Preliminary Study On The Gut Virome Of The Giant Pandas And The Red Pandas

Giant Panda is one of the most endangered species in the world.In recent years,China has taken various measures to protect giant pandas.Nowadays,Giant pandas have been well protected,their number have gradually increased.However,there are still some problems of protection of giant pandas from some unknown deseases.Red pandas(Ailurusfulgens)mainly distribute in the Himalayas and southern China,belonging to the second-class national protected animals.So far there have been many cases of giant panda pandas died of infection due to viral infectious diseases.For a long time,animal virology researches mainly focused on the economic value of domestic animals,while the main researches are always subjected on the known clinicopathological characteristics of the virus such as canine distemper virus(CMV),canine parvovirus(CPV),canine coronavirus(CCV),etc.,So viral disease researches on protected animals like giant pandas and red pandas are of very little.The traditional methods of molecular biology have many unavoidable limitations.They only can detect the current known virus,difficult to find unknown viruses.Nowadays,viral metagenomics research based on the second-generation sequencing technology is a good way to overcome this limitation,as it takes the whole viral nucleic acid of the given sample as the object of study,not only can effectively detect the presence of known viruses,but also can quickly find unknown viruses.Using viral metagenomics,this study conduct a preliminary study of the Gut virome of both giant pandas and red pandas.Main contents and results are as follows:Firstly,this research studied the composition of gut virus community of giant panda and red panda:In the rearch of the composition of gut virus community of giant pandas,the results showed that the composition of enterovirus community of captive giant panda in this study was mainly composed by: Unclassified virus 31.52%,Podoviridae 14.48%,Phycodnaviridae 11.35%,Miniviridae 6.20%,Picornaviridae 5.99%,Tombusviridae 5.83%,Iridoviridae 3.51%,Marseilleviridae2.71%,Baculoviridae 2.66%,Coronaviridae 1.57%,Partitiviridae 1.56%,Retroviridae 1.54%,Siphoviridae 1.48%,Parvoviridae 1.37%,Myoviridae 1.14%,Poxviridae 1.08%,etc.The viral composition of the main gut community of wild normal panda is: Unclaseeified virus 45.42%,Picornaviridae 39.27%,Microviridae 3.91%,Parvoviridae 2.15%,Circoviridae 2.12%,Reoviridae 1.15%,Caulimoviridae 1.03% and etc.Unclassified virus constitutes the main part of the giant panda gut virus community.The main components among the giant panda fecal eukaryotic host virus are as follows: Circoviridae(38.81%),Picornaviridae with Picorna-like viridae(30.07%),Plant virus families(19.53%),Insect virus families(11.46%)and Anelloviridae(0.13%),etc.Among them,Circoviridae accounts for the largest proportion of eukaryotic viruses.Comparison of the main eukaryotic virus communities between the sick wild giant panda and healthy giant panda showed that the ratio of Picornaviruses in diseased wild giant panda was 4.7%,which was higher than that in other healthy giant pandas(0.1%-0.98%).In the reaserch about the composition of enterovirus community in captive red panda,we found that the composition of enterovirus community of captive red panda was mainly composed by Unclassified virus and bacteriophage families(73.06%),followed by insect virus families(16.15%),mammalian viruses families(5.75%)and plant viruses families(5.05%).The composition of eukaryotic viruses form the viral libraries of red pandas was analyzed,the result showed that Genomoviridae took a possession of 62.03%,insect virus families 22.33%,plant viruses families 6.98%,Parvoviridae3.69%,circoviriade3.53%,Picorna-like viridae0.82% and Picornaviridae0.62%.Sencondly,this reaserch revealed the identification of the new intestinal viruses of the giant pandas and red pandasUsing of viral genomics and bioinformatics technology,four novel Circoviruses were found in the fecal samples of the giant pandas(named Gpc V1,Gpc V2,Gpc V3 and Gpc V4).Their whole genomes which were 1900-2800 kb in length were identified with sequence analysis also applied.All of the four viruses contained two main open reading frames(ORFs),Rep and Cap,and the stem-loop structure between the coding regions of Rep and Cap?The four Circoviruses were renamed as Gpc V1-4.Gpc V2 and Gpc V3 contained a putative protein of 570 bp and 417 bp,respectively.The construction of phylogenetic tree based on Rep amino acid sequence showed that four novel pandas Circoviruses were clustered with the Circoviruses from bat.Amino acid sequences Sequence analysis over the amino acid sequences of Rep shared identities were all below 70%,comparing with the Rep protein of relative virus in Gen Bank.Thus,the four Circoviruses are all new species of Circoviridae.Anellovirus is small,non-enveloped viruses containing a single-stranded,negative-sense circular DNA genome enclosed within icosahedral nucleocapsid,which has been identified in variety species of animals.Here,using viral metagenomic method,a novel Anellovirus strain(named AV-Chengdu1 and Gen Bank No.KX611132)was identified in feces from two red pandas.The genome of AV-Chengdu1 is of 2.9kb in length with the G+C content of 49.8%,containing three main ORFs(open reading frames)(ORF1: 581 aa,ORF2: 131 aa and ORF3: 133 aa,131aa).Sequence analysis based on the amino acid sequence of ORF1 showed that AV-Chengdu1 shared the highest identity(56.4%)with pine marten torque teno virus(TTV)(JN704611).Phylogenetic analysis indicated that this novel Anellovirus was clustered with the strain of pine marten TTV,belonging to a clade also contained two sea Anelloviruses and two canine TTVs.Our epidemiologic study indicated that the prevalence rate of this novel Anellovirus in the cohort of captive red pandas was 22.2%(2/9),indicating that this Anellovirus haven't widely spreaded.Conclusions: The composition of enteroviruses of giant panda and red panda was preliminarily studied by using the method of viral metagenomics,especially the composition of eukaryotic virus families,plant virus families and insect virus families;The compositions of intestinal virus community from both giant pandas and red pandas are complicated;This study has identified four novel Circoviruses from the giant pandas and a novel Anellovirus from the red pandas.