we purchased 853 freshly killed insectivorous bats in sedon and wutao counties in southeastern kachin state , myanmar ; the counties are adjacent to yunnan province , people s republic of china . the bats covered 6 species : miniopterus fuliginosus ( n = 640 ) , hipposideros armiger ( n = 8) , rhinolophus ferrumequinum ( n = 176 ) , myotis chinensis ( n = 11 ) , megaderma lyra ( n = 6 ) , and hipposideros fulvus ( n = 12 ) . the sampling of bats for this study was approved by the administrative committee on animal welfare of the institute of military veterinary , academy of military medical sciences , china . we used pcr to further study the prevalence of orthohepadnavirus in the 6 bat species ; the condition of the samples made serologic assay and pathology impracticable . viral dna was extracted from liver tissue of each of the 853 bats by using the qiaamp dna mini kit ( qiagen , hilden , germany ) . to detect virus in the samples , we conducted pcr by using the takara pcr kit ( takara , dalian , china ) with a pair of degenerate pan - orthohepadnavirus primers ( sequences available upon request ) . the pcr reaction was as follows : 45 cycles of denaturation at 94c for 30 s , annealing at 54c for 30 s , extension at 72c for 40 s , and a final extension at 72c for 7 min . positive results were obtained for 22 long - fingered bats ( miniopterus fuliginosus ) . of these bats , 2.19% ( 7/320 ) were from sedon county and 4.69% ( 15/320 ) from wutao county ; the viruses they harbored shared > 98% nt identity . no other species had positive amplification results , indicating that m. fuliginosus was the most likely species to harbor orthohepadnaviruses . of the 22 positive samples , 3 were randomly selected for full genome amplification : m086 from sedon county and 776 and m005 from wutao county . pcr was conducted by using the pcr protocol defined above with high - fidelity pfu dna polymerase ( promega , madison , wi , usa ) and 4 pairs of specific primers ( sequences available upon request ) . four overlapping amplicons were obtained , sequenced in both directions , and assembled into the full genomic sequence by using seqman , version 7.1.0 ( dnastar , madison , wi , usa ) . jx941466 jx941468 ) were 3,230 nt in length , which is close to the size of primate hepatitis viruses ( 3,200 nt ) but smaller than rodent hepatitis viruses ( 3,300 nt ) . we analyzed the genome structure by using vector nti advance 10 ( invitrogen , carlsbad , ca ) . the results showed that the bat hepatitis viruses ( bthvs ) contained the same circular and compact genomic structure as other orthohepadnaviruses , comprising 4 open reading frames encoding the multifunctional pol , pres1/pres2/s , prec / c , and x proteins in the same direction ( figure 1 , panel a ) . predicted schematic representation of the bat hepatitis virus ( bthv ) genome and its phylogenetic relationship with other hepadnaviruses . boxes and arrows represent the open reading frames encoding the main proteins : pol gene ( 2,3051,636 ) , pres1/s2 and s gene ( 2,864833 ) , prec / c gene ( 1,8152,468 ) and x gene ( 1,3781,812 ) . two 12-nt direct repeat sequences ( dr1 from 1,825 to 1,836 and dr2 from 1,594 to 1,605 ) , the encapsidation signal  ( 1,8481,903 ) , and ymdd domain ( 734745 ) are also depicted in the map . b ) phylogenetic analysis of bthvs and other hepadnaviruses based on amino acid sequences of pol genes . representatives of hepadnavirus species belonging to orthohepadnavirus and avihepadnavirus genera were used ; their genbank accession nos . genomic sequence comparison and phylogenetic analysis based on amino acids of the pol gene ( 2,562 bp ) were constructed with clustalw version 2.0 ( www.clustal.org/ ) and mega5 ( 5 ) . phylogenetic tree analysis showed that previously described orthohepadnaviruses formed 2 clusters , primate hepatitis viruses and rodent hepatitis viruses , whereas the 3 newly identified bthvs formed an independent cluster within the orthohepadnavirus genus ( figure 1 , panel b ) . sequence comparison showed that the full genomes of the bthvs were 63.1%65.3% and 33.9%34.8% identical to members of the orthohepadnavirus and avihepadnavirus genera , respectively . similar low identities were also observed separately in the 4 genes of the bthvs ( table ) . these results support the classification of the bthvs within the orthohepadnavirus genus , being distantly related to current species and likely to form a new species designated as bthv . * nt , nucleotide length ; % i d , percentage identity of nt and amino acid sequence between bthv and other viruses ; aa , amino acid length ; bthv , bat hepatitis virus ;  , not applicable ; hbv , hepatitis b virus ; wmhbv , woolly monkey hbv ; whv , woodchuck hepatitis virus ; ashv , arctic squirrel hepatitis virus ; dhbv , duck hbv ; na , not available . hepadnaviruses have not been grown in any available in vitro cell system ; thus , we did not attempt to isolate bthv in cell culture . to detect the presence of virus particles , we used pooled liver tissues from the 3 bats that were randomly selected for full genome amplification . we homogenized the pooled tissues in sm buffer ( 50 mm tris , 10 mm mgso4 , 0.1 m nacl ; ph7.5 ) , followed by clarification by low - speed centrifugation to remove cell debris . we then passed the pooled sample through a 0.22-m syringe filter ( millipore , carrigtwohill , ireland ) . polyethylene glycol 6000 was added , and the resulting precipitate was sedimented at 12,000  g in a desktop centrifuge ( eppendorf , hamburg , germany ) for 40 min at 4c . the pellet was resuspended and examined after negative staining in a jem-1200 exii transmission electron microscope ( jeol , tokyo , japan ) . the particles were morphologically similar to the australia antigens of hbv , the most abundant viral component found in hbv - infected humans and animals and also known as surface protein or s antigen ( 6,7 ) . pcr amplification of dna extracted from the virus pellet revealed the full genome of the bthv , with the expected size of 3200 bp ( image not shown ) . our observations provide strong evidence for the circulation of orthohepadnaviruses in at least 1 species of bats , m. fuliginosus , in myanmar . these bats have a wide distribution ( 8) , and increasing numbers of viruses , including coronaviruses and betaherpesviruses , are being isolated from them ( 9,10 ) . of the 6 bat species we sampled , only m. fuliginosus was positive for bthv . the prevalence of bthv - positive bats in the 2 counties from which we obtained bats , was 2.2% and 4.7% , respectively , indicating that this species is likely a natural reservoir host of bthv . the lack of detection of bthv in bats from the other 5 species may be due to the limited numbers of bats sampled ( although no evidence of hepadnavirus was found in any of the 176 r. ferrumequinum bats ) or to a narrow host range of the virus . further study is required to determine the tropism and prevalence of bthvs in other bat species .
during an analysis of the virome of bats from myanmar , a large number of reads were annotated to orthohepadnaviruses . 
 we present the full genome sequence and a morphological analysis of an orthohepadnavirus circulating in bats . 
 this virus is substantially different from currently known members of the genus orthohepadnavirus and represents a new species .