Epidemiological Investigation Of Bacteria And Viruses In Commensal Rat-shaped Animals Based On High-throughput Sequencing

1.Background and PurposeRat-shaped animals(rodent-like animals)refer to a large group of vector organisms,mainly included murine rodent in Rodentia and Soricidae in Eulipotyphla.Commensal rat-shaped animals,which mainly inhabit in residential areas in towns and villages with close contact with humans,represent asreservoirs of abundant human infectious diseases.It is important for the prevention and control of rodent-borne disease tounderstand the background information of pathogens carried by rat-shaped animals.At present,the investigation on rodent-borne disease is generallyfocused on some specific pathogens,which lack of overall understanding of the types and prevalence of pathogens carried by rat-shaped animals.In recent years,the emergence of high-throughput sequencing provideseffective means to elucidate the background compositionof bacterial and viral communities in specific human and animal environments.In this study,16S rDNA high-throughput sequencing and viral metagenomics methods were used to investigate the community composition,characteristics,and differences of bacteria and viruses carried by urban commensal rat-shaped animal in parts of China.At the same time,molecular epidemiological investigations were conducted on several important viruses with high abundance in rat-shaped animal samples based on the results of viral metagenomics.2.Methods2.1Animal capture and specimen collectionBetween August 2014 and May 2017,rat-shaped animals were captured using cage traps in six regions of four provinces in China:Guangzhou city,Shenzhen city and Maoming cityin Guangdong province;Xiamen city in Fujian province;Yiyang city in Hunan province;and Malipo country in Yunnan province.Among them,during the period from May 2015 to April 2016,a one-year sample collection was conducted in the communities of Southern Medical University of Baiyun district in Guangzhou City(including the Nanfang hospital,residential areas,and teaching areas).The samples were collected once a month at intervals.Serum,throat swabs,and rectal end stool samples were collected and stored at-80℃.2.2Library preparation,sequencing,and bioinformatics analysis of 16S rDNA high-throughput sequencingBetween May 2015 to April 2016,an one-year monitoring sample of rat-shaped animals in Baiyun district of Guangzhou City was selected as the study objects.According to the sampling time,a random sample of Rattus norvegicus and Suncus murinus feces was selected every month,resulting in a total of 24 samples.Bacterial V4 regions were amplified using bacterial 16S universal primers.After library construction performed,Illumina HiSeq was used for high-throughput sequencing and then further species annotations taxonomic were conducted.2.3Library preparation,sequencing,and bioinformatics analysis of viral metagenomicsBetween May 2015 to April 2016,an one-year monitoring sample ofrat-shaped animals in Baiyun district of Guangzhou City was selected as the study objects.All samples were pooled according to different seasons,animal species,and tissue types.According to the climate of Guangzhou,the seasons were classified into two group,summer/autumnand winter/spring.There are three species of animals:Rattus norvegicus,Rattus tanezumi,and Suncus murinus.Tissue types are classified into throat swabs,feces and serum.Finally,a total of 18 viral metagenomics sequencing samples are included.High-throughput sequencing was performed using Illumina HiSeq.After quality control and host sequence filtering of the raw data,BLAST comparisons were performed within NCBI’s MicroNR and MicroNT databases to obtain results of sequence annotation taxonomic.2.4Molecular epidemiological investigations of several important viruses in rat-shaped animalAccording to the preliminaryresults of the viral metagenomics,several viruses with high abundance were selected for further molecular epidemiological investigation.Viral nucleic acids of throat swab,fecal samples,and serum were extracted using Takara’s MiniBEST Viral RNA/DNA Extraction Kit.According to the reference sequence,Primer Primer 5 software was used to design specific primers and genomic amplification primers for different viruses.The products were all subjected to 1%agarose gel electrophoresis and positive samples were sent to Huada Gene(Guangzhou)for sequencing.Sequence splicing was performed using SeqMan software to obtain near full-length viral genome sequences.Multiple alignments were performed using ClustalW in MEGA6.Based on the alignment results,phylogenetic trees were constructed using the neighbor-joining(NJ)and maximum likelihood(ML)methods.Bootstrap values(1000 times)were used to examine the robustness