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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., Author summary Increasing evidence has been gathered to support the bat origin of SARS coronavirus (SARS-CoV) in the past decade. However, none of the currently known bat SARSr-CoVs is thought to be the direct ancestor of SARS-CoV. Herein, we report the identification of a diverse group of bat SARSr-CoVs in a single cave in Yunnan, China. Importantly, all of the building blocks of SARS-CoV genome, including the highly variable S gene, ORF8 and ORF3, could be found in the genomes of different SARSr-CoV strains from this single location. Based on the analysis of full-length genome sequences of the newly identified bat SARSr-CoVs, we speculate that the direct ancestor of SARS-CoV may have arisen from sequential recombination events between the precursors of these bat SARSr-CoVs prior to spillover to an intermediate host. In addition, we found bat SARSr-CoV strains with different S proteins that can all use the receptor of SARS-CoV in humans (ACE2) for cell entry, suggesting diverse SARSr-CoVs capable of direct transmission to humans are circulating in bats in this cave. Our current study therefore offers a clearer picture on the evolutionary origin of SARS-CoV and highlights the risk of future emergence of SARS-like diseases.

To assess the effect of Lep d2 from Lepidoglyphus destructor as a vaccine for specific immunotherapy on murine model of asthma.Thirty BALB/c mice (SPF) were randomly categorized into a PBS group, an asthma group, and a Lep d2 SIT group. The mice in the asthma group and Lep d2 SIT group were sensitized by intraperitoneal injection with extracts of dust mites on Days 0, 7th, and 14th, while those in the PBS group were injected with PBS. From the 21st day, the asthma group and Lep d2 SIT group exposed to the extracts of dust mites were stimulated by aerosol inhalation for 7 successive days. During the period of the 25th-27th Day, the mice in Lep d2 SIT group were injected intraperitoneally with Lep d2 allergen for SIT 30 min before nasal inhalation, whereas the PBS group and asthma group were treated with only PBS. Twenty-four hours after the final inhalation, all the mice were sacrificed, the bronchoalveolar lavage fluids (BALFs) were collected. The levels of IFN-γ, IL-5 and IL-13 in the BALF and the supernatant of splenocyte culture solution (SSCS) as well as the levels of specific IgE (sIgE) and sIgG2a in the sera were detected by ELISA. The lung tissues of the mice in the above 3 groups were stained by haematoxylin and eosin (HE) and observed by a microscope.The symptoms of acute asthma attack were observed in the mice of the asthma group and Lep d2 group, but not in the PBS group. The allergic inflammation changes in lung in the Lep d2 SIT group were significantly alleviated compared with those in the asthma group. The concentrations of IFN-γ in BALFs and SSCS of the mice in the Lep d2 SIT group were significantly higher than those in the asthma group (both P0.01), while the levels of IL-5 and IL-13 in the former group were significantly lower than those in the latter group (all P0.01). Meanwhile, the level of sIgE of mice in the Lep d2 SIT group was significantly lower than those in the asthma group (P0.01), while the level of sIgG2a of mice in the former group was higher than those in the latter group (P0.01).Lep d2 allergen as a vaccine can alleviate the allergic symptoms in the lung of mice effectively after allergen specific immunotherapy.