Application Of High-throughput Sequencing For Virus Detection And Analysis In Wild Animal Feces

With the fast development of bioscientific research and biological technology, new viruses, as well as some new types of popularly known viruses are discovered. A latest report by Anthony and his colleagues predicted that there might be over 320 thousand unknown viruses hiding in mammals. These newly emerging and re-emerging viruses, including Ebola virus, HCV, HIV, SARS and some other virus, pose a threat to human health. Therefore, in order to prevent these emerging viral infectious diseases cause by these newly emerging and re-emerging viruses, identifying the newly emerging viruses and validating the relationship between these viruses and the diseases has been the most emergent problem to be solved. There exist traditional methods to discover new viruses, including isolation of virus, nucleic acid testing,serologic test, etc. Viral Metagenomics, a new discipline based on High-throughput sequencing technology, has broken the limitations of traditional technologies, directly working on the genetic material of the pathogens in the specimens. This technique directly conducts a sequencing process after a slight processing on PCR products, which conveys the information of a large amount of nucleic acid sequences to identify the elements of viruses. This new technology has the advantage of lower cost and higher practical value with simple operatingprocedures.Some wild animals in special geographical climate conditions such as tropics and plateau regions might be the carried and spread sever pathogen. Researchers in western countries have been to study the viruses carried by wild woodchuck, and found some important virus caused sever disease, including Powassan Virus, Rabies virus, Woodchuck Hepatitis Virus, Hepatitis B virus, Hepatitis D virus, etc. However, no research has been done on studying the virus spectrum of Himalayan Marmot. Hence,the study on the virus spectrum of their stool specimens is also of great significance in predicting and warning new emerging virus-caused disease of human beings.This study explores virome of wild woodchuck stool specimen by processing method based on Miseq high-throughput sequencing technique and bioinformatics analysis of sequences, and further identify the potential emerging virus, by bioinformatics and molecular biology methods to explore the genetic structure, molecular epidemiology characteristics of the potential emerging virus, so as to conduct preliminary exploration on the infection of new viruses in human beings and animals. Through Miseq high-throughput sequencing, A total of5,742,980 reads were obtained, with unsorted, bacteria, phage sequences removed, and 10,074 sequences were annotated to viruses, of which5,836 reads were annoted to 30 viral families or subfamilies, including 10 vertebrate virus families(52%), 6 insect virus families(6.1%), 7 plant virus families(12.6%) and other viruses(27.3%). A novel Woodchuck Hepatitis A virus(WHAV) was found in the wild woodchuck stool samples by genetic sequences and evolutionary genetics analysis.Through further analysis, the length genomes, WHAV is 7566 bp,encoding a complete polyglutamine protein of 2252 amino acids long,which shared 67% amino acid homology with the known Hepataovirus,Simian HAV. The genetic characteristics and evolutionary analysis indicate that the virus might be a new species, within family Picornaviridae. Molecular epidemiological analysis of the viral 5′UTR region shows that the detection rate of this virus in healthy Qinghai-Himalayan Marmot stool specimens is 16.16%(16/19), while the virus is not detected in the serum of local people, sheep, pigs, cattle and chickens. This study shows that there emerge some novel types of viruses in stool specimens of woodchuck and it proves the diversity of these viruses,which will be used in predicting and warning potential virus-caused disease.