on october 27 , 2005 , a us soldier ( patient 1 ) assigned to camp hovey , dongducheon , exhibited signs and symptoms of hfrs ( table 1 ) . the patient was transferred to the brian allgood army community hospital ( baach ) , yongsan army garrison , seoul , south korea , and on october 28 , 2005 , was confirmed to be seropositive for hantavirus infection by elisa . a medical advisor initially suspected that the patient may have acquired the infection while sweeping out a dusty storage area at camp hovey , where he resided , 3 days before the onset of symptoms . a survey of the suspected storage room did not uncover any signs of rodent activity . these data along with the known incubation period of hantaviruses ( 4>50 days ) prompted a further search for the actual site of transmission . * tmc , troop medical clinic ; hfrs , hemorrhagic fever renal syndrome ; fp , firing point ; tbta , twin bridges training area ; s , south ; n , north . patients 1 and 3 were hospitalized for 8 days , patient 4 for 5 days , and patient 2 for 29 days . patient 2 was sent to samsung hospital for dialysis during part of his hospitalization at brian allgood army community hospital ( baach ) . confirmation of hantavirus type by reverse transcription  pcr , korea university . a blood sample from patient 1 , tested by both the indirect immunofluorescent antibody ( ifa ) technique and reverse transcription  pcr ( rt - pcr ) , confirmed that the patient was infected with htnv . an epidemiologic survey was conducted , and results showed that the patient had trained at 4 us- and south korea the patient had first trained at local training area ( lta ) 320 , then at lta 36/37 , and finally at firing point ( fp)-60 ( figure 1 ) . the training consisted of setting up firing positions , establishing a cantonment site , and performing other training activities for 5 days before moving on to fp-60 , where troops conducted firing exercises from september 2529 , 2005 , before returning to camp hovey . location of training sites where hemorrhagic fever with renal syndrome ( hfrs ) patients 14 conducted training exercises 50 days before the onset of illness . dmz , demilitarized zone ; lta , local training area ; solid circles , military training sites of patient 1 ; solid squares , military training sites of patients 2 , 3 , 4 ; star , base camp . on november 89 , 2005 , two soldiers ( patients 2 and 3 ) from camp casey , dongducheon , had signs and symptoms of hfrs and so were sent to the baach where they received a diagnosis of hfrs ( table 1 ) . patient 4 from the same unit sought treatment at the camp casey troop medical clinic on november 13 with low - grade fever , decreased appetite , abdominal pain , chills , low back pain , nausea , and vomiting , and was provided fluids and assigned to quarters ( home bed rest ) . on november 14 , laboratory results indicated a nonspecific proteinuria characteristic of hfrs infections , and the patient was transported to the baach , where hfrs was diagnosed . blood samples from all 3 patients were positive for htnv by ifa and rt - pcr . all 3 patients had trained at twin bridges training area ( tbta ) with potential incubation periods that ranged from 16 to 35 days , and 2 ( patients 2 and 4 ) had trained together at rodriguez and watkins ranges 3 days before conducting a tactical move to tbta ( table 1 ; figure 1 ) . patients 2 and 4 conducted training at tbta - north ( n ) and tbta - south ( s ) , and patient 3 only conducted training at tbta - n . preseasonal ( september ) a. agrarius mice trapping rates were relatively low at both fp-60 ( 11.4% ) and rodriguez range ( 7.6% ) , and although postseasonal trapping rates were high at fp-60 ( 42.3% ) , they remained relatively low at rodriguez range ( 12.1% ) ( table 2 ) . during the fall , hantavirus seropositive rates were high at fp-60 ( 20.0% ) and rodriguez range ( 31.3% ) . during the winter , seropositive rates increased at fp-60 ( 25.8% ) , but decreased at rodriguez range ( 15.7% ) . during the winter , seropositive rates at tbta - n ( 26.3% ) and tbta - s ( 37.8% ) were high , but were relatively low at other training sites ( lta320/36/37 ) surveyed . * fp , firing point ; ltas , local training area ; nd , not determined ; tbta , twin bridges training area ; n , north ; s , south blood samples from each of the 4 patients and lung tissues of seropositive rodents were assayed by rt - pcr . rna extracted by using rna - bee isolation kit ( tel - test inc . , friendswood , tx , usa ) was reverse transcribed by using the superscript ii rnase h - reverse transcriptase kit ( gibco - brl , gaithersburg , md , usa ) . primers ( outer primer set , 5-tgggctgcaagtgc-3 , 5-acatgc tgtacagcctgtgcc-3 ; inner primer set , 5-tgggctgcaagtgcatcagag-3 , 5-atggattacaaccccagctcg-3 ) amplified a 373-nt region of the hantavirus g2-encoding medium ( m ) segment ( 1,5,6 ) . amplified products were fractionated according to size by electrophoresis