in a survey for rodentborne hemorrhagic fever viruses , 612 small rodents representing 17 different genera ( most abundant were mastomys [ n = 325 ] , praomys [ n = 95 ] , and nannomys [ n = 83 ] ) were trapped in guinea from 2002 to 2004 and screened for hantavirus rna by reverse transcription  polymerase chain reaction ( rt - pcr ) .
we used a molecular genetic approach to screen the rodent population because hantavirus rna ( as shown for snv ) can be amplified from the blood of persistently infected mice by rt - pcr over a long period ( 6 ) . for this purpose
, we developed a nested rt - pcr assay to detect currently known and possible novel members of the genus hantavirus .
the assay was based on degenerated primers ( han - l - f1 : 5-atgtaygtbagtgcwgatgc-3  and han - l - r1 : 5-aaccadtcwgtyccrtcatc-3  for primary pcr , han - l - f2 : 5-tgcwgatgchaciaartggtc-3  and han - l - r2 : 5-gcrtcrtcwgartgrtgdgcaa-3  for nested pcr ) designed from an alignment of all available nucleotide sequences of the highly conserved l segment . for the rt - pcr
, total rna was extracted from wild - trapped rodent blood ( preserved in liquid nitrogen ) with the blood rna kit ( peqlab , erlangen , germany ) and reverse transcribed with random hexamers as primers . a sample ( designated sa14 ) obtained from 1 of 4 investigated african wood mice ( h. simus ) generated an l segment  derived pcr product of expected size .
this rodent was trapped in january 2004 in a forest habitat near the village of sangassou , near macenta , guinea ( 83649n , 92827w ) .
= 74 , autosomal fundamental no . = 70 ) and the complete cytochrome b gene was sequenced and compared with the genes of other hylomyscus species recognized in the most recent revision of the genus ( 7 ) ( genbank accession nos .
the 412-nucleotide ( nt ) sequence of the first pcr product was determined by amplification , cloning , and sequencing of overlapping fragments generated by 2 seminested pcrs ( genbank accession no .
additional s and m segment  specific nested pcr assays were developed to further characterize the novel virus .
pcr fragments of 837 nt and 694 nt could be analyzed ( genbank accession nos .
the table shows nucleotide sequence identity comparisons between sa14 and other members of the genus hantavirus .
murinae - associated hantaviruses ( htnv , dobv , seov ) showed the highest similarity to the sa14 sequence in all 3 genomic segments ( 71.3%77.1% for s , 72.9%77.9% for m , and 72.3%75.9% for l ) .
this similarity is consistent with the evolutionary relationship of their putative hosts . on the amino acid level
, corresponding sequences of deduced viral proteins showed highest similarity with those of other murinae - associated hantaviruses ( 81.7%88.5% for s , 82.2%89.6% for m , and 85.4%87.5% for l ) .
the amino acid sequence divergence between sa14 and most related hantaviruses corresponds to that typically found between different virus species , e.g. , snv and andv . *
s , small ; m , medium ; l , large ; htnv , hantaan virus ; seov , seoul virus ; dobv , dobrava virus ; puuv , puumala virus ; tulv , tula virus ; snv , sin nombre virus ; andv , andes virus . 
837 nucleotides ( nt ) of the s segment ( positions 3941230 ) , 694 nt of the m segment ( positions 22812974 ) , and 412 nt of the l segment ( positions 29563367 ) and the deduced amino acid ( aa ) sequences ( 279 aa , position 120398 of the nucleocapsid protein ; 231 aa , positions 748978 of the glycoprotein precursor ; 137 aa , positions 9741110 of the viral rna - dependent rna polymerase ) have been compared .
fragment positions were defined according to complete sequences of htnv strain 76 - 118 ( genbank accession nos .
the s- , m- , and l - segment  derived nucleotide sequences of sa14 were subjected to maximum likelihood ( ml ) and neighbor - joining ( nj ) phylogenetic analyses with available nucleotide sequences of other murinae - associated hantaviruses .
