Etiological Study Of Coronaviruses Carried By Representative Bat Species In Southwest China

Currently,more than half of the emerging infectious diseases in the world are animal-derived viral infectious diseases,and its proportion in emerging infectious diseases is increasing year by year.Bats are the only mammals that have the ability to fly,and they are widely distributed and numerous,and they are in close contact with humans.Bats are the natural hosts of many highly pathogenic zoonotic viruses that threaten human health,such as SARS-CoV,MERS-CoV,SADS-CoV and SARS-CoV-2 etc.According to reports,early cases of SARS and COVID-19 outbreaks were found in Guangdong and Hubei,respectively,but viruses highly homologous to them were found in Rhinolophus spp.in Yunnan province.Although SADS-CoV only broke out in swine herds in Guangdong Province,as a highly pathogenic pathogen of piglets recently spread from bats to herds,its threat to China's pig breeding industry has not disappeared.Sichuan Province is China's largest pig breeding base,and it is urgent to confirm whether local bats carry SADS-related-CoVs.Sichuan,Yunnan and Guangxi are geographically connected and have rich and diverse wildlife resources.Therefore,investigating the coronavirus carried by representative bat species in southwestern China is of great significance for early warning and traceability of emerging infectious diseases.Methods:(1)We used a viromics analysis method combining next-generation sequencing technology and bioinformatics analysis to investigate the coronavirus carried by representative bats in three provinces of Sichuan,Yunnan and Guangxi in China;(2)We use Nested PCR,Genome Walking and Rapid-amplification of cDNA ends technologies to obtain the full genome sequence of coronavirus in the positive samples of coronavirus;(3)We used Statistical methods and Similarity plot analysis to study the similarities and differences between newly identified coronaviruses and known coronaviruses;(4)We used Phylogenetic Analysis to study the genetic evolution characteristics,host characteristics and geographical distribution of newly identified coronaviruses in different bats in different regions;(5)We used Recombination analysis to study the possible recombination relationship between the newly identified coronavirus and known coronavirus,and then analyzed the problems of cross-species transmission and spillover risk of coronavirus.Results:(1)This study collected 895 bat anal-pharyngeal swab samples involving 24 bat species from three provinces in Sichuan,Yunnan and Guangxi,and amplified 11 complete sequence of coronaviruses from 22 positive samples involving 4 bat species;(2)The complete genome sequences of 4 coronaviruses(BtSk-AlphaCoV/GX2018A,B,C,D)amplified from Scotophilus kuhlii in Guangxi belong to the subgenus Pedacovirus of genus AlphaCoV,and belong to the same coronavirus species with BtCoV/512.BtSk-AlphaCoV/GX2018D is a recombinant sequence whose S1 subunit of S protein is more closely related to PEDV.This recombinant fragment may be derived from the ancestral sequence of PEDV;(3)BtRs-AlphaCoV/YN2018 amplified from the Rhinolophus sinicus in Yunnan belongs to a new coronavirus species of the subgenus Decacovirus of genus AlphaCoV.There are at least two traces of recombination in the S protein region;(4)Five SARSr-CoVs were amplified from Rhinolophus spp.in Sichuan and Yunnan,and belong to the subgenus Sarbecovirus of BetaCoV genus.The coronavirus sequence(BtR1-BetaCoV/SC2018)from Rhinolophus spp.in Sichuan Province is the first SARSr-CoVs discovered in Sichuan Province.BtRs-BetaCoV/YN2018B from Rhinolophus sinicus is closest to SARS-CoV and highly homologous to SARSr-CoV WIV1 and SARSr-CoV WIV16 that can utilize human ACE2;(5)The complete genomic sequence of BtCs-BetaCoV/GX2018 amplified from Rousettusleschenau belongs to a new species under the subgenus Nobecovirus of genus BetaCoV,which is far from other coronaviruses in the subgenus Nobecovirus.Conclusion:(1)The coronavirus found in Scotophilus kuhlii in Guangxi means that the BtCoV/512-related coronavirus has a wide geographical distribution and is closely related to PEDV.The recombinant sequence(BtSk-AlphaCoV/GX2018D)provides new clues that PEDV may have originated from bats;(2)The BtRs-AlphaCoV/YN2018 identified in Rhinolophus sinicus enriches the species of coronavirus carried by Rhinolophus sinicus,and the coronavirus of the subgenus Pedacovirus where the virus is located exhibits diverse host characteristics;(3)The BtRI-BetaCoV/SC2018 found in Rhinolophus spp.in Sichuan Province means that the distribution of SARSr-CoVs is more extensive than previously estimated.The SARSr-CoVs carried in Rhinolophus spp in Yunnan Province are more closely related to SARS-CoV,providing evidence that SARS-CoV originated from bats in Yunnan Province.The discovery of BtRs-BetaCoV/YN2018B means that SARSr-CoVs that can utilize human ACE2 continue to circulate in Rhinolophus sinicus in Yunnan Province;(4)BtCs-BetaCoV/GX2018 found in Rousettusleschenau in Guangxi belongs to the subgenus Nobecovirus is an independent evolutionary lineage.This study investigated the status of coronavirus carried by representative bat species in three provinces in Sichuan,Yunnan and Guangxi in southwestern China.The analysis of the newly identified coronavirus provided more evidence for the bats with SARS-CoV originating in Yunnan Province and a new clue to the origin of PEDV bats.The results of the study supplemented the baseline data on coronavirus carried by different bat species in key areas related to the epidemic,and provided background data to prevent potential emerging bat-derived infectious diseases.