Chinese Bats Research Articles

Isolation, characterization, and circulation sphere of a filovirus in fruit bats

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

Characterization of Pipistrellus pygmaeus Bat Virome from Sweden.

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78–99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88–94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

Characterization of Novel Rhabdoviruses in Chinese Bats.

Bats, the second largest order of mammals worldwide, harbor specific characteristics such as sustaining flight, a special immune system, unique habits, and ecological niches. In addition, they are the natural reservoirs of a variety of emerging or re-emerging zoonotic pathogens. Rhabdoviridae is one of the most diverse families of RNA viruses, which consists of 20 ecologically diverse genera, infecting plants, mammals, birds, reptiles, and fish. To date, three bat-related genera are described, named Lyssavirus, Vesiculovirus, and Ledantevirus. However, the prevalence and the distribution of these bat-related rhabdoviruses remain largely unknown, especially in China. To fill this gap, we performed a large molecular retrospective study based on the real-time reverse transcription polymerase chain reaction (RT-qPCR) detection of lyssavirus in bat samples (1044 brain and 3532 saliva samples, from 63 different bat species) originating from 21 provinces of China during 2006–2018. None of them were positive for lyssavirus, but six bat brains (0.6%) of Rhinolophus bat species, originating from Hubei and Hainan provinces, were positive for vesiculoviruses or ledanteviruses. Based on complete genomes, these viruses were phylogenetically classified into three putative new species, tentatively named Yinshui bat virus (YSBV), Taiyi bat virus (TYBV), and Qiongzhong bat virus (QZBV). These results indicate the novel rhabdoviruses circulated in different Chinese bat populations.

Seroreactive Profiling of Filoviruses in Chinese Bats Reveals Extensive Infection of Diverse Viruses.

Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs.IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.

Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats

SARS coronavirus (SARS-CoV), the causative agent of the large SARS outbreak in 2003, originated in bats. Many SARS-like coronaviruses (SL-CoVs) have been detected in bats, particularly those that reside in China, Europe, and Africa. To further understand the evolutionary relationship between SARS-CoV and its reservoirs, 334 bats were collected from Zhoushan city, Zhejiang province, China, between 2015 and 2017. PCR amplification of the conserved coronaviral protein RdRp detected coronaviruses in 26.65% of bats belonging to this region, and this number was influenced by seasonal changes. Full genomic analyses of the two new SL-CoVs from Zhoushan (ZXC21 and ZC45) showed that their genomes were 29,732 nucleotides (nt) and 29,802 nt in length, respectively, with 13 open reading frames (ORFs). These results revealed 81% shared nucleotide identity with human/civet SARS CoVs, which was more distant than that observed previously for bat SL-CoVs in China. Importantly, using pathogenic tests, we found that the virus can reproduce and cause disease in suckling rats, and further studies showed that the virus-like particles can be observed in the brains of suckling rats by electron microscopy. Thus, this study increased our understanding of the genetic diversity of the SL-CoVs carried by bats and also provided a new perspective to study the possibility of cross-species transmission of SL-CoVs using suckling rats as an animal model.

Receptor Usage of a Novel Bat Lineage C Betacoronavirus Reveals Evolution of Middle East Respiratory Syndrome-Related Coronavirus Spike Proteins for Human Dipeptidyl Peptidase 4 Binding.

Although bats are known to harbor Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses, the role of bats in the evolutionary origin and pathway remains obscure. We identified a novel MERS-CoV-related betacoronavirus, Hp-BatCoV HKU25, from Chinese pipistrelle bats. Although it is closely related to MERS-CoV in most genome regions, its spike protein occupies a phylogenetic position between that of Ty-BatCoV HKU4 and Pi-BatCoV HKU5. Because Ty-BatCoV HKU4 but not Pi-BatCoV HKU5 can use the MERS-CoV receptor human dipeptidyl peptidase 4 (hDPP4) for cell entry, we tested the ability of Hp-BatCoV HKU25 to bind and use hDPP4. The HKU25-receptor binding domain (RBD) can bind to hDPP4 protein and hDPP4-expressing cells, but it does so with lower efficiency than that of MERS-RBD. Pseudovirus assays showed that HKU25-spike can use hDPP4 for entry to hDPP4-expressing cells, although with lower efficiency than that of MERS-spike and HKU4-spike. Our findings support a bat origin of MERS-CoV and suggest that bat CoV spike proteins may have evolved in a stepwise manner for binding to hDPP4.

Group A Rotaviruses in Chinese Bats: Genetic Composition, Serology, and Evidence for Bat-to-Human Transmission and Reassortment.

