PMC:4504098 / 2911-9231 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4504098","sourcedb":"PMC","sourceid":"4504098","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4504098","text":"Results\nA total of 464 bats were captured, comprising 403 bats from 19 species at Bulacan and 61 bats from two species at Subic Bay (Fig. 1) (Table 1). Bulacan yielded 351 serum samples and 739 swab samples (148 pools) suitable for testing: 299 oropharangeal swabs (60 pools), 248 rectal swabs (50 pools) and 192 urine swabs (38 pools). A complete suite of samples was not collected from all bats. Subic Bay yielded 61 serum samples and 183 swab samples suitable for testing: 61 oropharangeal swabs, 61 rectal swabs, 31 urogenital swabs and 30 urine samples.\nFig. 1 Bat sampling locations in Bulacan Province and Subic Bay Freeport Zone on the Philippine island of Luzon\nTable 1 Details of 464 bats captured at two locations on the Philippine island of Luzon in 2010\nSpecies Count Location\nFruit-bats\n Pteropodidae\n   Acerodon jubatus 56 Subic Bay\n   Cynopterus brachyotis 37 Bulacan\n   Eonycteris robusta 11 Bulacan\n   Eonycteris spalea 1 Bulacan\n   Ptenochirus jagori 52 Bulacan\n   Pteropus vampyrus 5 Subic Bay\n   Rousettus amplexicaudatus 42 Bulacan\nInsectivorous bats\n Mollosidae\n   Chaerephon plicata 82 Bulacan\n Hipposideridae\n   Hipposideros ater 1 Bulacan\n   Hipposideros diadema 6 Bulacan\n   Hipposideros pygmeus 8 Bulacan\n   Hipposideros sp. 1 Bulacan\n Vespertillionidae\n   Miniopterus australis 70 Bulacan\n   Miniopterus schreibersii 44 Bulacan\n   Murina cyclotis 1 Bulacan\n   Myotis horsfieldii 5 Bulacan\n   Tylonycteris robustula 3 Bulacan\n Rhinolophidae\n   Rhinolophus arcuatus 31 Bulacan\n   Rhinolophus philippinensis 1 Bulacan\n   Rhinolophus rufus 6 Bulacan\n   Rhinolophus virgo 1 Bulacan\nTotal 464\nOf 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\nOf the Subic Bay samples, four sera were potentially positive on ELISA: three from Acerodon jubatus (s9, s21, s57), and one from Pteropus vampyrus (s53). Three (s9, s21, s57) were also positive on Western blot (Table 3). One sample (s57) showed a stronger response to EBOV than to RESTV antigen (Fig. 4). All samples and swabs were negative for RESTV RNA on qPCR.\nTable 3 Positive serologic findings in 61 flying-foxesa screened for anti-RESTV antibodies by ELISA and Western blot\nBat/sample ID Species Locality ELISA Western blot\ns9 A. jubatus Subic Bay + +\ns21 A. jubatus Subic Bay + +\ns53 P. vampyrus Tala +\ns57 A. jubatus Subic Bay + +b\naComprising 56 A. jubatus and 5 P. vampyrus from two locations\nbs57 showed a stronger reactivity to EBOV than to RESTV\nFig. 4 Western blot analysis. Recombinant nucleoproteins from RESTV (rN) and EBOV (zN) were used to probe for reactivity in four ELISA positive sera (s9, s21, s53 and s57) and one ELISA negative serum (s14). Anti-His tag monoclonal antibody (H) was used as a positive 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