Characterization Of Bat-borne Parvovirus Diversity And Construction Of Infectious Bat Circovirus DNA Clone

Bats belonging to Chiroptera,are the only mammals that really have the ability to fly.And they are one of the most species of mammal in nature.Bats are considered to be the natural hosts of many important zoonotic viruses,such as SARS-like coronavirus,filovirus,bunyavirus,calicivirus,hepatitis virus and so on.In the previous year,we studied the patho-ecology of viruses carried by bats all over China by means of viral metagenomics,and found a variety of parvovirus and circovirus,which showed rich diversity in the species and quantity.Therefore,this subject study the genetic diversity of parvovirus carried by bats in three provinces: Guangxi,Guangdong and Yunnan.At the same time,it is the first time that the infectious clones of bat circovirus have been constructed to study the special structure of its genome.Family Parvoviridae has a lot of members,there are 13 genera and 60 species in it.Its host range is extensive,including many insects and mammals.Researchers have found many bocaviruses and adeno-associated viruses in bats,but they have never been studied systematically.In this study,1311 bats were collected in three province: Guangxi,Guangdong and Yunnan,China,which belong to family Pteropodidae,Hipposideridae,Vespertilionidae,Rhinolophidae.High-throughput sequencing was used to study the viruses carried by bats,and a total of 285788 viral reads were annotated,including 32272 vertebrate viral reads,2049 plant viral reads,237912 phage reads and 13555 insect viral reads.In vertebrate viral reads,the largest number is parvovirus,annotated to 21809,8 genera in subfamily Parvovirinae.After sequencing,18 different parvoviruses were identified.PCR detection showed that the positive rate in bat group was 2.8%-100%.Identity analysis showed that the identity of amino acids between the 18 bat parvovirus and ICTV reference strain was less than 85%,all of them were new species of parvovirus family.Genomic amplification of 10 parvoviruses was carried out,and all or part of coding regions were obtained by PCR and genomewalking.Phylogeny analysis shows that 4 species of the ten new parvoviruses belong to the genus Dependoparvovirus.2 belong to the genus Bocaparvovirus,1 belongs to the genus Amdoparvovirus,one of the genus Tetraparvovirus,one of the genus Protoparvovirus,and one that may be a new genus of the other bats parvovirus.Circovirus is one of the smallest DNA viruses in the world.Its genome is only 1.7-2.2kb,which contains two main open reading frames.Circovirus is widely distributed,and can infect mammals,birds,fish,insects and so on.At the same time,some circovirus infections are also related to human diseases.Because of lack of suitable cell lines,circovirus is difficult to isolate and culture in vitro.We can not further study its life characteristics and pathogenicity.Therefore,we built the infectious clone of bat circovirus and studied the function of a special polypyrimidine structure(poly-T)in its genome.The poly-T consists of 25-29 consecutive thymidine deoxyribonucleotides,which is located in 3' intergenic region between the two open reading frames.We constructed two double-copy bat circovirus infectious clones: wild type(wtBatACV)and poly-T deletion(?TBatACV).By electron microscopy and RNA detection,two bat circovirus infectious clones were successfully rescued.Through the observation of in vitro transfection and indirect immunofluorescence assay(IFA),there was no significant difference in the speed and ability of the two infectious clones packaging virus particles.It was suggested that the poly-T structure had no effect on the replication of circovirus.Parvovirus and circovirus are widely distributed and diverse in nature as two kinds of small DNA viruses.This study systematically investigated the genetic diversity of parvovirus in China.Among the 4 families and 5 genera of 17 species of bats in three provinces of the southern coastal provinces of China,the existence of parvovirus in 8 genera of subfamily Parvovirinae was investigated in detail,18 new parvoviruses were found.We provided important basic data for the species and distribution of parvovirus in China.At the same time,this study first constructed the infectious clone of bats virus,successfully rescued the bats circovirus particles,identified the poly-T structure in its genome from function,and increased our understanding of the genome of the circovirus.