puuv , tulv , snv , and andv sequences were used as outgroups . in the s segment
ml phylogenetic tree ( figure ) , sa14 clustered with other murinae - associated viruses . as expected , 3 clades were formed by members of the 3 established hantavirus species ( htnv , dobv , and seov ) . within this well - supported cluster ,
the m segment analysis showed an identical placement of sa14 with strong statistical support ( puzzle   and bootstrap values above the threshold value of 70% , data not shown ) . in l - segment phylogeny , the resolution of the tree was decreased .
the sa14 l sequence did not join with statistical support any of the 3 groups but formed a fourth clade within the cluster of murinae - associated hantaviruses .
( puzzle and bootstrap values above the threshold for the placement of sa14 within the murinae - associated viruses but < 50% in both analyses for its clustering with any of these viruses , data not shown ) .
maximum likelihood phylogenetic tree of hantaviruses showing the placement of sa14 ( sangassou virus [ sangv ] , indicated by gray shading ) .
partial s segment genome sequences ( 837 nucleotides , positions 3941230 ) were used to calculate the tree with tree - puzzle program ( 8) . the tamura - nei evolutionary model was used ; the values above the branches represent puzzle support values .
the values below the branches represent bootstrap values of the corresponding neighbor - joining tree computed with paup * program ( 9 ) using 10,000 bootstrap replicates .
the scale bar indicates an evolutionary distance of 0.1 nucleotide substitutions per position in the sequence .
htnv , hantaan virus ; dobv , dobrava virus ; seov , seoul virus ; puuv , puumala virus ; tulv , tula virus ; andv , andes virus ; snv , sin nombre virus .
extended fragments of novel hantavirus s , m , and l genome segments were recovered from an arboreal african rodent .
they clearly represent genetic material of a novel hantavirus species because their amino acid sequence is significantly ( 15% ) divergent from those of other hantaviruses , they form a distinct clade in phylogenetic trees , and they were detected in a rodent species previously not recognized as a natural host of hantaviruses .
we propose to name the new species sangassou virus ( sangv ) after the locality where it was detected .
although hantaviruses are emerging viruses circulating in asia , europe , and the americas , our study represents the first genetic evidence for hantaviruses in africa .
however , apodemus agrarius , the natural host of htnv , is not found in africa .
based on the putative cross - reactivity of antigens from htnv , sangv , and other murinae - associated viruses , human infections , at least in tropical forest parts of africa where hylomyscus species are prevalent , could be caused by sangv or other murine - associated hantaviruses .
to our knowledge , 1 case of hfrs has been reported in central africa ( 14 ) .
although hfrs is not a known disease in west or central africa , one can not ignore the potential pathogenicity of sangv or other african hantaviruses .
hfrs may be confused with other severe diseases ( leptospirosis , rickettsiosis , other viral hemorrhagic fevers , plague , severe pneumonia , sepsis ) or may be unrecognized because of poor health care .
one should remember that hps and sigmodontinae - associated hantaviruses were not recognized until 1993 , even in such a highly developed country as the united states .
further studies are needed to verify the presence and distribution of hantaviruses in africa and their potential impact on human health .
these studies should focus on areas with forest activities , such as logging , which may bring humans into contact with viral reservoirs ( 15 ) .
our data justify inclusion of hantavirus infection in the differential diagnosis of patients from africa with unexplained febrile nephropathies or noncardiogenic pulmonary edema .
 hantaviruses are rodentborne , emerging viruses that cause life - threatening human diseases in eurasia and the americas . 
 we detected hantavirus genome sequences in an african wood mouse ( hylomyscus simus ) captured in sangassou , guinea . 
 sequence and phylogenetic analyses of the genetic material demonstrate a novel hantavirus species , which we propose to name " sangassou virus . " 
The Study
Conclusions