of this subspecies can be ruled out. On the other hand, we found a clear signature of asymmetric mtDNA gene flow from septentrionalis into central populations of sinicus yet no nuclear gene flow, thus strongly pointing to historical mtDNA introgression. We suggest that the observed deeply divergent lineages within R. sinicus probably evolved in isolation in separate Pleistocene refugia, although their close phylogeographic correspondence with distinct eco-environmental zones suggests that divergent selection might also have promoted broad patterns of population genetic structure.

The karyotype of R. sinicus was 2n = 36, FN = 60 (Fig. 3), consisting of 13 large-to-small metacentric or submetacentric pairs and four medium-sized to small acrocentric pairs of autosomes, a medium-sized subtelocentric X chromosome, and a small subtelocentric Y chromosome. This species had a prominent secondary constriction involving one pair of medium-sized acrocentric chromosomes. The karyotype of R. sinicus from Hainan Island (2n = 36, FN = 60) does not differ from conspecific populations in the Chinese provinces of Anhui [6] (reported as R. rouxii sinicus), Sichuan [14], and Guangdong [15] in conventional karyotype and that from "south-western China" also in G-banding pattern [17], but does differ from the 2n = 56, FN = 60 karyotype of R. rouxii reported from India and Sri Lanka [31]. These karyological differences between R. sinicus and R. rouxii support the view that these two taxonomically confused taxa both represent distinct species, as previously proposed [14, 15, 26]., Published as part of Wu, Yi, Motokawa, Masaharu, Li, Yu-Chun, Harada, Masashi, Chen, Zhong & Lin, Liang-Kong, 2009, Karyology of eight species of bats (Mammalia: Chiroptera) from Hainan Island, China, pp. 659-666 in International Journal of Biological Sciences 5 (7) on page 660, DOI: 10.7150/ijbs.5.659, http://zenodo.org/record/4266204, {"references":["6. Zhang WD. A study on the karyotypes in four species of bat (Rhinolophus). Acta Theriologica Sinica 1985; 5: 95 - 101.","14. Wu Y, Harada M, Li YH. Karyology of seven species bats from Sichuan, China. Acta Theriologica Sinica 2004; 24: 30 - 35.","15. Wu Y, Harada M. Karyology of five species of the Rhinolophus (Chiroptera: Rhinolophidae) from Guangdong, China. Acta Theriologica Sinica 2005; 25: 163 - 167.","17. Mao X, Nie W, Wang J, Su W, Ao L, Feng Q, Wang Y, Volleth M, Yang F. Karyotype evolution in Rhinolophus bats (Rhinolophidae, Chiroptera) illuminated by cross-species chromosome painting and G-banding comparison. Chromosome Res 2007; 15: 835 - 847.","31. Zima J, Volleth J, Horacek I, Cerveny J, Cervena A, Prucha K, Macholan M. Comparative karyology of rhinolophid bats (Chiroptera: Rhinolophidae). In: Horacek I, Vorhali V, eds. Prague Studies in Mammalogy. Prague: Charles University Press; 1992: 229 - 236.","26. Simmons NB. Order Chiroptera. In: Wilson DE, Reeder DM, eds. Mammal Species of the World: A Taxonomic and Geographic Reference, 3 rd ed. Baltimore: Johns Hopkins University Press; 2005: 312 - 529."]}

The Chinese rufous horseshoe bat (Rhinolophus sinicus) has been suggested to carry the direct ancestor of severe acute respiratory syndrome (SARS) coronavirus (SCoV), and the diversity of SARS-like CoVs (SLCoV) within this Rhinolophus species is therefore worth investigating. Here, we demonstrate the remarkable diversity of SLCoVs in R. sinicus and identify a strain with the same pattern of phylogenetic incongruence (i.e. an indication of recombination) as reported previously in another SLCoV strain. Moreover, this strain possesses a distinctive 579 nt deletion in the nsp3 region that was also found in a human SCoV from the late-phase epidemic. Phylogenetic analysis of the Orf1 region suggested that the human SCoVs are phylogenetically closer to SLCoVs in R. sinicus than to SLCoVs in other Rhinolophus species. These findings reveal a closer evolutionary linkage between SCoV in humans and SLCoVs in R. sinicus, defining the scope of surveillance to search for the direct ancestor of human SCoVs.

