Virome Analysis Of Novel Mammalian Viruses In Bats And Characterization Of Bat Coronavirus From Southeast China

In recent years,the frequent occurrence of new infectious diseases has seriously threatened human health and social stability.According to incomplete statistics,more than 60% of new infectious diseases originate from animals.Bats,the second largest mammal in the world after rodents,play an important role in the occurrence,epidemic and transmission of many important zoonoses,especially as natural reservoir of a large number of viruses.The southeastern coastal area of China is an important foreign exchange market which is located in the southeastern border of China.The coastal wetlands southeast of China consist of growing ports,industrial districts,and port cities with warm climate and abundant rainfall,the natural environment is suitable for the multiplication of a variety of medical organisms.It is known that more than 2000 species of wildlife are abundant in host animals and medical vector communities.Studies show the occurrence of natural focal diseases,such as Dengue fever and hemorrhagic fever with renal syndrome,in species-rich and densely populated southeast of China.It is important to widely conduct surveillance on virus carrying situation in different animals across different habitats.An understanding of the natural habitat of bat-associated viruses can prevent the cross-species transmission of virus from wildlife and newly emerging and re-emerging zoonoses.With the rapid development of biotechnology,the use of viral metagenomics based on second-generation sequence technology allowed us directly carries out high-throughput sequencing of all nucleic acid sequences in the environment,provides an efficient tool for large scale investigating species diversity of environmental communities,and becomes an effective means of discovering new viruses.In order to understand the viral spectrum information of bats in southeastern coastal areas of China,viral metagenomics technology was used to investigate the virome of bats collected in this area from 2015 to 2017,and to carry out in-depth research on bat-origin SARS-like coronavirus of this area.The main research contents and results are as follows:1.We combined second-generation sequencing technology with metagenomics to understand the outbreak of new infectious diseases caused by animal-origin pathogens and explore the unknown viruses from the natural environment,humans,and animals.In this study,we collected 235 bats from six locations in the southeastern coastal area of China from July 2015 to August 2015.Using Illumina platform for sequencing gut and lung tissue from bats,Viral metagenomic analyses were carried by bats in southeast coastal China.A total of 5990261 high-quality reads were obtained,of which 297531 were spliced and merged into tag sequences and 6314901 overlapping sequences were successfully predicted to be ORF,of which 2.37% of the ORF(15012/631490)were matched the virus.Further classification revealed that the virus sequence involved 25 virus families.It includes 16 vertebrate viruses,4 plant viruses and 5 insect viruses.Through BLAST comparison and evolutionary analysis,it is suggested that novel and genetically diverse viruses may be found.This investigation of virus spectrum in bats in the southeastern coast of China enriched the understanding of bats as virus reservoir,and provided a theoretical basis for further isolation and culture of bat-borne viruses and further analysis of the possible transmission of viruses from bats to humans.2.To verify and analyze important pathogens.In order to verify the reliability of viral metagenomic results,six viruses(Coronavirus,Mastadenovirus,Mamastrovirus,Circovirus,Norovirus,Bocaparvovirus)closely related to human pathogenicity were selected for PCR amplification and evolutionary analysis.Results among the different bat species,Rhinolophus pusillus carried the broadest spectrum of viruses including coronavirus,adenovirus,norovirus,circovirus,and astrovirus.Of the remaining four bat species,bocavirus was detected in the bat species Myotis davidii for the first time(n =2),coronavirus was detected in the bat species Rhinolophus ferrumequinum(n= 2),and astrovirus was detected in bat species Myotis formosus(n = 1).However other viruses were absent.Among them,coronavirus carried the highest rate,with the detection rate of 6.8%-18.2% in Zhoushan,Changle and Shishi groups.The results of PCR validation were basically consistent with those of viral metagenomic,suggesting that viral metagenomic has guiding significance for the discovery of new viruses in the environment.3.Identification of coronavirus in bats in Zhoushan area.In this study,334 bats collected from July 2015 to February 2017 were investigated by RT-PCR.The results showed that the total carrying rate of coronavirus was 26.65%(89/334).Two full-length sequences of SARS-like coronavirus were successfully obtained by overlap PCR and terminal RACE amplification.Phylogenetic analysis showed that the two viruses belonged to a new branch of ?coronavirus.The two new bat SL-CoVs shared 97% genomic sequence identity among themselves.The overall nucleotide sequence identity of these two genomes with civet SARS-CoV(SZ3 strain)was 81%,which was lower than the previously reported observations associated with bat SL-CoVs collected from China(88–92%).4.Study on virulence of newly identified SL-CoVs virus strains.Infection of suckling mice with supernatant of ZC45 bat tissue showed that inflammation occurred in various tissues and organs of suckling mice.Electron microscopic observation of brain tissue showed that coronavirus particles were detected.The anti-polypeptides were derived from the ZC45 N protein antibodies from six different sources of N proteins(50kDa),including the intestinal tissues,brain tissues and lung tissues of infected rats.The results indicated that the polypeptide antigen was synthesized correctly,and the polyclonal antibodies produced against this polypeptide could react with the N proteins of the bat SL-CoV.This observation highlights the possibility of cross-species transmission of these viruses.In summary,through the metagenomic sequencing of bat samples collected in the past three years in southeast China and RT-PCR confirmation,this study enriched the knowledge of the virus spectrum carried by bats in this areas of China,and also greatly promoted our understanding of the diversity of virus pools carried by bats.The original identification study and systematic elaboration of the evolutionary relationship of important pathogens provide clues for the basic data of bat-origin viruses in this area.This study involved continuous surveillance of the SL-CoVs that originated from the bats of this region.Diverse bat SL-CoVs were identified in this region,and the SL-CoVs in this region remained stable and could be transmitted to each other.Although there were several differences between the SARS-CoVs and the bat-SL-CoVs procured from this region based on the two-full-length samples obtained in this study,especially pertaining to the S protein region,and more importantly,this strain could still cause infection in suckling mice.This observation highlights the possibility of cross-species transmission of these viruses.These findings strongly suggest the need for continued surveillance of viruses originating from wild animals and promote further research to study the possibility of cross-species transmission of these viruses.