on 1.5% agarose gels containing ethidium bromide ( 0.5 mg / ml ) . dna sequencing was performed in both directions , by using the dye primer cycle sequencing ready reaction kit ( applied biosystems , foster city , ca , usa ) on an automated sequencer ( model 3730xl ; applied biosystems ) . phylogenetic analysis , both neighbor - joining and maximum - parsimony methods , based on the 320-nt region of the g2 glycoprotein  encoding m segment of the 4 hfrs patients , and htnv sequences amplified from a. agrarius mice captured at fp-10 , fp-60 , tbta - n , and tbta - s demonstrated that the htnv sequence amplified from patient 1 was identical to sequences from the a. agrarius htnv strain ( 041325 ) at fp-60 . the analyses also demonstrated that the htnv sequences from patients 2 and 4 were identical to a. agrarius htnv sequence ( 051437 ) at tbta - s , and the htnv sequence from patient 3 was identical to a. agrarius htnv sequences ( 07196 and 051465 ) at tbta - n . these data demonstrate the most likely site of infection for the 4 hfrs patients ( figure 2 ) . phylogenetic tree by maximum parsimony method , rooted at the midpoint , based on the 320-bp region of g2 glycoprotein  encoding medium segment of 4 hemorrhagic fever with renal syndrome patients who were us soldiers in south korea ( patients 14 ) , 2005 ( genbank accession nos . htnv sequence amplified from patient 1 was identical with a htnv sequence ( aa041325 ) from a. agrarius mice captured at firing point ( fp ) 60 . htnv sequences from patients 2 and 4 were the same as 3 htnv sequences ( aa051437 , aa06119 , aa06171 ) from a. agrarius mice captured at twin bridge training area  south ( tbta - s ) , and the htnv sequence from patient 3 was identical to 11 htnv sequences ( aa051465 , aa072 , aa0721 , aa07196 , aa07206 , aa07223 , aa07280 , aa07284 , aa07288 , aa07306 and aa07318 ) from a. agrarius mice at twin bridge training area - north ( tbta - n ) . branch lengths are proportional to the number of nucleotide substitutions , while vertical distances are for clarity only . the numbers at each node are bootstrap probabilities ( expressed as percentages ) , as determined for 100 iterations by using paup version 4.0b ( http://paup.csit.fsu.poly ) . the relationships between rodent density , the proportion of hantavirus - seropositive rodents , and incidence of human infection are complex and poorly understood ( 7,8 ) . previous literature indicates that a prevalence of hantavirus seropositivity > 20% among a. agrarius mice greatly increased the risk for transmission ( 911 ) . although monitoring rodent populations may provide some warning , the most effective means of controlling hantavirus infections is limiting human contact with rodents and the inhalation of dust with virus - laden rodent excreta ( 12 ) . the large patches of tall dense grasses narrowly separated by barren ground where artillery firing is conducted at fp-60 provide harborage for a. agrarius mice and , during the winter trapping period , yielded 20% capture rates ( 10 ) . eliminating these large grassy islands , and thus the rodents that inhabit these areas , and cutting the tall grasses and scrub vegetation to < 10 cm along the training site perimeter would decrease hantavirus infection risks by reducing the rodent populations . efforts to mitigate disease risks through modernization of training sites , cutting of vegetation that increases predation of small mammals , and habitat reduction within 50 m of military operations , where possible , are being instituted at selected us - operated training sites as a result of consolidation and modernization of large , multipurpose range complexes . previously , phylogenetic analysis of a 324-nt region of the g2 glycoprotein - encoding m genomic segment has been shown to be representative of the entire m segment ( 6 ) . this region may be useful for classifying newly identified hantaviruses ( when cross - neutralization data are unavailable ) or for further analysis of molecular phylogeny of hantaviruses spatially . the genome of htnv sequences obtained from patient 1 was identical to a viral sequence from an a. agrarius field mouse captured at fp-60 and those from patients 2 and 4 were identical to 3 viral sequences from a. agrarius at tbta - south , and patient 3 was identical to 11 viral sequences from a. agrarius field mice at tbta - north . these data showed the epidemiologic link between us soldier patients and rodent hosts at the training sites near the demilitarized zone in south korea .
four us soldiers acquired hemorrhagic fever with renal syndrome while training near the demilitarized zone , south korea , in 2005 . 
 hantaan virus sequences were amplified by reverse transcription 
 pcr from patient serum samples and from lung tissues of striped field mice ( apodemus agrarius ) captured at training sites . 
 epidemiologic investigations specified the ecology of possible sites of patient infection .