Rhinolophus sinicus K. Andersen, 1905 Chinese rufous horseshoe bat The species is most easily confused with R. affinis, from which it is best distinguished by its straight-sided lancet and the relatively short second phalanx of the third digit (et al., 2003). Unequivocal separation can seemingly be performed by sequencing the control region of mtDNA (authors’ unpublished data). Echolocation call frequencies overlap with those emitted by R. affinis. Rhinolophus affinis is also typically a larger species, though overlap occurs with R. sinicus at forearm lengths between 50–53 mm (Fig. 4). Rhinolophus sinicus is very similar to R. thomasi from Myanmar, Vietnam, Lao PDR and Thailand, to which it is closely related and possibly conspecific. Call frequency for R. thomasi in Lao PDR is reported as 76–86 kHz (Francis and Habersetzer 1998), and so the two taxa overlap considerably in call frequency. Rhinolophus thomasi is reported to have smaller upper canines than R. sinicus (Francis 2008). FA — 42.6–52.6 mm, mass — 8.5–14.3 g. Seventy-four males and 43 females were captured from Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hubei, Jiangxi, Sichuan, and Yunnan. The echolocation calls vary over the geographic range, from 73.4–88.5 kHz. The call frequencies (0 ± SD) of a sample of males (82.0 ± 2.0 kHz, n = 38) were lower than females (85.4 ± 1.9, n = 29 — Mann- Whitney W = 839, P ♂♂: 46.9 ± 1.5, n = 37; ♀♀: 46.6 ± 2.0, n = 29; Mann-Whitney W = 1325, P > 0.05). Previous records from China:, Anhui, Fujian, Gansu, Guangdong, Guangxi, Guizhou, Hainan, Hubei Jiangsu, Jiangxi, Shaanxi, Sichuan, Tibet, Yunnan, Zhejiang (Zhang, 1997; Wang, 2003). Ecological Notes Widespread in caves in southern China, but never abundant. Maximum count was 24 bats in one cave in Jiangxi. An abandoned brick kiln was used by a solitary male in Xinyi County., Published as part of Zhang, Libiao, Jones, Gareth, Zhang, Jinshuo, Zhu, Guangjian & Parsons, Stuart, 2009, Recent surveys of bats (Mammalia: Chiroptera) from China. I. Rhinolophidae and Hipposideridae, pp. 71-88 in Acta Chiropterologica 11 (1) on page 80, DOI: 10.3161/150811009X465703, http://zenodo.org/record/3944511, {"references":["FRANCIS, C. M., and J. HABERSETZER. 1998. Interspecific and intraspecific variation in echolocation call frequency and morphology of horseshoe bats, Rhinolophus and Hipposideros. Pp. 169 - 179, in Bat biology and conservation (T. H. KUNZ and P. A. RACEY, eds.). Smithsonian Institution Press, Washington, D. C., 365 pp.","ZHANG, Y. Z. (ed.). 1997. Distribution of mammalian species in China. China Forestry Publishing House, Beijing, 280 pp.","WANG, Y. X. (ed.). 2003. A complete checklist of mammal species and subspecies in China: a taxonomic and geographic reference. China Forestry Publishing House, Beijing, 394 pp."]}

Bats are a unique but enigmatic group of mammals and have a world-wide distribution. The phylogenetic relationships of extant bats are far from being resolved. Here, we investigated the karyotypic relationships of representative species from four families of the order Chiroptera by comparative chromosome painting and banding. A complete set of painting probes derived from flow-sorted chromosomes of Myotis myotis (family Vespertilionidae) were hybridized onto metaphases of Cynopterus sphinx (2n = 34, family Pteropodidae), Rhinolophus sinicus (2n = 36, family Rhinolophidae) and Aselliscus stoliczkanus (2n = 30, family Hipposideridae) and delimited 27, 30 and 25 conserved chromosomal segments in the three genomes, respectively. The results substantiate that Robertsonian translocation is the main mode of chromosome evolution in the order Chiroptera, with extensive conservation of whole chromosomal arms. The use of M. myotis (2n = 44) probes has enabled the integration of C. sphinx, R. sinicus and A. stoliczkanus chromosomes into the previously established comparative maps between human and Eonycteris spelaea (2n = 36), Rhinolophus mehelyi (2n = 58), Hipposideros larvatus (2n = 32), and M. myotis. Our results provide the first cytogenetic signature rearrangement that supports the grouping of Pteropodidae and Rhinolophoidea in a common clade (i.e. Pteropodiformes or Yinpterochiroptera) and thus improve our understanding on the karyotypic relationships and genome phylogeny of these bat species.

Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horse-shoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals. © 2005 by The National Academy of Sciences of the USA., link_to_OA_fulltext

Rhinolophus sinicus (Andersen, 1905 a) Chinese horseshoe bat Rhinolophus rouxii sinicus Andersen 1905 a: 98 ; Chinteh, Anhwei, China. External characters (measurements included in Table 4): A medium-sized Rhinolophid, with an average forearm length of 47.4 mm (range 45.5-50.0 mm). The ears average 17.8 mm in length (l15.8 5 8 —-20.0 20.0 mm), being smaller than those of R. rouxii. The noseleaf is shorter and narrower than in R. rouxii, averaging 12.5 mm in maximum height and 7.7 mm in maximum width. The lancet is broad and short with a well-defined ti tip? (F ig 6a). The base of the sella is broad. In side view, the superior connecting process of the sella is bluntly rounded off, with the base of the sella projecting slightly forwards and downwards. The wing morphology differs significantly to that of R. rouxii. The forearms and metacarpals of R. sinicus average 4.7 % shorter than in R. rouxii. However the phalanges, with the exception of the second phalanx of the fifth finger, average 4.5 % longer. This wing structure is similar to that of R. qfiìnís, although when these two species are found sympatrically, as at Nala Pani Cave in Mussoorie (30.27' N 78.06 'E), R. affinis is absolutely larger in all respects. The pelage is soft and silky, and is generally rich russet brown on the back and paler on the belly. Cranial and dental characters: R. sinícııs has characteristically small cranial measurements, with condylocanine length ranging from 16.7—18.4 mm (Table 4). The skull is narrow, having an average zygomatic breadth of 10.3 mm, and a mastoid breadth of 9.4 mm. The palate is short, its anterior border lies adjacent to the metacone of the first upper molar ml; it averages 1.9 mm in length (Fig. 7 a). This is in contrast to the longer palate ofR. rouxii which averages 2.4 mm in length. The dentition is not as robust as rouxii, having short upper and lower toothrows, averaging 7.5 mm and 8.1 mm respectively. The upper canine is not in contact with the second upper premolar (pm4) and the first upper premolar (pmz) is usually situated in the toothrow. The second lower premolar (pmz) is often displaced from the toothrow, with the first (pmz) and third (pm 4) premolars in contact. Bacular morphology“ The shaft of the baculum is long, parallel-sided and generally straight, thickening towards the base. The base is slightly expanded, and the tip is simple and unexpanded (Fig. 8 a). The average length is 2.1 mm, and the average width 0.5 mm. Distribution. R. sinicus ranges from southern China, through Nepal and into northern India (Fig. 9). Localities include: India: Himachal Pradesh: Solan (Das 1986); Uttar Pradesh: Mussoorie (Blandford 1888 -91, HZM); Dhakuri (Wroughton 1914); West Bengal: Darjeeling (BMNH); Pashok (Sinha 1973); Sikkim: Tashiding (Bhat 1974). Nepal: Sipuri (Fry 1925); Thankot; Parchung (BMNH); Godavari; Pulchowki (HZM); Num (FMNH). China: Sichuan Province: Wanhsien; Fujian Province: Yungan; Nanping; Hebei Province: Ichang; Zheijiang Province; Tunglin; Yunnan: Likiang (Allen 1938). Hong Kong: Lam Tao Island (BMNH). Variation. At present, all specimens are referred to the nominate race R. s. sinicus. However, those from northern India and Nepal average slightly larger in body and skull size than individuals from China, particularly in condylocanine length, the length of the upper and lower toothrows and the length of the mandible. In addition, the noseleaf in individuals from China is slightly shorter and narrower. Specimens examined. (S) denotes inclusion in discriminant analysis, (D) denotes inclusion in molecular analysis. India: Darjeeling, BM. 21.1.17.2 (S), BM. 79.11.21.57; Mussoorie, HZM. 22.28154 (S, D), HZM. 23.28155, MM. 85 (S), MM 86 (S), HZM. 21.28153 (S, D), BM. 79.11.21.146 (S), BM. 79.11.21.149. Nepal: Godavari, HZM. 1.16291 (S), HZM.2.16292, HZM. 3.16293 (S), HZM.4.16294 (S); HZM. 5.162895 (S), HZM. 6.16296 (S), HZM. 7.16297 (S), HZM.8.16298, HZM.9.16296; Parchung, BM. 21.5.1.3 (S); Thankot, BM. 22.5.16.6. China: Ichang, AM. 60217; Nanping, AM. 47997, AM. 48006, AM. 48012 (S), AM. 48015 (S), AM. 48018 (S), AM. 48019, AM. 48020 (S), AM. 56944, AM. 56946; Wanhsien, AM. 59607 (S); Yungan, AM. 60225 (S), AM. 84857 (S), AM. 84859 (S). Hong Kong: Lam Tao Island, BM. 66.24 (S). Habits: In China, R. sínicus is common over the southem half of the country. In the southeast it is found at relatively low altitudes of up to 200 metres (656 feet), being recorded from Hebei Province, F ujian Province, Zheijiang Province and Sichuan Province. In the south-west, it is found at higher altitudes of up to 2000 metres (6562 feet), such as a series of specimens collected from southern Yunnan (Allen 1938). In northern India and Nepal, it is restricted to higher elevations. It has been collected at an altitude of 500 metres (1625 feet) in Arunachal Pradesh (Lal 1982); 550 metres (1788 feet) in West Bengal (Bhat, 1974); 862 metres (2800 feet) in Nepal (FMN H) and at 2769 metres (9000 feet) in Uttar Pradesh (Wroughton 1914). In Mussoorie, Uttar Pradesh (1910 metres / 6208 feet), individuals were found by the author roosting in a cave with R. ferrumeqııínum and R. afiinis. In cold regions, R. sinicus hibernates during winter (Blanford 1888-91). It is a largely social species, but segregation of males and females occurs when the females are having their young (Allen 1938)., Published as part of Thomas, Nikky M., 2000, Morphological and mitochondrial-DNA variation in Rhinolophus rouxii (Chiroptera), pp. 1-18 in Bonner zoologische Beiträge 49 (1) on pages 8-10, DOI: 10.5281/zenodo.3759227, {"references":["Andersen, K. (1905 a): On some bats ofthe genus Rhinolophus, with remarks on their mutual affinities, and descriptions of twenty-six new forms. B Proc. Z 001. Soc. London 2 (10): 75 - 145.","Das, P. K. (1986): Studies on the taxonomy and geographical distribution of the species of bat obtained by the Silent Valley (Kerela, India) expedition, 1980. Records Zool. Surv. India 84: 259 - 276.","Blandford a, W. T. (1 1888 888 - - I 1891 89 l)): 1 The fauna of British India, Mammalia. - Taylor and Francis, London.","Wroughton, R. C. (1914) Report No. 16: Kumaon. Bombay Natural History Societj mammal survey of India, Burma and Ceylon. B J. Bombay nat. Hist. Soc. 22 (4): 282 - 3 (","Sinha, Y. P. (1973): Taxonomic studies on the Indian horseshoe bats of the genus Rhino]: phiis. Mammalia 37 (4): 603 - 630.","Bhat, H. R. (1974): Records and observations on bats of Himalayan region of Uttar Pradesh and West Bengal, India. J. Bombay nat. Hist. Soc. (1): 51 - 57.","Fry, T. B. (1925): Report No. 37 a: Nepal. Bombay Natural History Society's Mammal Survey of India, Bunna and Ceylon. J. Bombay nat. Hist Soc. 30: 525 - 530.","Allen, G. M. (1938): The mammals of China and Mongolia. - American Museum ofNatural History, New York.","Lal, J. P. (1982): Andersen's rufous horseshoe bat, Rhinolophus rouxii sinicus Andersen (Chiroptera: Rhinolophidae) from Arunachal Pradesh, India. - J. Bombay nat. Hist. Soc. 79: 402."]}

Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses., Author Summary Hantaviruses are important human pathogens, occasionally emerging from animal reservoirs. However, both the biodiversity of hantaviruses in nature, as well as the frequency with which they have jumped species barriers in the past, are unclear. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) that were sampled from bats and shrews in China. These viruses are different from known hantaviruses, with each representing a novel species. An evolutionary analysis of all known hantaviruses including the novel viruses described here reveals the existence of four distinct phylogenetic groups of viruses that infect a range of mammalian hosts, and which have sometimes exchanged genes through segment reassortment. Our analysis also suggests that hantaviruses might have first appeared in bats or insectivores, before spreading to rodents, even though rodents are currently the best documented hosts of hantaviruses. Because the phylogenetic trees of the hantaviruses do not always match those of their mammalian hosts, we conclude that both host-jumping and co-divergence have played important roles in hantavirus evolution. Overall, our study shows that bats are likely to be important natural reservoir hosts of hantaviruses from which novel hantaviruses may emerge in the future.

Potorolepis gulyaevi sp. n. (Cestoda: Hymenolepididae) is described from the Chinese horseshoe bat, Rhinolophus sinicus Andersen (Chiroptera: Rhinolophidae), from southern China. The new species differs from known species of the genus by the shape, number and size of rostellar hooks, the relative position and length of the cirrus-sac and the morphology of gravid uterus. This is the first report of a member of the genus from non-marsupial mammals and the first record of a Potorolepis Spassky, 1994 from eastern Asia. The generic diagnosis of Potorolepis is amended.