and feed upon a variety of ectothermic hosts, but seem to prefer snakes over amphibians.25 feeds preferentially upon amphibians and targets other host classes much less frequently.25 If snakes are indeed the major competent ectothermic reservoir hosts for EEEV at Tuskegee National Forest, this partitioning of feeding by the different mosquito species may limit the exposure of frogs to EEEV. Because of their relatively low body temperatures and inefficient antibody responses,14 ectotherms can support prolonged viremias compared with birds and mammals,15 and previous laboratory studies have suggested a role for ectotherms as overwintering hosts for arboviruses.20C22 These studies suggest that ectotherms may symbolize an over-looked reservoir host, and perhaps an important over-wintering refuge for EEEV. preparation was stored at 4C for 24 hours and viral inactivation was confirmed by plaque assay.30 Once viral inactivation was confirmed, the protein concentration in the antigen preparation was determined by using the Bradford method.31 Set 15 Luminex beads coated with monoclonal antibody 2A2C-3 against alphaviruses (Radix Biosolutions, Georgetown, TX) were then coated with the EEEV antigen preparation by mixing 50 L of the beads with 1 g of EEEV antigen in a final volume of 500 L of phosphate-buffered saline (PBS). The combination was placed on a shaker at room temperature for 1 hour. The bead answer was added to 9.5 mL of PBS containing 1% BSA (bovine serum albumin (w/v) and stored on ice. Serum samples (1.5 L per reaction) were biotinylated with approximately a 50-fold molar excess of biotin by using the EZ-Link Sulfo-LC-Biotin Kit (Pierce Biotechnology, Rockford, IL) according to the manufacturers instructions. The biotinylated antibodies were exceeded through a 100-kD MW cutoff filter (Acroprep 96 Omega 100K; VWR Scientific, San Francisco, CA). The 12.5 L of retentate was washed twice with PBS and diluted with 62.5 L of PBS, 0.2% BSA to produce Bavisant dihydrochloride a final dilution of 1/50 relative to the original serum sample. To bind the biotin-labeled antibody preparations, 100 L per well of the antigen-coated bead preparation was placed into each well of a 96-well 1.2-m filter plate (MultiScreen-BV, 1.2 m, Millipore, Billerica, MA). The beads were washed twice in PBS, 1% BSA by vacuum filtration and resuspended in 50 L of the biotinylated serum sample. The plate was shaken for 45 moments at room temperature. The plate was removed from the shaker, the supernatant was removed by vacuum filtration, and the retained beads were washed twice PBS, 1% BSA. The beads were resuspended in 50 L of a solution consisting of 4 g/mL of streptavidin-phycoerythrin (Jackson Immunoresearch, West Grove, PA) in PBS, 1% BSA and the plate shaken for 15 minutes at room temperature. The solution was removed by vacuum filtration, and the beads were washed twice in PBS, 1% BSA Thbd and resuspended in 100 L of PBS, 1% BSA. The samples were analyzed by using a Bio-Rad (Hercules, CA) BioPlex instrument. Results were expressed as mean fluorescent intensity (MFI) of two replicates per sample tested. All plates tested included a series of three known positive and negative serum samples. Positive serum samples were derived from garter snakes (= 0.14). Thus, for any seasonal analysis of the prevalence of seropositivity, data from all three years were combined and compared across months. When all amphibian and reptile species were combined and compared across months, the proportion of seropositive individuals was relatively constant from April through July and then increased in August and September (Physique 1). In contrast, when cottonmouths were considered alone, the seasonal pattern of the proportion Bavisant dihydrochloride of seropositive individuals exhibited a bimodal pattern, with peaks in the early spring and the fall (Physique 1). Open in Bavisant dihydrochloride a separate window Physique 1. Monthly variance in Eastern equine encephalitis computer virus seroprevalence of tested amphibians and reptiles from Tuskegee National Forest, Alabama. Conversation The data offered show that antibodies against EEEV are commonly found in some ectothermic species, particularly snakes residing at Tuskegee National Forest. These findings corroborate previous studies on the reservoir competence of these species for EEEV.20 In the reservoir competency studies, green treefrogs (and and taken from collected at the Tuskegee National Forest TNF site (Graham SP, unpublished data). The serologic data offered suggest that seroprevalence in frogs was quite low. There may be two explanations for this obtaining. First, because frogs are refractory to contamination,20 it is possible that the computer virus does not replicate much, if at all, in these hosts, resulting in a lack of any antibody response. If this is the case, frogs may serve as a dilution host, particularly early in the year, when frogs are an important host for mosquitoes at Tuskegee National Forest.25 Alternatively, this finding may also reflect a.
- du Bois A, Weber B, Rochon J, Meier W, Goupil A, Olbricht S, Barats JC, Kuhn W, Orfeuvre H, Wagner U, Richter B, Lueck HJ, Pfisterer J, et al
- The specificity of the shift was confirmed using antibodies against the nuclear protein ENP1 (Figure?7E; ENP1), which is not related to protein translocation into chloroplasts (Missbach et al