Of the Bulacan samples, all sera were negative on ELISA, and all rectal and urine swabs pools were negative for RESTV RNA on qPCR. Five oropharangeal swab pools returned potentially positive results on qPCR (Table 2). Each of the 25 component individual samples of the five pools was then tested individually. Three of these individual samples (from the same pool) yielded positive results (Table 2). All three samples were from Miniopterus schreibersii caught in the same cave on the same day. In the conventional PCR, all three samples yielded a product whose sequence differed by one nucleotide from a pig isolate sequence from Farm A [14] in Bulacan Province (Fig. 2). Likewise, in the phylogenetic analysis, the three bat-derived PCR product sequences are most related to the Reston isolate from Farm A (Fig. 3). Subsequent testing of 23 duplicate and five additional (M. schreibserii) oropharangeal swabs held by the PAHC laboratory in the qPCR yielded six samples with potentially positive results (four of which were Miniopterus species), including two of the three previously identified positives (Table 2). Conventional PCR was unable to generate a clean PCR product for direct sequencing of the PAHC duplicate samples because of the small sample volume and limited RNA present. Table 2 qPCR results on original and archived PAHC duplicate oropharangeal swabs from five pools screening potentially positivea Pool Animal ID Species Location Original sample pool Ct Original sample: individual Ct Duplicate sample: individual Ct 2 U95 C. brachyotis Puning Cave 42.0/ND 41.2/ND U96 Pt. jagori Puning Cave U97 Pt. jagori Puning Cave U98 Pt. jagori Puning Cave U99 Pt. jagori Puning Cave 3 R1 M. australis Biak na Bato 43.0/ND R2 M. australis Biak na Bato R3 M. australis Biak na Bato R4 M. australis Biak na Bato 39.3/ND R5 M. australis Biak na Bato 4 T67 M. australis Biak na Bato 40.6/41.9b T68 M. australis Biak na Bato T69 M. schreibersii c Biak na Bato 33.6d T70 M. schreibersii c Biak na Bato 37.7d 39.9/ND T71 M. schreibersii c Biak na Bato 32.9d 40.1/ND T72 M. schreibersii e Biak na Bato T73 M. schreibersii e Biak na Bato T74 M. schreibersii e Biak na Bato 40.5/ND T75 M. schreibersii e Biak na Bato T76 M. schreibersii e Biak na Bato 6 T56 Ch. plicata Biak na Bato 39.7/40.1b T57 Ch. plicata Biak na Bato T58 Ch. plicata Biak na Bato T59 Ch. plicata Biak na Bato T60 M. schreibersii Biak na Bato 12 U21 M. australis Biak na Bato 40.2/ND 42.2/ND U22 M. australis Biak na Bato U23 Ch. plicata Biak na Bato U24 Ch. plicata Biak na Bato U25 Ch. plicata Biak na Bato aAll samples were tested in duplicate. Positive samples were confirmed in triplicate bPools 4 and 6 had repeatable results on the original pooled sample cT69, T70 and T71 yielded a product on hemi-nested PCR whose sequence differed by one nucleotide from a pig isolate in Bulacan province dMean value of the duplicates eAdditional M. schreibersii samples from a pool which tested negative in the original round Fig. 2 Comparison of sequencing trace files showing the 1-nt difference. (a) Sequence from the earlier Bulacan Farm A pig isolate; (b) Sequence from bat oropharangeal swab T69. Identical sequences were obtained from bat oropharangeal swabs T70 and T71 (not shown). The single nucleotide difference is highlighted in bold and red, which corresponds to nt residue 1,274 of the Reston ebolavirus isolate RESTV/Sus-wt/PHL/2009/09A Farm A (GenBank accession number JX477165.1) Fig. 3 Phylogenetic analysis by maximum likelihood method, based on partial NP sequences (519 bp) obtained from hemi-nested PCR. Bat-derived RESTV sequence are shown in red