the first occurred in april  may 2003 in 2 communities ( vila sanso , 140 inhabitants , and vila paulo fontelles , 835 inhabitants).in the municipality of parauapebas ( 64s , 4954w ) .
august 2004 in 1 community ( vila tapara , 2,000 inhabitants ) in the municipality of porto de moz ( 145s , 5214w ) ( figure 1 ) .
map of brazil showing locations where oropouche fever outbreaks were identified during 20032004 and previous locations of this disease .
a total of 125 and 109 serum samples were collected from residents of parauapebas and porto de moz , which represented 12.8% and 5.45% of all inhabitants , respectively .
criteria for sampling were a history of acute fever several weeks before or during the survey or clinical symptoms similar to those of oropouche fever .
all serum samples were analyzed by hemagglutination inhibition ( hi ) test ( 7 ) and immunoglobulin m  elisa ( 8) for specific hi and igm antibodies to orov .
hi titers > 20 and elisa results greater than the cut - off value ( optical density > 0.200 ) were considered positive ( 8).virus isolation was conducted by intracranial injection of newborn mice with a 1:10 ( v / v ) suspension of serum samples in phosphate - buffered saline , ph 7.4 , as described elsewhere ( 9 ) .
fifty - four and 11 serum samples from parauapebas and porto de moz , respectively , were used for virus isolation .
two orov strains were isolated from patients in parauapebas , and 2 strains were isolated from patients in porto de moz .
to genetically characterize the viruses , 2 isolates were selected from parauapebas ( brazil 2003a and brazil 2003b ) and 2 from porto de moz ( brazil 2004a and brazil 2004b ) .
viral rna was extracted from vero cells infected with human samples , and s rna was amplified by using a 1-step reverse transcription 
phylogenetic trees were constructed for nucleocapsid gene nucleotide sequences by comparison with other orov nucleocapsid gene sequences in genbank ( table 1 ) ; neighbor - joining analysis ( 10 ) implemented in mega version 2.1 ( 11 ) was used .
bootstrap analyses were performed on 1,000 replicates to generate confidence for groupings ( 12 ) .
of 125 serum samples from patients in parauapebas , hi results were positive for 16 ( 12.7% ) from vila sanso , 6 ( 4.8% ) from paulo fontelles , and 4 ( 3.2% ) from other localities .
igm was detected in 16 ( 12.7% ) , 8 ( 4.8% ) , and 6 ( 4.8% ) serum samples from these 3 areas , respectively .
of 117 serum samples from patients in porto de moz , 56 ( 46.7% ) had hi antibodies and 61 ( 52.1% ) had igm to orov . a total of 71.9% of female patients in parauapebas and 59% in porto
although all age groups were affected , persons 514 years of age had the highest frequency of symptoms ( 30.4% ) and those < 14 years of age had the lowest frequency ( 4.8% ) ( table 2 ) .
symptoms most frequently reported were fever ( 100% ) , headache ( 79.3% ) , joint pain ( 68.7% ) , and muscle pain ( 30% ) .
seventy percent of patients reported 1 episode of recurrence of fever , characterized by fever , headache , and other symptoms 23 weeks after onset of initial symptoms ( 2,3 ) .
full - length s rna of the 4 orov strains contained 754 nt and encoded 2 overlapping open reading frames , the nucleocapsid ( 693 nt and 231 aa ) and nonstructural protein ( 273 nt and 91 aa ) .
two small noncoding regions were also found at the 3 and 5 ends of these reading frames , spanning nt positions 144 and 741754 , respectively .
phylogenetic analysis of brazil 2003 and 2004 isolates grouped strains from parauapebas ( brazil 2003a and brazil 2003b ) into orov genotype i and strains from porto de moz ( brazil 2004a and brazil 2004b ) into orov genotype ii ( figure 2 ) .
comparative small ( s ) rna phylogenetic tree constructed by using the neighbor - joining method for oropouche virus strains isolated in parauapebas and porto de moz , par state , brazil .
bootstrap values were placed over the 3 nodes for each main group ( i , ii , and iii ) .
oropouche fever is the second most common arboviral disease ( after dengue fever ) in the brazilian amazon region . from 1960 to 1980
, oropouche fever outbreaks were detected only in par state , mainly in belm and neighboring areas , where thousands of people were infected ( 2,3 ) .
orov was then detected in other amazonian states including amazonas , amap , acre , rondnia , and tocantins ; and non - amazonian states , including maranho in northeastern brazil and tocantins in central brazil ( 3,8 ) .
recently , orov isolated from callithrix sp.in arinos , minas gerais state , southeastern brazil was characterized as genotype iii , which indicated the presence of this genotype in brazil ( 6 ) .
orov from this species has been identified only in panama ( 5 ) . from 1980 to 2005
, sporadic cases or self - limited outbreaks of oropouche fever were reported in areas of the brazilian amazon , which suggested silent endemic circulation of the virus ( 13 ) . in 2003 and 2004 , several cases of oropouche fever were detected in parauaebas and porto de moz in par state .
parauaebas is located in the carajs mineral province and porto de moz is located in the altamira region
. genetic characterization of strains indicated the presence of genotype ii in the eastern amazon region .
this genotype had been associated with cases of oropouche fever in restricted western amazonian areas ( rondnia state ) , as well as in peru ( 5 ) .
this finding suggests movement of orov genotype ii across the amazon region from western to eastern areas or emergence of this genotype after silent circulation for several years .
genotype i ( brazil 2003a and brazil 2003b ) found in parauapebas was closely related to trinidadian and brazilian isolates obtained from 1955 through 1960 ( trinidad 55 and brazil 60 ) ( 5 ) .
genotype ii strains isolated in porto de moz were genetically related to strains isolated in peru during the 1990s ( peru 92 , 93 , 97 , 98a , 98b ) and rondnia state in 1991 ( brazil 91a , 91b ) , as reported by saeed et al .
these data indicate that parauapebas and porto de moz orov isolates are genetically distinct and have different ancestor viruses ( figure 2 ) .
recognition of different orov genotypes in the brazilian amazon , as well as new genetic information , is useful for understanding the epidemiology and genetic diversity of this emergent human pathogen .
 oropouche fever has reemerged in parauapebas and porto de moz municipalities , par state , brazil . 
 serologic analysis ( immunoglobulin m  
 elisa ) and virus isolation confirmed oropouche virus ( orov ) in both municipalities . 
 nucleotide sequencing of 2 orov isolates from each location indicated genotypes i ( parauapebas ) and ii ( porto de moz ) in brazil . 
The Study
Conclusions