Novel Virus Discovery In Bat And The Exploration Of Receptor Of Bat Coronavirus HKU9

The outbreak and pandemic of infectious diseases have presented as greatest threat to public health. Under the background of globalization and industralization,pathogens quickly spread all over the world wherever they outbreak. Meanwhile, in recent decades, the rate of occurrence of novel pathogens also seems to exceed any other time in the past.Wildlife plays an important role in the emerging and spreading of zoonoses.Current researches in the public health community give rise more concerns on the detection, isolation and identification of pathogens that wild animals harbor, and the relationship with diseases.The modern molecular biology methods have played a vital role in the clinical detection and identification of pathogens. Metagenomics and next-generation sequencing (NGS) focus on the direct analyses of genetic materials (DNA or RNA)from clinical samples. The advances in technologies play an increasingly important role in the rapid detection and identification of pathogens and further for the prevention and control of diseases.The outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 aroused immense attention to biodiversity, genomics and the possibility of cross-species transmission of coronavirus, especially the coronaviruses from bats, the second largest group of mammals. Bats are particularly notable in this respect because they are known to harbor a diverse range of pathogens, and are known to be reservoir hosts of human coronavirus 229E and SARS-CoV, and are closely related to MERS-CoV. As a consequence, bats have been prioritized for surveillance of emerging zoonotic diseases.In the current study, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples from Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3'-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus.Subgenomic mRNA and cellular-level findings demonstrated that the p10 gene is functional and may induce the formation of cell syncytia. This is the first report of heterologous inter-family recombination between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, and as such provides insights into the fundamental mechanisms of viral evolution.Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (BetaCoV)that is phylogenetically affiliated to the same genus as MERS-CoV. The bat-surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential of cross-species transmision. The receptor binding domain (RBD) of the coronavirus spike (S) recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other BetaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither the SARS-CoV receptor of ACE2 (Angiotensin-Converting Enzyme 2) nor the MERS-CoV receptor of CD26. We further solved the atomic structure of HKU9-RBD,which is expectedly composed of a core and an external subdomain. The core subdomain fold resembles those of other BetaCoV RBDs; whereas the external subdomain is structurally unique with a single helix, explaining the inertness of HKU9-RBD to react with either ACE2 or CD26. Via comparison of thus-far available RBD structures, we further proposed a homologous inter-subdomain binding mode in BetaCoV RBDs that anchors the core subdomain to the external subdomain. The revealed RBD features would shed light on the BetaCoV evolution route.