of the phylogenetic tree.3.Results3.1The result of rat-shaped animals capturedA total of 1,072 rat-shaped animals were captured in this study,including 643Rattus norvegicus(60.0%),68Rattus tanezumi(6.3%),48Rattus losea(4.5%)and 31 3Suncus murinus(29.2%).3.2The results of 16S rDNA high-throughput sequencing3.2.1Overview of sequencing and species annotation resultsAccording to sequencing,24 fecal samples obtained averaged about 90,000 Raw Reads,with an average of 576 operational taxonomic units(OTUs).The 24 samplesannotated203 families,420 genera,and 197 species of bacteria.The top five most abundant abundances of the 420 bacterial genera included Clostridium,Lactobacillus,Enterococcus,Escherichia,and Prevotella.In addition,there are a large number of other bacterias,such asStaphylococcus,Streptococcus,Pseudomonas,Proteus,Vibrio,Neisseria,Bacillus and Corynebacterium.3.2.2Comparison of bacterial community structure between differentspecies of animals(Rattus norvegicus and Suncus murinus),and different seasonal samplesThe bacterial community structure of Rattus norvegicus and Suncus murinus had an independent feature,and there were significant differences between dominant and distinctgenera.Rattus norvegicus annotated89orders,157 families,167 genera and 110 species of bacteria.Correspondingly,Suncus murinus annotated 110orders,196 families,189 genera and 110 species of bacteria.At genus level,Faecalibacterium,Anaerovibrio,Oribacterium,Subdoligranulum,Alloprevotella,Anaerobiospirillum,Megamonas,Mitsuokella and Lawsoniaare abundant in the fecal samples of Rattus norvegicus.Conversely,Romboutsia,Turicibacter,Rickettsiella,Listeria,Stenotrophomonas,Peptoclostridium,Mycoplasma,Pseudomonas,Cetobacterium and Cetobacteriu are abundant in the fecal samplesof Suncus murinus.At the same time,the numbers of bacteria and dominant bacteria carried by the Rattus norvegicus and Suncus murinus intestine in different season were also different.The four froups of Rattus norvegicus(R1-4)carried 506 same OTUs,and carried 74,84,72 and 52distinct OTUs.Correspondingly,four froups of Suncus murinus(S1-4)carried 342 same OTUs,and carried 111.29,165 and 315distinct OTUs.3.3The results of viral metagenomics3.3.1Overview of sequencing and species annotation resultsA total of 92.3G of sequencing data was obtained from 18 samples,which annotated213 viral generaincluded animal viruses,insect viruses,plant viruses,and algae viruses.The top 5 viral genera with the highest abundance were Bocaparvovirus,Dependoparvovirus,Unclassified genus in Parvoviridae,Unclassified genus and Ambidensovirus in Parvovirida.There are many high abundant mammalian virus including Cytomegalovirus,Alphapapillomavirus,Hepacivirus,Pestivirus,Sapovirus,Orthopoxvirus,Circovirus,Arterivirus,Orthohepadnavirus,Simplexvirus,Rotavirus and Lentivirus.3.3.2Comparison of community characteristics of viruses carried by different species of rat-shaped animals(Rattus norvegicus,Rattus tanezumi and Suncus murinus),different climates(summer/autumn and winter/spring),and different tissues(throat swab,fecal samples,and serum)The viral communities of the three different species of rat-shaped animalsweresome differences.Rattus norvegicus,Rattus tanezumi and Suncus murinus carried 97 same viralgenera.However,they carried 22,13 and 33 distinctviral genera,respectively.There were major differences in the virus communities of samples collected in different seasons of Rattus norvegicusand Rattus tanezumi.The samples of summer/autumn carriedhigh abundances of Dependoparvovirusand Unclassified virus.The samples in the winter/spring carried a large number of Bocaparvovirus.However,virus communities ofSuncus murinus were similar.The absolute abundance of viruses in fecal samples was much higher than that of throat swabs and serums.The fecal samples carried abundantly distinct viral genera.The mainly distinct mammalian viruses in fecal samples includedSapovirus,Circovirus,Kobuvirus and Hunnivirus.3.4Molecular epidemiology of four viruses3.4.1 Torque teno virusThe totalpositive rate of nucleic acidsof rodent torque teno virus(RoTTV)in throat swabs,feces,and serum of rat-shaped animals was 22.8%(113/496),29.1%(142/488),and 23.8%(54/227),respectively.Samples collected in Guangdong,Fujian,Hunan,and Yunnan Province were all detected positive for RoTTV in throat swabs,feces,and serum samples.At the same time,one throat swab and one serum sample of Suncus murinus were positive for RoTTV.Twelve near full-length RoTTV genomes isolated from throat swabs samples of Rattus norvegicus were acquired,which were between 2245bp and 2399bp in length.Phylogenetic tree showed that the 12 RoTTV sequences obtained in this study formed a distinct clade,which was distinguished to the published RoTTV1,RoTTV2,and other