Investigation On Bat Virome, Discovery And Characterization Of Novel Bat Viruses

Bats, comprising the second largest mammalian population in the world anddistributing globally with the exception of two polar areas, are important virusreservoir animals. Yunnan, an important border province of China adjacent to VietNam, Laos and Myanmar, is a main epicenter of emerging/re-emerging viral diseases.Studies have shown that such many viruses pathogenic to human and other animals,as Nipah virus, Japanese encephalitis virus, Chikungunya virus, circovirus, and rabiesvirus, are present in bats in these areas. To control the outbreaks of emerginginfectious diseases (EID) and prevent the cross-species/border transmission of virusesfrom wildlife, particularly bats, to humans, surveillance on the virus carrying situationin natural hosts and vector animals is important and should be widely conducted.Availability of next generation sequencing-based viral metagenomics (ViralMeta) inrecent years has provided a powerful tool for large-scale detection of known andunknown viruses existing in host animals.In this study,1,219health-looking bats were collected from six counties inYunnan province and two counties in Myanmar during2008and2011. These batscovered11species from5genera within families Vespertilionidae, Rhinolophidae andMegadermatidae. Viral metagenomic analyses showed that a total of5,864,636readswere generated and further overlapped into3,605,620contigs. After bioinformaticanalysis,2%(59,661/3,605,620) contigs were related to viruses from27families(including Parvovirinae and Densovirinae). These viral-like contigs can be furtherclassified into mammalian virus (16families), insect virus (5families), plant virus (3families) and phages (3families). We mainly focus on mammalian viruses which areAdenoviridae (Mastadenovirus), Herpesviridae (Cytomegalovirus, Mardivirus,Roseolovirus), Alloherpesviridae (Ictalurivirus), Papillomaviridae(Alphapapillomavirus), Polyomaviridae (Polyomavirus), Picobirnaviridae(Picobirnavirus), Hepadnaviridae (Orthohepadnavirus), Circoviridae (Circovirus),Poxviridae (Orthopoxviridae), Retroviridae (Deltaretrovirus, Gammaretrovirus),Parvoviridae (Dependovirus, Bocavirus), Astroviridae (Mamastrovirus), Reoviridae (Rotavirus A), Coronaviridae (Alphacoronavirus, Betacoronavirus), Picornaviridae(Kobuvirus, Enterovirus), and Flaviviridae (Hepacivirus). Data comparison showedthat there were dramatic differences in virus species, the identities of viral-likesequences to known viruses, and the abundance of viral-like sequence betweendifferent locations and bat species. A general viromes of these bats from involvedlocations were presented to us.According to the results of ViralMeta, PCR/RT-PCR methods amplifyingadenovirus, orthohepadnavirus, astrovirus, coronavirus, rotavirus, and iflavirus wereestablished and used to screen these viruses in bat samples. Detection results were inline with ViralMeta and showed that1.2%(1/84）Hipposideros armiger from WutaoMyanmar positive for adenovirus,2%(7/320) Miniopterus fuliginosus from Sedonand5%(15/320) from Wutao as well as20%(4/20) Hipposideros pomona from PuerYunnan positive for orthohepadnavirus,2%(2/84) Rhinolophus ferrumequinum fromWutao positive for astrovirus,11%(13/120),7%(7/100) and6%(2/34) Myotis batsfrom Yunnan, Bingchuan, Jinghong, respectively, positive for alphacoronavirus,14%(2/14) Rhinolophus affinis from Baoshan positive for betacoronavirus,6%(1/16)Rhinolophus hipposideros from Dehong positive for rotavirus, while iflavirus widelypresent in these bats. Amplicon sequences analyses showed that all these virusesshared less than85%nucleotide identities with currently known viruses, indicatingthat these viruses were novel bat viruses.Among these novel viruses, we further studied the selected orthohepadnavirus,coronavirus and rotavirus in full genomic, antigenic, and pathogenic ways. We foundnovel hepatitis viruses in Miniopterus bats from Myanmar as well as Hipposiderosbats from Yunnan, which was the first report that bats carry orthohepadnavirus. Fullgenomic analysis showed that these viruses were less than60%nucleotide (nt)identical to currently known orthohepadnaviruses, suggesting they would be a newspecies within the genus Orthohepadnavirus. Through antigenic prediction of Cprotein, we found that BtHV had the potential to cross-react with HBV. To confirmthat bats were infected with BtHV, we observed Australia antigen-like particlesexisted in bat livers, which further mean that bats did carry BtHV. Diversealphacoronaviruses were discovered in Bingchuan, Xiangyun and Jinghong with viralmetagenomic and RT-PCR methods, these alphacoronaviruses can grouped into5clades and all of them shared limited identity with approved species. A new betacoronavirus was found in Rhinolophus affinis from Dehong, this virus (LYRa11)was much more identical to SARS CoV, and especially in the Spike gene whichdetermine viral host tropism by attaching to human ACE II. Antigenic analysisshowed that it can bind to SARS convalescence serum as efficiently as SARS CoVdoes, but other bat SARS-like CoVs do not. Recombinational analysis along withBayesian analysis showed that SARS CoV was recombinated from three bat viruses,Rp3, LYRa11and Rf1. We provide further evidence that bats are natural host ofSARS CoV. We isolated a group A rotavirus from a lesser horseshoe bats in Yunnanprovince, China, named RVA/Bat-tc/MSLH14/2012/G3P[3]. Full genomic sequencinganalyses showed that MSLH14possessed the following genotype constellation:G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is akin to human and animalrotaviruses believed to be of feline/canine origin. Phylogenetic analysis indicated thatVP7was most closely related to bovine RVA strains from India, whereas VP4wasmost closely related to an unusual human RVA strain CMH222with animalcharacteristics isolated in Thailand. The remaining gene segments were only distantlyrelated to a range of animal RVA strains, most of which are believed to be related tofeline/canine RVAs. Experimental infection showed that bat RVA strain MSLH14washighly pathogenic to suckling mice, causing100%mortality when inoculated orallywith a titer as low as5×102TCID50virus. As this virus is not closely related to anyknown RVA strain it is tempting to speculate that this is a true bat RVA strain ratherthan an interspecies transmitted virus. However, further screening of bat populations,preferentially juvenile animals, will be crucial to determine whether or not this virusis widely distributed into the bat population.In conclusion, we established Solexa sequencing-based viral metagenomicmethod with specific PCR confirmation and successfully applied it to virome in batsfrom Yunnan and Myanmar. Results elicited many significant findings on novel batviruses, enhanced our knowledge of bat viruses as well as described virus diversityand composition in these areas. Our results also show that the composition of batviromes differs depending on geographical location and bat species. Current batviromes have therefore uncovered only the tip of the iceberg as regards virus florapresent in these mammals, and future studies involving a wider sampling of batspecies in different locations will undoubtedly increase our understanding of theglobal diversity of bat viruses. Furthermore, here we give a paradigm to investigateviral epidemiology in many aspects: viral metagenomics plus specific PCR confirmation. We firstly obtain the virus spectrum by viral metagenomics, and thenidentify the selected virus according to the results of viral metagenomics toinvestigate their epidemiology in bat population. By this way, we can shoot the arrowat the target with saving labor, saving time. The method will play a key role inmicrobe-alarm, diagnosis on emerging and re-emerging infectious diseases as well asinvestigation on viral ecology in kinds of environment.