RESEARCH Results highest dilution inhibiting >80% of approximately 100 PFU of virus. Distribution and Characterization of Human VEEV Cases in Iquitos Studies to identify the cause of human febrile illness in the Amazon region of Peru since 1995 (Watts et al., unpub. data) identified at least 183 cases of VEEV on the basis of immunoglobulin (Ig) M antibody detection, virus isola- tion, or both. The VEEV cases in the Iquitos area were localized into two major clusters: the region surrounding Lake Morona Cocha, which typically floods during the rainy season, and cases near the Itaya and the Amazon rivers (Figure 2). Cases were also observed to the north and southwest of Iquitos. Most patients with VEEV did not report travel outside their home region, suggesting that many infections occurred in the city of Iquitos. Common signs and symptoms of VEEV infection included fever, headache, chills, malaise, diarrhea, vomiting, arthralgia, and abdominal pain. Clinical information obtained by NMRCD provided no indication of neurologic disease, and no deaths were reported. Sequencing and Phylogenetic Analysis An 856-bp fragment from the N terminus of the PE2 gene was obtained for most of the VEEV subtype ID iso- lates using primers V8369(+)/V9207(–). This region was chosen because it has been used previously in similar phy- logenetic analyses, resulting in a large database that allows for genetic comparison with new isolates (4,9,25). In addi- tion, the fragment sequenced contains the N-terminal por- tion of the E2 glycoprotein gene, which has been shown to undergo critical amino acid substitutions associated with epizootic VEEV emergence (18). To screen the amplified isolates for genetic differences and to eliminate the cost and effort in sequencing each virus isolate, SSCP analyses were performed as previously described (19). At least 2–3 representatives of each SSCP genotype were selected at random for sequence and phylo- genetic analysis. Some isolates could not be amplified with primers V8369(+)/V9207B(–), and later phylogenetic analyses indicated that they corresponded mainly to sub- types other than ID. For these isolates, primer V9257(–) was substituted for V9207B(–) in the cDNA syntheses and PCR amplifications. In addition, alphavirus consensus primers (20) that amplify a portion of the nsP1 gene were used to confirm the identity of these isolates and to look for evidence of recombination within the VEE complex (data not shown). Phylogenetic analyses performed using maximum par- simony, neighbor-joining, and distance-matrix methods Figure 2. Map of Iquitos showing the locations of human Venezuelan equine encephalitis virus cases in the city. generated similar tree topologies. The neighbor-joining tree based on the PE2 gene (Figure 3) showed that the newly sequenced VEEV Peruvian strains grouped into four major clades: the subtype ID Colombia/Venezuela geno- type, the ID Panama/Peru genotype, subtype IIIC isolated in 1971 in the Iquitos area of Peru from a mosquito, and a newly identified group of viruses that fell within subtype III but were distinct from IIIC, which we propose as a new genetic subtype IIID. The greatest number of human isolates from Peru fell into VEEV subtype ID. In agreement with previous studies (9), genetic analysis of the ID strains delineated two dis- tinct genotypes that differ by approximately 5% in their nucleotide sequences: the Colombia/Venezuela genotype, which is believed to have generated the epizootic IAB and IC viruses (15–18), and the Panama/Peru genotype. The Colombia/Venezuela ID genotype was represented by iso- lates in Peru during the 1970s and two isolates made from Pantoja in 1994. Previous data suggested that this geno- type was no longer circulating in Iquitos and had been replaced by the Panama genotype; however, the evidence that these viruses continued to circulate in Iquitos was sup- ported by human isolates from 1998 and 2002, which grouped into the Colombia/Venezuela clade with strong 884 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 5, May 2004