RESEARCH Figure 4. Unrooted phylogenetic tree of the Venezuelan equine encephalitis virus (VEEV) complex and other representative alphaviruses derived from complete structural polyprotein sequences using the neighbor joining program implemented in PAUP 4.0 (22). Viruses are labeled by species according to the International Committee for Taxonomy of Viruses (27). VEEV sub- type IIIC and IIID strain names are in parentheses. Numbers indi- cate bootstrap values for clades to the right. of enzootic VEEV in many other locations. Several differ- ent species (Culex [Mel.] gnomatos, Cx. [Mel.] pedroi, Cx. [Mel.] vomerifer, and Psorophora albigenu) are competent laboratory vectors of the ID viruses currently circulating in the Iquitos area (29,30). Further studies are required to determine whether these mosquitoes, other species, or both transmit VEEV to humans in Peru. This information will be important in understanding human exposure and infec- tion with these different VEE complex strains. Epidemiologic information available from 1995 through 2002 suggests that many VEE cases occurred within the city of Iquitos. Interviews indicated that many patients had not traveled during the time of probable infec- tion, suggesting urban transmission. Studies carried out in other VEEV-enzootic areas indicate that transmission is confined to rural and forest habitats (30–32). Whether the viruses have adapted to infect peridomestic mosquitoes or whether Amazonian deforestation and urbanization is increasing the risk for VEEV transmission to humans through changes in Cx. Melanoconion spp. host preference and larval habitats remains to be determined. Subtype III VEE Complex Strains Before the strain reported herein, the only isolate of subtype IIIC was obtained in 1971 from a pool of mosqui- toes collected near Iquitos (12,13). Although epidemiolog- ic and ecologic studies were conducted in the same area between 1971 and 1995, no evidence was obtained that the IIIC virus was still circulating in the Peruvian Amazon until we isolated it from a sentinel hamster in 2002. The 2002 IIIC isolate did not produce fatal disease in the sen- tinel animal nor in other hamsters infected experimentally in our laboratory (data not shown). The failure to isolate the subtype IIIC virus from febrile patients in Iquitos sug- gests that it is not transmitted readily to humans, that the viremia generated in humans is relatively low and usually undetectable by virus isolation, or that the virus simply does not cause clinical human disease. We propose that the newly identified subtype III strain be defined as a new genetic variety: subtype III, variety D in the VEE complex. These strains do not represent a new antigenic subtype (28) because they exhibit a twofold dif- ference in homologous versus heterologous PRNT anti- body titers when compared to the IIIC strain. Arbovirus varieties are described as “isolates differentiable only by the application of special tests or reagents [kinetic hemag- glutination inhibition (HI), monoclonal antibody assays, etc.] (28).” We propose that this definition of special tests for variety assignment be extended to include sequence and phylogenetic differentiation of major lineages, such as IIIC versus the proposed IIID genetic variety. These line- ages are equivalent in genetic divergence to other VEEV varieties defined previously based on serologic reactions using kinetic HI (8,33). Both subtype III variants isolated in the Iquitos area are antigenically distinct from Mucambo (IIIA) and Tonate (IIIB) viruses, based on greater than fourfold endpoint antibody titer differences in both directions. The variants are also quite distinct genetically from Mucambo and Tonate viruses (23% nucleotide and 14% amino acid sequence divergence in the structural protein genes) and appear to be geographically distinct as well. We do not have enough information available to know whether the Table 4. Results of the plaque reduction neutralization tests between members of Venezuelan equine encephalitis subtype IIIa Antiserum (subtype/variety) Virus strain (subtype/variety) Mucambo (IIIA) Tonate (IIIB) 71D1252 (IIIC) PE407660 (IIID) Mucambo (IIIA) 1 2 16 8 Tonate (IIIB) 4 1 >160 8 71D1252 (IIIC) >32 4 1 2 PE4.07660 (IIID) 8 16 2 1 aNumbers indicate ratios of homologous to heterologous reciprocal titers. 886 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 5, May 2004