Viral Metegenomic Analysis Of Viromes Of Brain,Serum And Rectum Samples And An Epidemiological Investigation Of Rabies Virus In Bats

Bats(order Chiroptera,suborders Megachiroptera and Microchiroptera)are abundant,diverse,and geographically widespread.To date,more than 70 kinds of viruses from bats have been isolated or tested.It is necessary to research the role of bats in ecosystems ecology,and their importance as reservoir hosts of viruses of proven or potential significance for human and veterinary health.With the development of molecular biology techniques and the needs of identifing new viruses,Viral metagenomics is used as a technology to study a population of viruses in a specific circumstance.This technology emphasizes sonsequence-independent amplification and shows grat advantages in identifying unknown viruses,which compensates the deficiency of classcal methods.But there is a challenge to analyse large-scale high-throughput sequencing data,many researchers have to discard most of the raw data or just perform a preliminary analysis,as lacking of processing power and storage capacity.In the past five years,there have been five studies that viral metagenomics was used in bats,including two studies from the United States,three from China.The five studies have found a lot of new viruses in bats by 454 sequencing or Solexa sequencing,although they made people have a far greater understanding about the diversity of the virus bats carried,they are still very limited on research the type and number of bats.Since 2006,our research team has been devoted to conducting epidemiology study about bat-associated viruses in regions of South China.In this study we intend to establish a viral metagenomics analysis method in bats,then this method is used to have a background study on bat-associated viruses of Myotis ricketti and Miniopterus schreibersii.Several important viruses in bats samples will be analysed preliminarily,based on the results of high-through sequencing.At last,a molecular epidemiology investigation of bat-associated viruses will be performed in bats from Hainan,Guangdong provinces.1 Methods1.1 Collection of bat samplesBetween August 2011 to November 2013,545 bats from 11 species were captured and sampled from their natural habitats in some regions of Hainan,Guangdong provinces.Bat species were identified by Prof.Wu Yi(Bats expert,academy for life science,Guangzhou University).The brain tissue,rectum and serum of each bat were collected in sterile tube and store immediately in RNAlater,transport to lab at 4℃,test immediately or store it at-800 C for long-term storage.1.2 Viral metagenomics methodsPurified virus was treated with RNase A and DNase I to remove background nucleic acid,The viral nucleic acid(DNA,RNA)was extracted with kit according to the manufacturer’s protocols.Random PCR was performed in the mixture viral nucleic,after second-strand viral cDNA/DNA synthesized.The PCR products were used to construct library.The sequencing procedure was conducted according to the Solexa sequencing protocol(8)at the Beijing Institute of Genomics.Firstly,a series of DNA fragments less than 800bp were get by ultrasound method(Covaris).The purified DNA were sequenced by the Illumina Genome Analyzer to get the original Reads data.After removing the contaminated reads,the clean reads were were first aligned against the GenBank nt database.The reads and Scaffold with no similarity to the GenBank nt,scaffold database were then aligned against Joint Genome Institute JGI database.The sequences with similarity to eukaryotes,fungi and bacteria were removed.1.3 Detection of specific viruses in bat fecal samplesSpecific primers were designed based on the sequences of target viruses(the human immunodeficiency virus,Semliki Forest virus,human papillomavirus,sugarcane mosaic virus),The cDN A transcribed was used as a template,PCR reaction conditions were differented with primers.PCR products were sequenced Beijing Institute of Genomics,The open reading frames(ORFs)of the viral genomes were predicted using NCBI ORF finderCompare sequences of the PCR products with HIV(or SFV or HPV or ScMV)known sequences of in GeneBank using on line server BLAST.Mutiple sequence alignments were performed by MEGA 4.0 and percentage identities between aligned nucleotide were carried out using MegAlign.MEGA 4.0 was used for phylogenetic analysis.The robustness of phylograms was evaluated by 1,000 bootstrap replicates.Nucleotide distances were estimated from bootstrapped datasets with the method of Maximum composite likelihood.1.4 Detection of rabies virusFirtsly,RT-LAMP method,established by our past research research,was uesed to rapid-screen the brain tissue samples of bats.Then,a nested RT-PCR method was used to detect rabies virus,enlarging for all of the samples.PCR products were sequenced Beijing Institute of Genomics.The acquired sequencers were used to align with the standard sequence.1.5 Analysis of rabies virus N gene fragmentRabies tissue-culture inoculation techniques and mouse inoculation techniques were used to increasing the copies of RV.The filtrated bat brain tissues supernatants of the suspected positive RV strains were