Methods

Ethics Statement
All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was covered by an IACUC protocol from the Lubee Bat Conservancy (USDA Research Facility #58-R-0131).

Animals and sample collection
Subjects for this study included 10 male Large Flying Foxes (Pteropus vampyrus) Some of the animals were wild caught, and some were laboratory born; all were reproductively mature, ranging in age from 4 to 21 yrs. Animals were housed in captivity at the Lubee Bat Conservancy in Gainesville, Florida, USA. Animals were housed together with conspecifics in an indoor/outdoor circular flight enclosure and were fed a mixture of fruit, vegetables, commercial primate chow, and a vitamin supplement. Water was provided ad libitum. The bats were captured manually, anesthesia was mask-induced with 5% isoflurane in oxygen (3 L/min), and the bats were maintained with 2.5% isoflurane. Blood was collected from the left and right brachial veins using 3-ml syringes and 25-gauge needles. Blood samples were transferred to heparanized tubes, maintained and shipped at room temperature (22°C) via overnight courier.

Cloning and Sequencing
Genomic DNA was prepared from whole unfractionated peripheral blood from Pteropus vampyrus bats. Using primers derived from the inferred interferon gene sequences and encoded for restriction enzyme cloning sites, the genes were PCR amplified from the genomic DNA. The forward and reverse primers encoded restriction sites for NheI and XhoI respectively, which allowed cloning of the full length gene into mammalian expression vector pcDNA3.1(+). The PCR program consisted of an initialization step at 94°C for 5 minutes, then 30 cycles of amplification consisting of denaturation at 94°C for 1 minute, annealing at 55°C for 30 seconds, and elongation at 72° for 1 minute. Amplification was followed by a final elongation step at 72°C for 5 minutes and samples held at 4°C. PCR was performed on a P × 2 Thermal Cycler from ThermoElectron Corporation (Waltham, MA).
After purification, the inserted genes were sequenced using vector primers downstream and upstream of the insert site. Pteropus vampyrus IFNB, IFNK, IFND, and IFNA sequences have been submitted to Genbank (GU126493, HM63650, HM636501, and HM636502).

Quantitative RT-PCR
Whole anticoagulated blood was collected from the brachial vein and layered over lymphocyte separation medium (Lympholyte M, Cedarlane Labs). Cells were collected from the interface, washed, and plated on 24 well flat-bottom tissue culture plates (2 × 106 cells/well) in a total volume of 250 μl. Cells were treated as indicated with either poly(I:C)(Invivogen), LPS (Sigma), or VSV (Ramsburg lab stocks confirmed endotoxin free using LAL assay). Stimulants were diluted such that the addition of 250 μl stimulant would give a final concentration of 10 μg/ml poly I:C, 1 μg/ml LPS, or an MOI of 5 for VSV. 500 μl of complete medium was added to the unstimulated control wells. Cells were incubated at 37' and 5% CO2 for the indicated times, after which cells were harvested into RNA lysis buffer (Qiagen RNEasy kit) according to the manufacturer's instructions.
Purified RNA was prepared from these whole-cell lysates as described in the protocols accompanying the Qiagen RNeasy kit, Qiashedder, and Qiagen RNeasy Mini kit and Qiagen DNase-Free DNase Set. cDNA synthesis was performed using the Invitrogen two-step qRT-PCR kit according to the manufacturer's protocol. Quantitative real-time PCR was performed in an Eppendorf Mastercycler ep realplex thermal cycler using SYBRGreenER qPCR Supermix and primers designed from the inferred gene sequences (additional file 3).
Independent biological replicates were prepared for each treatment and time point. The log2 expression level of IFNB and OAS2 relative to the housekeeping gene PPIA was estimated by subtracting the mean Ct value for each gene and treatment/time point combination from the corresponding mean Ct value for PPIA.

Estimation of Gene Family Size
The likelihood function for the observed set S of sequences is a function of the true number, g, of genes and the sample size, n, by summing over the unobserved number, c, of distinct genes sampled.14
The pmf on c is given by the recursion15
with Pc (1 | g,1) = 1 and Pc (c | g,1) = 0 for c ≠ 1.
PS (S | c) is approximated by minimizing the number, m, of mutations over the assignments of observed sequences to c groups. PS is then the posterior probability of getting m mutations given the observed number of mutations in the IFNB and IFNK datasets.
Note that this technique is independent of the assembly methods described elsewhere in the paper.

Phylogenetic Inference
The set consisting of type-I interferon amino acid sequences from human (Homo sapiens), domestic pig (Sus scrofa), duckbill platypus (Ornithorhynchus anatinus) and the amino acid sequences corresponding to the Chiropteran genes inferred in this paper was submitted to multiple sequence alignment by CLUSTALW 2.0.10 [36] and ProbCons 1.12 [37]. Maximum likelihood and minimum evolution phylogenies were inferred from each resulting multiple sequence alignment. Maximum likelihood phylogenies were inferred with proml found in the PHYLIP 3.68 package [38]. Minimum evolution phylogenies were inferred using fastme[39] based on the distance matrix calculated with protdist found in PHYLIP. In both cases, 1000 boot-strap replications were generated using seqboot and the consensus phylogeny was assembled with consense, both from the PHYLIP package. FigTree 1.2.1 http://tree.bio.ed.ac.uk/software/figtree/ was used for initial visualization and final production of figures of the resulting phylogenetic trees. Alignments of the bat DNA sequences and of all the amino acid sequences are available in additional files 4.