Chua KB, et al

Chua KB, et al. 2002. reservoirs of other important pathogens. Examples of viruses for which bats are reservoirs MBX-2982 include rabies computer virus and related lyssaviruses, henipaviruses, filoviruses, and coronaviruses causing severe acute respiratory syndrome (4, 8, 15, 25, 28). Little is known about virus-host interactions in bats because bats have not been extensively analyzed in regard to their physiology, including immunology and responses to infections. Bats of at least some species may become persistently infected with viruses without apparent ill effect (4, 7), and because of a general lack of knowledge of bat biology, the mechanism by which this occurs is usually unknown. Tacaribe computer virus (TCRV) is an arenavirus (family and cells (Invitrogen) that were subsequently plated and produced on LB-ampicillin plates. Bacterial-colony screening was carried out using PCR to verify plasmid inserts. Colonies with the TCRV place were then incubated at 37C overnight in 4 ml of LB broth with 50 g/ml ampicillin. The plasmids were purified using a QIAprep spin Miniprep kit (Qiagen) according to the manufacturer’s directions. Plasmid recovery was confirmed using a 1% agarose gel. Sequencing reactions were performed using T7 and T3 primers with BigDye Terminator (Applied Biosystems, Foster City, CA). Sequencing reactions were carried out by CSU Macromolecular Services. Sequencing files were edited using Sequencher (GeneCodes, Ann Arbor, MI), and BLAST was utilized for gene identification. Virus isolation. Organs that experienced previously been frozen at ?80C were thawed in 500 l of BA1 medium containing 5% FBS. Approximately 12 2.3-mm zirconia/silica beads were added to each tube, and tissues were homogenized using a mini-bead-beater. The tubes were centrifuged at 13,000 rpm for 5 min to separate virus-containing supernatant from tissue debris. The supernatants were diluted in 96-well tissue culture plates in a log10 dilution series beginning with neat supernatant and leaving the last row uninoculated as unfavorable controls. The supernatants were added to a corresponding plate made up of confluent Vero E6 cells. The cells were incubated with 100 l of computer virus dilutions for 24 h, at which point 200 l of new medium was added to replace the aged medium. The cells were kept at 37C and 5% CO2 for 7 days. Cells were evaluated for the presence of computer virus using immunofluorescent-antibody staining. The medium was removed, and the cells were fixed by incubation with chilly acetone for 2 h. They were then incubated with mouse anti-TCRV ascites fluid (kindly provided by R. Tesh) diluted 1:200 in PBS for 30 min at 37C. The cells then were washed with PBS and incubated with a 1:1,600 MBX-2982 dilution of DyLight goat anti-mouse IgG conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA) and incubated for 30 min at 37C. The cells were again washed, stored in PBS, and guarded from light until they were evaluated with a fluorescence microscope. The same protocol for computer virus isolation was used on terminal serum samples from each experiment. Wells were considered positive for computer virus isolation if a large proportion of Vero E6 cells were fluorescing. Neutralizing antibody. Terminal serum MBX-2982 samples from each bat were screened for anti-TCRV neutralizing antibody and by immunofluorescence. Fifty MBX-2982 microliters of each serum sample was diluted with 75 l of BA1 cell culture medium and warmth inactivated in a water bath for 10 min. The serum MBX-2982 samples were further diluted 1:2 with BA1 cell culture medium and placed in the first well of a 96-well tissue culture plate. A log2 dilution series was prepared for each sample. A dilution series with mouse anti-TCRV ascites fluid in BA1 cell culture medium was carried out without the addition of serum to serve as an anti-TCRV positive control. A negative-control sample was included by using a dilution series with TCRV in BA1 cell culture medium without the addition of serum. Fifty microliters made up of 10 TCID50 TCRV was added (1:2 dilution). The plates were incubated at 37C for 60 min, and 50 l made up of 4 105 Vero E6 cells was then added to each well. The plates were incubated at 37C and 5% CO2 for 7 days, and the cells were then screened for the presence of TCRV using immunofluorescence, as explained above for organ sample computer virus isolations. Histopathology. Formalin-fixed specimens were prepared and sectioned by Colorado Histprep, Inc. (Fort Collins, CO), for evaluation by one of the authors, a veterinary pathologist (D.G.). RESULTS Experimental infections. In the first experiment, 12 bats were inoculated with 106 TCID50 (100 l LEFTY2 s.c.) and 2.5 105 TCID50 (25 l i.n.) to determine susceptibility to TCRV. For the initial experiment, we had planned to euthanize two bats each on days 4, 8, 12, 16,.