Bats are natural reservoirs for many pathogenic viruses, and increasing evidence supports the notion that bats can also harbor group A rotaviruses (RVAs), important causative agents of diarrhea in children and young animals. Currently, 8 RVA strains possessing completely novel genotype constellations or genotypes possibly originating from other mammals have been identified from African and Chinese bats. However, all the data were mainly based on detection of RVA RNA, present only during acute infections, which does not permit assessment of the true exposure of a bat population to RVA. To systematically investigate the genetic diversity of RVAs, 547 bat anal swabs or gut samples along with 448 bat sera were collected from five South Chinese provinces. Specific reverse transcription-PCR (RT-PCR) screening found four RVA strains. Strain GLRL1 possessed a completely novel genotype constellation, whereas the other three possessed a constellation consistent with the MSLH14-like genotype, a newly characterized group of viruses widely prevalent in Chinese insectivorous bats. Among the latter, strain LZHP2 provided strong evidence of cross-species transmission of RVAs from bats to humans, whereas strains YSSK5 and BSTM70 were likely reassortants between typical MSLH14-like RVAs and human RVAs. RVA-specific antibodies were detected in 10.7% (48/448) of bat sera by an indirect immunofluorescence assay (IIFA). Bats in Guangxi and Yunnan had a higher RVA-specific antibody prevalence than those from Fujian and Zhejiang provinces. These observations provide evidence for cross-species transmission of MSLH14-like bat RVAs to humans, highlighting the impact of bats as reservoirs of RVAs on public health.IMPORTANCE Bat viruses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), Ebola, Hendra, and Nipah viruses, are important pathogens causing outbreaks of severe emerging infectious diseases. However, little is known about bat viruses capable of causing gastroenteritis in humans, even though 8 group A viruses (RVAs) have been identified from bats so far. In this study, another 4 RVA strains were identified, with one providing strong evidence for zoonotic transmission from bats to humans. Serological investigation has also indicated that RVA infection in bats is far more prevalent than expected based on the detection of viral RNA.

Close genetic relatedness of picornaviruses from European and Asian bats.

Our investigation of 1004 faecal specimens from European bats for picornaviruses by broadly reactive nested reverse transcription-PCR found picornaviral RNA in 28 samples (2.8 %). Phylogenetic analysis of the partial 3D genomic region suggested that one bat virus belonged to the species Enterovirus G (EV-G, formerly Porcine enterovirus B). Bat infection was supported by relatively high EV-G concentrations of 1.1×106 RNA copies per gram of faeces. All other bat viruses belonged either to the bat-associated genus Mischivirus, or to an unclassified Picornaviridae group distantly related to the genus Sapelovirus. Members of this unclassified sapelovirus-related group had RNA secondary structures in their 3'-nontranslated regions that were typical of enteroviruses and that resembled structures that occur in bat-associated coronaviruses, suggesting ancient recombination events. Based on sequence distances, several picornaviruses from European and Chinese bats were likely conspecific, suggesting connectivity of virus populations. Due to their high mutation rates and their diversity, picornaviruses may be useful tools for studies of bat and virus ecology.

SARS-CoV ancestor found in Chinese bats

A new study reports the isolation of a novel coronavirus (CoV) from Chinese horseshoe bats, which is more closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) than any previously identified bat CoV, providing strong evidence that SARS-CoV might have originated in bats. Whole-genome sequencing of the novel CoV revealed that it shares 95% sequence identity with human SARS-CoV, with the receptor-binding domain (RBD) in the spike protein from both viruses showing an identical amino acid sequence. The RBD of human SARS-CoV uses the cellular receptor angiotensin-converting enzyme 2 (ACE2) for cell entry, and infectivity assays confirmed that the novel CoV also uses human ACE2 for cell entry, suggesting that it might be capable of directly infecting humans. Furthermore, preliminary in vitro data suggest that the new virus has broad species tropism.

Shandong University—Virginia Tech Biosonar Research in China

China is home to a diverse and in many places abundant bat fauna. Among the most diverse and prominent bat families in the country are the horseshoe bats (Rhinolophidae) which also have one of the most specialized and capable sonar systems found in nature. Biosonar research at the Shandong University - Virginia Tech International Laboratory seeks to understand the capabilities and the diverse adaptations in the sonar systems of Chinese bats. A common thread of this research is the analysis of the natural variability in biosonar solutions. For example, the International Laboratory has been compiling a digital database of the noseleaves and outer ear shapes of bats from China and neighboring regions. Current research explores biological adaptation pattern in these shapes and their acoustic properties. Other research explores sound production across different species of Chinese bats. The International Laboratory also conducts behavioral experiments, in particular with horseshoe bats. This research has characterized novel dynamic features on the emission as well as on the reception side of the horseshoe bats' biosonar system. The International Laboratory collaborates with a sister lab at Virginia Tech (GLOBES) on the engineering analysis of the biosonar properties it discovers as well as the development of bioinspired devices.

