three hps cases were confirmed in misiones province , argentina , in the following patients : a 14-year - old boy from santa ana in november 2003 , a 28-year - old man from leandro n. alem in december 2003 , and a 12-year - old boy living in the dos arroyos locality of alem city . in january 2005 , hps was confirmed in a 19-year - old girl living in pirapo , a rural area of itapua department , paraguay .
figure 1 shows the approximate geographic location of exposure sites for patients included in this study .
clinical manifestations in the 4 patients studied were similar to those reported for andv infections : fever , myalgia , headache , and vomiting , soon followed by pulmonary edema .
thrombocytopenia and hemoconcentration were reported , renal involvement was minimal , neither oliguria nor renal failure was observed in any case - patient , but all case - patients showed petechiae .
the 4 hps case - patients had immunoglobulin m ( igm ) and igg antibodies to andv n recombinant protein by elisa ( 13 ) and all survived .
these cases led us to investigate reservoirs for hantaviruses in misiones by using sherman live - capture traps ( h.b .
a total of 59 rodents were trapped at 2 study sites in misiones , where o. nigripes was the most frequently captured rodent ( 42 specimens ) , followed by akodon montensis ( 11 specimens ) .
the rodent species was identified by morphologic features , in particular , qualitative external and cranial characteristics .
animals were tested for andv igg antibodies ( table 1 ) . the capture done in august 2005 in santa ana and leandro n. alem cities found 5 ( 11.9% ) andv - positive o. nigripes rodents of 42 tested from this species .
misiones province , argentina , and eastern paraguay , where cases of hantavirus pulmonary syndrome have occurred and rodents were trapped for testing . *
rt , reverse transcription . before we discovered the case in itapua , we had conducted a serosurvey of rodents in 3 departments in eastern paraguay ( table 1 ) .
fifty - one rodent specimens were collected , of which 32 were sigmodontine rodents and 20 were akodon cursor .
only 1 a. cursor rodent obtained from the limoy biologic reserve , paraguay , was seropositive . because of the diversity of akodontine rodents , precise diagnosis , based solely on morphometric characteristics , is not always possible . to confirm the morphologic identification of the a. cursor rodent , we compared the mitochondrial control region ( fragment of 245 nt ) with that of an a. montensis rodent used as a control .
mitochondrial dna sequencing of a. cursor from limoy found higher identity ( 94% ) with an a. cursor from paraguay ( af296264 ) than with a. montensis ( 90% ) .
positive voucher specimens were archived at the museo argentino de ciencias naturales and at the university of buenos aires .
pcr was performed on the 4 human blood samples and on the lung tissues of the 6 seropositive rodents .
initially , a substantial portion of the nucleoprotein n coding region of the s segment ( nt 50954 ) and different fragments of the encoding region of the m segment : g1 glycoprotein ( nt 41443 ) , g1g2 glycoprotein ( nt 1,7281,976 ) , and g2 glycoprotein ( nt 2,7152,941 ) were amplified and subsequently sequenced .
comparison of the viral 905-nt n fragment sequence from case - patient 1 showed the highest degree of identity , 90% , with lec ( table 2 ) .
the strains from case - patients 2 and 3 showed little genetic variation between them and were 95% identical to arav from parana city , brazil ( 12 ) , in the same fragment .
comparison of a g1g2 fragment available for juqv strain ( nt 1,8671,976 ) with that from case - patient 2 showed a 93.6% identity .
the g2 fragment from case - patient 3 was 95.6% identical to that of juqv .
thus , these results suggest that the strains from case - patients 2 and 3 are juqv , although they demonstrate that the strain called araucaria would also be juqv . * orf , open reading frame ; lec , lechiguanas virus ; orn , oran virus ; bmj , bermejo virus ; and , andes virus ; ara , araraquara virus ; cas , castelo dos sonhos virus ; juq , juquitiba virus ; ln , laguna negra virus ; rime , rio mearim virus ; anaj , anajatuba virus ; riom , rio mamor virus ; map , maporal virus ; sn , sin nombre virus .  comparison done on 636 nt .
sequences from the 5 positive o. nigripes from misiones showed little variability between them and the n fragments were 98.3% identical to those in strains from case - patients 2 and 3 . the case - patient from itapua , paraguay , showed the greatest nucleotide identity ( 93.5% ) with bmj lineage , in the n fragment ; identity at the amino acid level was 99.3% .