animal-derived TTV.Further analyses of the nucleic acid and amino acid similarities suggested that the Torque teno virus obtained in this study represent a new viral genotype(RoTTV3).3.4.2 Porcine bocavirusThe positive rate of nucleic acidsof porcine bocavirus(PBoV)nucleic acid in stool was 60.5%(309/511),which was higher than that of serum(22.9%,52/227)and throat swab(7.5%,39/519).The overall positive rate of Rattus norvegicus and Rattus tanezumi,was higher than that of Rattus losea andSuncus murinus.At the same time,PBoV was detected in samples collected in Guangdong,Fujian,Hunan,and Yunnan Province,which suggested that PBoV has a wide distribution in rat-shaped animals in ChinaFive near full-length PBoV genome sequences were obtained from throat swabs samples of Rattus norvegicus,ranging in length from 4715bp to 4798bp.Phylogenetic tree results showed that the five PBoV sequences obtained in this study formed a distinct clade,which was distinguished to PBoV G1-3 and other animal-derived bocavirus virus sequences,suggesting that the PBoV virus obtained in this study is a new viral gene group(PBoV G4).3.4.3Rat bocavirusThe total positive rate of rat bocavirus(RBoV)in throat swabs and feces was 23.7%(123/519)and 60.0%(286/511),but it was not detected in serum samples.RBoV was detected in throat swabs(29.2%,119/408 and 10.0%,4/40)and feces(65.7%,266/405 and 51.3%,20/39)in both Rattus norvegicus and Rattus tanezumi.However,the virus was not detected in Rattus losea and Suncus murinus.RBoV was detected in samples collected in four provinces,suggesting that RBoV has a wide distribution in rat-shaped animals in China.Eleven and one near full-length RBoV genome sequences were obtained from fecal samples of Rattus norvegicus and Rattus tanezumi,respectively,which ranging in length from 5110 bp to 5148 bp.Phylogenetic tree showed that the RBoV sequences obtained in this study were similar to the reported RBoV sequence and all clustered into one clade,which was distinguished from PBoV and other animal-derived bocaviruses.3.4.4Rat adeno-associated virusRat adeno-associated virus(Rat AAV)was positive in 54.7%(267/488),14.3%(70/488)and 18.4%(41/223)of throat swabs,feces,and serum,respectively.The results showed that Rattus norvegicus,Rattus tanezumi,Rattus losea and Suncus murinus were able to carry Rat AAV.But the overall positive rate of Rattus losea is relatively low.The samples collection from four provinces,including Guangdong,Fujian,Hunan,and Yunnan Province,were all detected positive for Rat AAV,suggesting that Rat AAV is widely distributed in rat-shaped animals in China.The phylogenetic tree based on the Cap gene showed the AAV sequence obtained in this study was similar to reported Rat AAV,which was distinguished from the primate AAV type 1-8 and other animal sources AVV.4.Conclusions4.1 This study preliminarilyclarified the characteristics of intestinal flora ofRattus norvegicus and Suncus murinus,as well as the divergence in these two species and different seasons.A large number of pathogenic or conditional pathogenic bacteria were carried byRattus norvegicus and shrews.The abundance of pathogenic bacteria carried by Suncus murinus was higher than that of Rattus norvegicus.A large number of distinct bacteria were carried by by the Rattus norvegicus and Suncus murinus intestine in different season4.2 This study preliminarily established a baseline database of viralcommunitisin pharynx,intestineand serum of commensal rats and shrews.The results showed that there are some differences in the viral community among urban rat-shaped animals in different season(summer/autumn and winter/spring),different tissues(throat swabs,feces,and serum)and different species(Rattus norvegicus,Rattus tanezumi andSuncus murinus)4.3Themolecular epidemiological analysis of four viruses with high abundanceshowed(1)A novel RoTTV(RoTTV 3)was discovered in both commensal rats and shrews.In addition,RoTTV 3 was had a high carrier rate in common murines(Rattus norvegicus,Rattus tanezumi and Rattus losea)in China.(2)This is the first reported that thecommensal rats and shrews could serve as the natural reservoirs of PBov,which represented a novel genotype(PBoV G4).The results showed that PBoV4 was widely distributed in common murine species(Rattus norvegicus,Rattus tanezumi and Rattus losea),suggesting that murine rodents play an important role in the storage,spread and evolution of PBoV.(3)RBoV is widely distributed in murine rodents with a high infection rate in China,and firstly reported the infection in Rattus tanezumi.(4)Molecular epidemiological investigation was firstly conducted to detect the prevalence of AAV in rat-shaped animals in China.The results showed that Rat AAV was widely distributed in murine rodents with high infection rate.