Research Of Intestinal Viral Metagenomics And Identification Of Trichomonas From Tupaia Belangeri

Tupaia belangeri belongs to the familu tupaiidae of Order Scandentia,is similar in appearance to squirrels.Compared with rodents,genome of tree shrews are similar to primates.The tree shrew has been widely used in the study of infection of hepatitis virus,Staphylococcus aureus and Trypanosoma,but the tree shrews used in reports were mostly wild type,and have not been standardized,resulting in the reproducibility and stability of experimental results were not good.Therefore,the standardization of tree shrew quality is extremely important for its application in life science research,and the infection and pathogenicity of pathogenic microorganisms and parasites are the focus of monitoring.Herpesviruses,adenoviruses,and reoviruses have been detected from the tree shrews using traditional methods,but the coverages are still less and incomplete,and the discovery of viruses are limited to the traditional method.Viral metagenomic analyses of uncultured viral communities circumvent these limitations and can provide insights into the composition and structure of environmental viral communities.Therefore,to obtain an unbiased measure of viral diversity in the enteric tract of tree shrews by viral metagenomic approach,we deep sequenced the viral nucleic acid extracted from stool collected from 50 wild tree shrews.In this paper,the best enrichment method of virus from fecal supernatant was obtained by comparing the recovery efficiency of PEG precipitation combined with ultrasonic separation,iron ion flocculation,GBviroFast virus concentration kit and ultra-high speed centrifugation with Q-PCR.Using the Illumina HiSeq 4000 sequencing and bioinformatics analysis,fecal virome of 50 wild Tupaia belanger chinensis were analyzed.The morphological observation of trichomonas in tree shrews was carried out by direct smear and iodine staining.The total DNA of the parasites was extracted by the transmission method.The 18S rRNA and 5.8S rRNA of the trichomoniasis were amplified by PCR.Then,BLAST is used for homology analysis,and MEGA6.06 is used to map phylogenetic tree.The results showed that:(1)The concentration efficiency of PEG precipitation virus was 6.13%,iron ion flocculation was 49.57%,GBviroFast26.12%,ultra high speed centrifugation was 29.9%,iron ion flocculation concentration was the best,and later used in concentration of virus in fecal supematant.(2)In total 20,195,972 reads were generated,which were compared with virus-related databases(Virus Refseq,VirusNT and Acalme),of which 1.177.922 reads were associate with viruses(including phages)Sequences that are homologous to animal viruses include:Picornaviridae(16%)and Herpesviridae(6%),Others also matched Torque teno tupaia virus,tree shrews adenovirus,(21%);and phages of Siphoviridae(27%).Microviridae(26%);Futher,several tree shrew virome sequences matched the insect virus(1%)and the plant virus(1%),as Densoviridae and Caulimoviridae.In addition to the viral sequence,sequences of protozoa,such as Acanthainoeba,Plasmodium falciparum and Trichomonas vaginalis were obtained.(3)Morphological observation showed that the flagellates were Tritrichomonas and Pentatrichomonas in the tree shrew.The sequencing analysis showed that the 18S rRNA of the tree shrew tritrichomonas was similar to tritrichomonas foetus 18S rRNA(AY754332.1)99%;5.8S rRNA was similar to tritrichomonas foetus 5.8S rRNA(AF466749.1)99%.In addition,the 5.8S rRNA of tree shrew Pentatrichomonas was homologous to Trichomonas japonicum(KT203771.1)99%,and 97%with the homology of Pentatrichomonas(AF156964.1).This study expands the knowledge about the spectrum of viruses harboured in feces of wild Tupaia belanger chinensis in Yunnan.Insect virus and plant virus reflect the diet and habitation of tree shrew.Genetic identification of trichomonas in tree shrews revealed that there may be simultaneous Tritrichomonas and Pentatrichomonas.The results of this study provide important data for further study and analysis of the zoonotic virus,which has potential public health significance,as well as the quality control of tree shrews and the establishment of viral infectious disease model,and provide the basis for the quality control of parasites in tree shrews.