Recent Surveys of Bats (Mammalia: Chiroptera) from China II. Pteropodidae

We review the distribution and conservation status of Old World fruit bats (Pteropodidae) in tropical and subtropical China. Our results are based on a series of surveys conducted between 1999 and 2008 by Chinese researchers and international collaborators, spanning almost the entire range of pteropodids within China over nine provinces. Additionally we provide new information on morphometrics and notes on ecology. We also review earlier Chinese literature on fruit bats because much of this has previously been inaccessible to western scientists, and we evaluate the reliability of some of these older records. Thirteen species of fruit bats have been reputed to occur in China, including one species restricted to Taiwan. We classified Chinese fruit bats according to distribution and status as follows: Resident (six species, including one species formerly found in Taiwan and now restricted to its neighbouring islands), Marginal (three species), Questionable (one species) and Alien (three species). Consequently, only five species are encountered with any regularity in mainland China and Hainan Island: Cynopterus sphinx, Eonycteris spelaea, Macroglossus sobrinus, Rousettus leschenaultii, and Sphaerias blanfordi. Three species in the genus Pteropus are referred to in old records, but these bats are clearly not native to mainland China.

Seroprevalence of Rabies Virus Antibodies in Bats from Southern China

Members of the Order Chiroptera are the natural reservoirs of lyssaviruses and play an important role in the transmission of rabies to animals and humans. In this present study, the seroprevalence for rabies virus was determined for bats sampled from four southern provinces on the Chinese mainland. A total of 685 bats of 8 species representing 4 families were collected from 10 sites, and were tested by the indirect fluorescent antibody test using fluorescein isothiocyanate (FITC)-conjugated protein A/G mixture and viral neutralization test. Rabies antibody response was only detected from three bat species (Rousettus leschenaulti, Rhinolophus blythi, and Rhinolophus ferrumequinum). The overall rabies seroconversion rate was only 2.2% (15/685). Of the 15 positive sera, 13 (12 fruit bats and 1 insectivorous bat) were indirect fluorescent antibody test positive, and two insectivorous bats were virus neutralization positive when tested by the modified fluorescent antibody viral neutralization test, albeit extremely low. To our knowledge, this is the first published report describing rabies seroprevalences from Chinese bats. These results suggest that bats may play a role in the ecology of lyssaviruses in China, and further surveillance for the presence of lyssaviruses in bats should be undertaken throughout the country and extended to other species.

Analysis of Putative Virulence Factors in Enterococcus faecium Isolated from Different Sources in Iran

Background: Although the finding of SARS coronavirus SARS-CoV) in caged palm civets suggested that wild animals re the origin of SARS-CoV, subsequent studies suggested hat civet may have served only as an amplification host. n 2005, we identified a coronavirus closely related to SARSoV (bat-SARS-CoV) in Chinese horseshoe bats. However, it emains to be determined if bat-SARS-CoV or other coronviruses in bats are the direct progenitor of SARS-CoV. Methods: To understand the diversity and evolution of oronaviruses in bats, a 2-year surveillance study for coroaviruses was conducted in bats from various rural areas n Hong Kong. As coronaviruses are known to have high ecombination frequency, the genomes of the identified ovel coronaviruses were also sequenced and analyzed to etermine possible recombination events responsible for nterspecies transmission. Results: Among 1389 bats of 16 species from 24 different ocations, coronaviruses were identified from anal swabs of 32 (9.5%) bats by RT-PCR. Phylogenetic analysis revealed t least seven novel coronaviruses from seven different bat pecies, in addition to bat-SARS-CoV. Five of them belonged o group 1 coronaviruses while two belonged to group 2 coroaviruses. Besides bat-SARS-CoV, Chinese horseshoe bats ere found to harbor another novel group 1 coronavirus. he genome of this virus represents the smallest coronavirus enome and possessed a unique spike protein evolutionarly distinct from the rest of the genome and containing a 5-amino acid peptide homologous to a corresponding pepide within the RBM of spike protein of SARS-CoV, suggesting common evolutionary origin in the spike protein of this roup 1 bat coronavirus, bat-SARS-CoV, and SARS-CoV. Conclusion: Bats are important reservoir for a huge diverity of coronaviruses, including SARS-CoV-like viruses. The nding of another group 1 coronavirus in Chinese horsehoe bats with a homologous peptide to SARS-CoV warrants urther investigations on the origin of the SARS-CoV spike rotein.