inoculated into BHK-21 monolayer.The observation lasted for7 days.2-3 day old NIH rats were inoculated suspected virus supernatants,the observation lasted for 14 days.Supernatants with virus solution were obtained and frozen at-80℃.Nested RT-PCR was used get Long fragment sequence of RV,then the sequences identity comparisons were per-formed by aligning sequences with BLAST.Mutiple sequence alignments were performed by MEGA 4.0 and percentage identities between aligned nucleotide were carried out using MegAlign.MEGA 4.0 was used for phylogenetic analysis.The robustness of phylograms was evaluated by 1,000 bootstrap replicates.Nucleotide distances were estimated from bootstrapped datasets with the method of Maximum composite likelihood.2 Results2.1 Libraries of samples and sequenced resultsFive libraries were constructed,According to viral metagenomics methods.Two libraries were constructed with the brain tissue of Myotis ricketti(HZ47),the other three libraries were constructed brain,rectum,serum of Miniopterus schreibersii(HK69).The Solexa technique was used to sequence the five librarie.There were some reads of each library aligned with virus gene in the GenBank virus databse,There also were some Scaffold,assembled by reads,annotated to virus in Joint Genome Institute(JGI)database.2.2 Metagenomic Analysis of the K6 sample(culture suspension of bat brain tissues)After Solexa sequencing,a total of 1175Mb raw sequence data were obtained from K6 sample,including 5,797,406 clean reads.Among them,8408(0.15%)reads had similarity to viruses(E value<0.00001 for BLASTn).Viral sequences can be divided into three categories:Reverse Transcribing Viruses,dsDNA Viruses,ssRNA Viruses,including 9 Viraceae,they were mainly align to Retroviridae,Polydnaviridae,Herpesviridae,Alloherpesviridae.The Scaffold had similarity to many viruses and phages.Enterobacteria phage lambda,as one of them,had a 1233 nt annotated length.2.3 Metagenomic Analysis of the K3 sample(culture-befor supension of bat brain tissues)After Solexa sequencing,a total of 1,083Mb raw sequence data were obtained from K6 sample,including 5,071,276 clean reads.Among them,5058(0.10%)reads had similarity to viruses(E value<0.00001 for BLASTn).Viral sequences can be divided into four categories:Reverse Transcribing Viruses,dsDNA Viruses,ssRNA Viruses,dsRNA viruses,including 13 Viraceae,they were mainly align to Retroviridae,Polydnaviridae,Herpesviridae,Flaviviridae,Picornaviridae,Secoviridae,T ogaviridae.The Scaffold had similarity to 35 viruses or phages.Among them Enterobacteria phage lambda had a 4705 nt annotated length,Murine leukemia virus had a 1994 nt annotated length2.4 Metagenomic Analysis of the K26 sample(culture-befor supension of bat brain tissues)After Solexa sequencing,a total of 1,826Mb raw sequence data were obtained from K6 sample,including 8,864,191 clean reads.Among them,513(0.01%)reads had similarity to viruses(E value<0.00001 for BLASTn).Viral sequences can be divided into five categories:Reverse Transcribing Viruses,dsDNA Viruses,ssRNA Viruses,dsRNA viruses,Viroids,including 6 Viraceae,they were mainly align to Herpesviridae,Flaviviridae,Picornavirida,Potyviridae.The Scaffold had similarity to7 viruse or phages.Among them,Sugarcane mosaic virus had a 4113 nt annotated length.2.5 Metagenomic Analysis of the K24 sample(culture-befor supension of bat serum)After Solexa sequencing,a total of 1,470Mb raw sequence data were obtained from K24 sample,including 6,632,091 clean reads.Among them,318(0.0048%)reads had similarity to viruses(E value<0.00001 for BLASTn).Viral sequences can be divided into five categories:Reverse Transcribing Viruses,dsDNA Viruses,ssRNA Viruses,dsRNA viruses,including 7 Viraceae,they were mainly align to Picornaviridae,Polydnaviridae,Herpesviridae,Togaviridae,Retroviridae,Flaviviridae.The Scaffold had similarity to 9 viruses or phages.Among them,Enterobacteria phage P1 virion had a 770 nt annotated length.2.6 Metagenomic Analysis of the K25sample(culture-befor supension of bats rectum)After Solexa sequencing,a total of 1,622Mb raw sequence data were obtained from K24 sample,including 7,564,963 clean reads.Among them,322(0.0043%)reads had similarity to viruses(E value<0.00001 for BLASTn).Viral sequences can be divided into five categories:Reverse Transcribing Viruses,dsDNA Viruses,ssRNA Viruses,dsRNA viruses,including 7 Viraceae,they were mainly align to Herpesviridae,Picornaviridae.among them,there 58 reads similarity to HPV,36 reads to HIV,15 reads to ScMV.The Scaffold had similarity to 17 viruses or phages.Among them,Enterobacteria phage PI virion had a 771 nt annotated length.2.7 Preliminary analysis of important viruses2.7.1 PCR amplification and sequence analysis of Human papillomavirusA sequence was got by sequencing the products of PCR amplification,the sequence had a 100%identity amino acid identities of the strain(GeneID:392790),when compare to BLASTn of NCBI,but there was no open reading frame(ORF)of HIV found in the sequence.2.7.2 PCR amplification and sequence analysis of Human papillomavirusTwo pairs of primers was designed to amplify the PCR,one of them amplifed successfully,it’s product had 846bp length.ORF of Protein