PMC:4504098 / 4527-8192 JSONTXT

{"project":"2_test","denotations":[{"id":"26184657-19590002-143716469","span":{"begin":639,"end":641},"obj":"19590002"}],"text":"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.\nTable 2 qPCR results on original and archived PAHC duplicate oropharangeal swabs from five pools screening potentially positivea\nPool Animal ID Species Location Original sample pool Ct Original sample: individual Ct Duplicate sample: individual Ct\n2 U95 C. brachyotis Puning Cave 42.0/ND 41.2/ND\nU96 Pt. jagori Puning Cave\nU97 Pt. jagori Puning Cave\nU98 Pt. jagori Puning Cave\nU99 Pt. jagori Puning Cave\n3 R1 M. australis Biak na Bato 43.0/ND\nR2 M. australis Biak na Bato\nR3 M. australis Biak na Bato\nR4 M. australis Biak na Bato 39.3/ND\nR5 M. australis Biak na Bato\n4 T67 M. australis Biak na Bato 40.6/41.9b\nT68 M. australis Biak na Bato\nT69 M. schreibersii c Biak na Bato 33.6d\nT70 M. schreibersii c Biak na Bato 37.7d 39.9/ND\nT71 M. schreibersii c Biak na Bato 32.9d 40.1/ND\nT72 M. schreibersii e Biak na Bato\nT73 M. schreibersii e Biak na Bato\nT74 M. schreibersii e Biak na Bato 40.5/ND\nT75 M. schreibersii e Biak na Bato\nT76 M. schreibersii e Biak na Bato\n6 T56 Ch. plicata Biak na Bato 39.7/40.1b\nT57 Ch. plicata Biak na Bato\nT58 Ch. plicata Biak na Bato\nT59 Ch. plicata Biak na Bato\nT60 M. schreibersii Biak na Bato\n12 U21 M. australis Biak na Bato 40.2/ND 42.2/ND\nU22 M. australis Biak na Bato\nU23 Ch. plicata Biak na Bato\nU24 Ch. plicata Biak na Bato\nU25 Ch. plicata Biak na Bato\naAll samples were tested in duplicate. Positive samples were confirmed in triplicate\nbPools 4 and 6 had repeatable results on the original pooled sample\ncT69, T70 and T71 yielded a product on hemi-nested PCR whose sequence differed by one nucleotide from a pig isolate in Bulacan province\ndMean value of the duplicates\neAdditional M. schreibersii samples from a pool which tested negative in the original round\nFig. 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)\nFig. 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\n"}