another different strain was obtained from the a. cursor rodent captured in limoy reserve ; this was the most distinct strain .
the g1 fragment from this strain was compared with the closest related hantavirus and showed 67% identity with strains isolated from o. nigripes , case - patients 2 and 3 , and lnv .
all virus sequences from misiones and the itapua case form a monophyletic group together with andv lineages , nonpathogenic pergamino ( prg ) and maciel ( figure 2 ) , and casv and arav ( done in a 643-nt parsimonious tree , data not shown ) .
the other south american clade was formed with rio mearim virus and anajatuba virus from brazil , together with lnv , htn virus 007 , and rio mamor virus from paraguay , peru , and bolivia , respectively , with a moderate support of 63% .
sequences from case - patients 2 and 3 from misiones grouped together with juqv ( araucaria ) to form a separate clade , since they were from the most divergent strains . the sequence from case - patient 1 grouped with lec lineage from central argentina .
different alignment parameters and phylogenetic methods produced the same results in trees with similar topology ; however , bootstrap supports were moderate or low level for some lineages .
the sequence from the a. cursor rodent was a quite genetically distinguishable virus lineage , separated and apart from choclo and maporal viruses .
phylogenetic relationships among the nucleotide sequences of the n protein of different hantaviruses from north america .
a maximum parsimonious phylogenetic tree was generated on the basis of nucleotide sequence differences in the 904-nt region of the n gene open reading frame , which is available for south american strains by using phylip version 3.57c .
bootstrap values > 50% , obtained from 1,000 replicates of the analysis , are shown for the branch points .
the following published s - segment sequences were included in the analysis ( genbank accession no . ) : hantaan ( htn ; u37768 ) , seoul ( seo ; ab027522 ) , prospect hill ( ph ; z49098 ) , puumala ( puu ; x61035 ) , black creek canal ( bcc ; l39949 ) , bayou ( bay ; l36929 ) , sin nombre ( sn ; l25784 ) , new york ( ny ; u36801 ) , el moro canyon ( elmc ; u11427 ) , ro segundo ( rios ; u18100 ) , cao delgadito ( cdg ; af000140 ) , choclo ( choclo ; do285046 ) , maporal ( map ; ay267347 ) , htn-007 per ( htn-007 per .
af133254 ) , anajatuba ( anaj ; dq451829 ) , rio mearim ( rime ; dq451828 ) , ro mamor bolivia ( riom ; u52136 ) , laguna negra ( ln ; af005727 ) , araucaria ( juq ; ay740633 ) , maciel ( mac ; af0482716 ) , pergamino ( prn ; 482717 ) , andes ( and ; af324902 ) , oran ( orn ; af028024 ) , hu39694 ( hu39694 ; af482711 ) , bermejo ( bmj ; af482713 ) , lechiguanas ( lec ; af482714 ) .
hps is an emerging disease in south america , and investigations strengthen the belief that the disease is underestimated . despite being surrounded by hps - endemic countries ,
two pathogenic hantaviruses that cause hps have so far been proven to circulate in misiones : lec and juqv .
we confirmed o. nigripes as the reservoir species associated with 2 juqv cases in misiones . in eastern paraguay , itapua department , bmj lineage produced hps .
lec was characterized originally from an o. flavescens mouse trapped in the rio de la plata river , an area where several hps cases had occurred ( 6 ) .
, no akodon - borne hantavirus has been reported to be associated with cases in south america .
a. azarae is the most abundant sigmodontine species widely distributed in rural and peridomestic habits of central argentina . in buenos aires province ,
prg was characterized in a. azarae populations . despite the absence of reported hps cases associated with this species in the studied area ,
we characterized a distinct akodon - borne hantavirus at the biologic reserve of paraguay , although we did not investigate whether this virus can produce illness .
a prior study analyzed a collection of sigmodontine rodents from the major biomes of paraguay where 1 a. montensis and 2 o. nigripes were positive for viral rna ( 15 ) .
precise identification of source populations in the reservoir and collection of quantitative data on their relative contribution to hantavirus transmission will be essential for disease control in the 3-country frontier .
 we describe the first , to our knowledge , cases of hantavirus pulmonary syndrome in northeastern argentina and eastern paraguay . 
 andes and juquitiba ( juq ) viruses were characterized . 
 juqv was also confirmed in 5 oligoryzomys nigripes reservoir species from misiones . 
 a novel akodon - borne genetic hantavirus lineage was detected in 1 rodent from the biologic reserve of limoy . 