LI was found According to the RefSeq(GeneID:1489082)in the sequence.The sequence had a 100%identity amino acid identities of the strain(GeneID:399525615)and it were closed to the same strain by phylogenetic tree analysis based on Protein LI.2.7.3 Nucleic acid amplification and sequence analysis of Semliki Forest virusThe agarose electrophoresis showed that all were negative in 10 samples,when PCR performed to amplifier SFV gene According to the primers based on the SFV standard strain.HZ1 and HZ47 were positive when PCR performed to amplifier SFV gene According to the primers based on the SFV standard strain.A sequence was got by sequencing the products of PCR amplification,the sequence had a 100%identity amino acid identities of the strain(Gene ID:Z48163.2),when compare to BLASTn of NCBI,but there was no open reading frame(ORF)of HIV found in the sequence.2.7.4 Sequence analysis of sugarcane mosaic virusA scaffold,lenth 4113bp,was was found In a sample of K26 and has 99%identity with Zhejiang strain(Gene ID:18652414)isolated from China.This scaffold can code protein NIa-VPg and NIa-Pro etc.phylogenetic tree analysis was performed,after ClustalW analysising of multiple sequence alignment,which showed that the scaffold were closed to the Hebei strain(Gene ID:AJ310109).2.8 Detection of rabies virus in batA total of 88 bats of 5 species in 3 families were screened for the presence of rabies virus in Huizhou city of Guangdong province.Reverse transcription loop-mediated isothermal amplification(RT-LAMP)was employed as a simultaneous detection method for rabies virus and 20 of 88 bat samples(22.73%)were examinedpositive.Among these,32.26%(10/31)of Hipposideros larvatus bat samples were in active surveillance.In total 21 bats of Myotis rickettispecies captured from a cavern in Longmen of Huizhou city,only 8 samples(38.1%)were screened positive with RT-LAMP method while in total 6 bats of Scotophilus kuhli species captured from a Trachycarpus fortunei tree beside a hotel in Huizhou,only 2 samples(6.06%)were positive.545 bats of 11 species in 5 families were analyzed for the presence of rabies virus with nested-PCR method beween August,2011 and November,2013.In 2011 August,88 bat samples were captured and all 32 positive findings(30.68%)of rabie virus in the first collection were from Hipposideros larvatus and Hipposideros larvatusspecies.In October 2011,114 bat samples were collected and 32 of 104(30.77%)Miniopterus schreibersi bat samples were examined positive.In total 96 bat samples,11 of 64 Miniopterus schreibersi bat samples were positive and 8 of 27 Rousettus leschenaultia bat samples were positive in August 2013 and the positive rates were 17.19%and 29.63%,respectively.In 2013 November,three bat species were succeeded positive findings and many of findings were Miniopterus schreibersi species with the highest positive rate 25.77%(42/163).In total 88 bat samples collected in 2011 August,the positive rate were 22.73%using RT-LAMP method while the positive rate 30.68%using nested RT-PCR.There was no significant deference between two methods(P=0.092)and the detection of rabies virus among different species bats was similar.2.9 Sequence analysis of long nucleotide sequence of rabies virusFour samples were amplified with the primerof nucleotide sequence and then obtained nucleotide sequence.Thehomologiesof rabies virus’nucleotidesranging from 97%to 109%revealed that there was no significant difference of nucleotide sequence of rabies virus in bats.According to homology analysis using an online software in GenBank,the rabies virus’ nucleotides from these four samples were closely related to that nucleotides isolated from canine brain in Guangdong China in 2008 and the nucleotide sequence similarity was over 99%.The nucleotides sequence of N gene of rabies virus were constructed a phylogenetic tree and the analysis result suggested that the rabies virus detected from all four samples in our study were belonged to genotype I of rabies virus.Specifically,the HZ8 strain and YF15 strain were of similarity in our study.Meanwhile,the sequence analysis of HZ47 strain revealed a close genetic relationship to the strain reported by a French scholar,while HZ1 strain was similar to the strain isolated from canine brain in Henan China.3 Conclusions3.1 Viral metagenomics method,basing on high-throughput sequencing,to detect bat virome was established.It was applied to brain,serum and rectum were successfully.A large number of animal viruses and plant viruses were detected.3.2 Though application of viral metagenomics,we found BHK-21 cells are not suitable for double stranded RNA virus,revealed that Cell culture can influenced the species and abundance of the virus.3.3 Bats have the Possibilities of carring HIV,SFV,HPV,ScMV,for open reading frame of HPV and ScMV and some untranslated region sequences of HIV,SFV were found by preliminary analysis3.4 The rate of bat carrying rabies virus is 23.49%with nested RT-PCR method,higher than in previous research report.3.5 The similarity of rabies virus sequence was likelihood in this study is high,all the sequences were Genotype lof rabies virus genes which is a popular genotype in Asian.