Ben Hu

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

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.

Bat origin of human coronaviruses.

Bats have been recognized as the natural reservoirs of a large variety of viruses. Special attention has been paid to bat coronaviruses as the two emerging coronaviruses which have caused unexpected human disease outbreaks in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are suggested to be originated from bats. Various species of horseshoe bats in China have been found to harbor genetically diverse SARS-like coronaviruses. Some strains are highly similar to SARS-CoV even in the spike protein and are able to use the same receptor as SARS-CoV for cell entry. On the other hand, diverse coronaviruses phylogenetically related to MERS-CoV have been discovered worldwide in a wide range of bat species, some of which can be classified to the same coronavirus species as MERS-CoV. Coronaviruses genetically related to human coronavirus 229E and NL63 have been detected in bats as well. Moreover, intermediate hosts are believed to play an important role in the transmission and emergence of these coronaviruses from bats to humans. Understanding the bat origin of human coronaviruses is helpful for the prediction and prevention of another pandemic emergence in the future.

Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT We report the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.

Detection of diverse novel astroviruses from small mammals in China

Astroviruses infect humans and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive-bred porcupines and a nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of the complete ORF2 sequence suggested that this strain may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astroviruses in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft

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.

Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses

This is the first country-wide surveillance of bat-borne viruses in Kenya spanning from 2012–2015 covering sites perceived to have medium to high level bat-human interaction. The objective of this surveillance study was to apply a non-invasive approach using fresh feces to detect viruses circulating within the diverse species of Kenyan bats. We screened for both DNA and RNA viruses; specifically, astroviruses (AstVs), adenoviruses (ADVs), caliciviruses (CalVs), coronaviruses (CoVs), flaviviruses, filoviruses, paramyxoviruses (PMVs), polyomaviruses (PYVs) and rotaviruses. We used family-specific primers, amplicon sequencing and further characterization by phylogenetic analysis. Except for filoviruses, eight virus families were detected with varying distributions and positive rates across the five regions (former provinces) studied. AstVs (12.83%), CoVs (3.97%), PMV (2.4%), ADV (2.26%), PYV (1.65%), CalVs (0.29%), rotavirus (0.19%) and flavivirus (0.19%). Novel CalVs were detected in Rousettus aegyptiacus and Mops condylurus while novel Rotavirus-A-related viruses were detected in Taphozous bats and R. aegyptiacus. The two Rotavirus A (RVA) strains detected were highly related to human strains with VP6 genotypes I2 and I16. Genotype I16 has previously been assigned to human RVA-strain B10 from Kenya only, which raises public health concern, particularly considering increased human-bat interaction. Additionally, 229E-like bat CoVs were detected in samples originating from Hipposideros bats roosting in sites with high human activity. Our findings confirm the presence of diverse viruses in Kenyan bats while providing extended knowledge on bat virus distribution. The detection of viruses highly related to human strains and hence of public health concern, underscores the importance of continuous surveillance.

Longitudinal surveillance of SARS-like coronaviruses in bats by quantitative real-time PCR

In previous studies, the authors have found diverse SARS-like coronaviruses (SL-CoVs) in a single bat colony. To investigate the spatio-temporal dynamics of these viruses, a longitudinal surveillance of bat SL-CoV from a total of 431 bat fecal samples collected during 2011 to 2014 from the Yunnan in China using quantitative real-time PCR (qRT-PCR) by targeting the nucleocapsid (N) and RNA dependent RNA polymerase (RdRp) genes was conducted in this study. The authors demonstrated that the detection rate of the SL-CoV was higher during July to October providing useful information that might be helpful for surveillance of potential transmission of bats in the future.

Longitudinal Surveillance of Betacoronaviruses in Fruit Bats in Yunnan Province, China During 2009–2016

Previous studies indicated that fruit bats carry two betacoronaviruses, BatCoV HKU9 and BatCoV GCCDC1. To investigate the epidemiology and genetic diversity of these coronaviruses, we conducted a longitudinal surveillance in fruit bats in Yunnan province, China during 2009–2016. A total of 59 (10.63%) bat samples were positive for the two betacorona-viruses, 46 (8.29%) for HKU9 and 13 (2.34%) for GCCDC1, or closely related viruses. We identified a novel HKU9 strain, tentatively designated as BatCoV HKU9-2202, by sequencing the full-length genome. The BatCoV HKU9-2202 shared 83% nucleotide identity with other BatCoV HKU9 stains based on whole genome sequences. The most divergent region is in the spike protein, which only shares 68% amino acid identity with BatCoV HKU9. Quantitative PCR revealed that the intestine was the primary infection organ of BatCoV HKU9 and GCCDC1, but some HKU9 was also detected in the heart, kidney, and lung tissues of bats. This study highlights the importance of virus surveillance in natural reservoirs and emphasizes the need for preparedness against the potential spill-over of these viruses to local residents living near bat caves.

Correction to: Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses.

The affiliation listed for Cecilia Waruhiu is incorrect. The byline and affiliation line should appear as shown above.