Collection of plasma from snakes at the Tuskegee National Forest has been described in detail in a previous publication.10
In brief, samples were collected from an EEEV-endemic area in Tuskegee National Forest, located in east-central Alabama. Samples were collected during April–September 2007–2009 during surveys of the herpetofauna present at the site, as described.10
Procedures used for collection of blood samples were approved by the Institutional Review Board for Animal Use and Care of Auburn University. Blood samples (1 mL) were collected from the caudal sinus with a 26-gauge heparinized syringe. At the time of blood draw, body size and sex of each snake were recorded. Animals were marked to prevent re-sampling and released at the point of capture. Blood samples were transferred to 1.5-mL microcentrifuge tubes, placed on wet ice, and transported to the laboratory. Samples were centrifuged briefly and the plasma was decanted from the cell pellet. The luminex assay (recognizing antibodies to EEEV) was then used to determine snake exposure to EEEV only, as described.10
Samples determined to be antibody positive by luminex were then tested by qRT-PCR for EEEV RNA. In addition, samples from 66 randomly selected seronegative snakes (11 per month, April–September) were tested by qRT-PCR for EEEV RNA.
Total RNA was prepared from 140 μL of plasma by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. The process was automated with the Qiacube system (QIAGEN), and isolated RNA (60 μL) was stored at –80°C. Each batch of 12 samples processed in the Qiacube consisted of 11 serum samples and one sham extraction as a negative control.
The qRT-PCR was performed using the iScript one step RT-PCR kit for probes (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. Primers and reaction conditions used to detect EEEV RNA were those recommended by Lambert and others,12
with the exception that reactions were performed in a final volume of 25 μL and used 5 μL of the RNA template. These primers produced an amplicon spanning positions 9298–9456 in the EEEV genome sequence (GenBank accession no. X67111).13
Samples (and associated sham extractions) were run in a 96-well plate format, with each plate containing two qRT-PCR negative control wells. Amplicons were detected by using a 5′ 6-FAM, 3′ BHQ1a-Q probe spanning positions 9411–9431 in the EEEV genome. Samples producing a signal at a cycle threshold (Ct
) value ≤ 37 were considered putatively positive.
RNA samples found to be putatively positive in the screening assay were subjected to a confirmatory qRT-PCR assay provided by the Centers for Disease Control and Prevention (Atlanta, GA) to state Department of Health Laboratories conducting arboviral surveillance activities.14
The confirmatory assay used two primers (5′-ACCTTGCTGACGACCAGGTC-3′ and 5′-GTTGTTGGTCGCTCAATCCA-3′), which produced an amplicon spanning positions 9428–9497 in the EEEV genome. The sequence of the probe used in this assay was 5′-CTTGGAAGTGATGCAAATCCAACTCGACA-3′, which spanned positions 9449–9477 in the genome. The probe contained the same fluorescent and quencher molecules as the first qRT-PCR. Putatively positive samples that produced a detectable amplicon in the confirmatory assay (Ct
< 40) were considered confirmed as positive for EEEV RNA. The biochemical limit of detection of both assays was determined to be ≤ 1 plaque-forming unit when assayed against cultured viral stocks of known titers.
Virus isolation was attempted from all confirmed qRT-PCR–positive samples by inoculating individual T-25 flasks of confluent Vero cell cultures with 1 mL of plasma. Flasks were incubated for two hours at 37°C, with gentle rocking every 15 minutes. After the incubation, 9 mL of maintenance media (1× Earle's minimal essential medium, 2% fetal bovine serum, 200 U/mL penicillin, 200 μg/mL streptomycin, and 2.5 μg/mL amphotericin B) were added to each flask. Cells were then monitored daily for a cytopathic effect.
Fisher's exact test was used to test for the significance of the proportion of qRT-PCR–positive samples in seropositive and seronegative snakes. The Pearson's chi-square test was used to test differences in sex ratio in EEEV qRT-PCR–positive and EEEV qRT-PCR–negative snakes. Statistical differences in body sizes of infected and uninfected snakes (separated by sex) were determined using a t-test. All analyses were performed by using SAS version 9.1 statistical software (SAS Institute, Cary, NC).