Endemic Venezuelan Equine Encephalitis in Northern Peru Table 3. Oligonucleotides used for polymerase chain reaction amplification and sequencing analysis Primer (genetic sense) Sequence (5′→3′) V8369(+) GAGAACTGCGAGCAATGGTCA V9207B(–) TRCACTGGCTGAACTGTT V9257B(–) TACACCCAYTTRTCRTTCTG V8659(+) AATTGAGGCAGTGAAGAGCGAC V8953(–) CTGCCTACAGGATTAAAT E/V 7514(+) ACYCTCTACGGCTRACCTRA VIIID 10471(–) CCTTCCGGTCGAACGGGGTCC α10247A(+) TACCCNTTYATGTGGG Mlu-T25 (–) TTACGAATTCACGCGTTTTTTTTTTTTTT TTTTTTTTTTT Medical Research Center Detachment, Lima, Peru (NMRCD). Most of the viruses were isolated in Vero cells from serum samples of febrile patients, sentinel hamsters, and mosquitoes. These viruses were passaged once in Vero cells, and supernatant fluid was stored at –70°C for subse- quent extraction of viral RNA. Extraction of Viral RNA and cDNA Synthesis Viral RNA was extracted from cell culture media as described elsewhere (19). Briefly, 250 µ L of infected cell culture supernatant was mixed with 750 µ L of Trizol LS (Gibco BRL, Bethesda, MD), and RNA was extracted fol- lowing the manufacturer’s protocol. For cDNA synthesis, 0.05% xylene cyanol). To denature the DNA, the reaction 5 µ L of the extracted RNA was mixed with 1 µ mol of was heated to 95°C for 5 min and immediately cooled on reverse primer V9207B (Table 3), 1x First Strand Buffer ice, loaded onto an 8% polyacrylamide gel, and elec- (Gibco BRL, Bethesda, MD), 1 mmol deoxynocleoside trophoresed in 1 x Tris-borate-EDTA buffer at room tem- triphosphate (dNTPs), 80U RNAsin (Promega, Madison, perature for 20 h at 8 mA. The single-stranded DNA WI), and 200 U SuperScript II Reverse Transcriptase products were visualized with silver staining (21). Single- (Gibco BRL, Bethesda, MD), and then incubated at 42°C strand conformational polymorphism genotypes were for 1 h. determined by comparing the migration patterns of the double-stranded DNA of the various isolates with one PCR Amplification another and against a standard DNA ladder. Unique migra- Primers used for the polymerase chain reaction (PCR) tion patterns were designated as distinct SSCP genotypes. and sequencing are listed in Table 3. For amplification of the N-terminus of the PE2 envelope glycoprotein precur- sor gene, primers V8369(+) and V9207B(–) were used, Sequencing and Phylogenetic Analysis Two or three representatives of each SSCP genotype and the resulting product was sequenced with primers were selected randomly for sequencing and phylogenetic V8659(+) and V8953(–) (19). PCRs included 2.5 U of Taq analysis. Most PCR products were sequenced directly with polymerase (Promega, Madison, WI), 1X Promega Taq an Applied Biosystems (Foster City, CA) Prism automated buffer, 300 nmol of each primer, 1 mmol MgCl2, 0.2 mmol DNA sequencing kit according to the manufacturer’s pro- dNTPs, and 10 µ L of the cDNA reaction; 30 amplification tocol. In some cases, PCR products were cloned into the cycles included heat-denaturation at 95°C for 30 s, primer PCR II vector (Invitrogen, Carlsbad, CA), and at least two annealing at 48°C for 30 s, and extension at 72°C for 1 clones were sequenced by using vector-specific primers. min. A final extension of 10 min was used to ensure com- Sequences were aligned by using the Clustal program in plete double-stranded DNA synthesis. In the case of the the MacVector (Oxford Molecular Group, Campbell, CA) proposed subtype IIID strains, the reverse primer used for software package, and phylogenetic analyses were con- cDNA synthesis and PCR was V9257B(–). In some cases, ducted by using maximum parsimony, neighbor-joining, PE2 amplicons were not obtained, and those isolates were and maximum likelihood programs implemented in the amplified by using alphavirus-specific nsP1 primers PAUP 4.0 software (22). For the neighbor joining analysis, described previously (20). The complete 26S gene of one the HKY85 distance formula was used. Bootstrap analyses subtype IIID strain (human isolate FSL190) was amplified were conducted with 1,000 replicates to place confidence and sequenced for further comparison with other VEE values on groupings within trees (23). complex subtypes; two overlapping PCR amplicons were obtained by using primer pairs E/V 7514(+)/VIIID 10471 (–), and α10247A(+)/Mlu-T25(–), and sequenced by gene Plaque Reduction Neutralization Tests To obtain immune sera against VEEV subtypes IIIA, walking. IIIB, IIIC and IIID, cotton rats (Sigmodon hispidus, Single-Stranded Conformation Polymorphism (SSCP) Harlan, Indianapolis, IN) were infected subcutaneously SSCP analysis was performed as previously described with 1,000 PFUs of virus. Three weeks after injection, the (16). PCR products were purified with the Qiaquick gel animals were bled for antibody testing using plaque reduc- extraction kit (Qiagen, Valencia, CA), and 2 µ L of the puri- tion neutralization tests (PRNT) with Vero cells (24). fied PCR amplicon DNA was mixed with 8 µ L of SSCP Viruses were tested against all antibody preparations, and loading buffer (95% formamide, 0.05% bromophenol blue, the endpoint titers were defined as the reciprocal of the Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 5, May 2004 883