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1、BRIEF REPORT Genomic and serological detection of bat coronavirus from bats in the Philippines Shumpei TsudaShumpei WatanabeJoseph S. MasangkayTetsuya Mizutani Phillip AlviolaNaoya UedaKoichiro IhaSatoshi TaniguchiHikaru Fujii Kentaro KatoTaisuke HorimotoShigeru KyuwaYasuhiro YoshikawaHiroomi Akashi
2、 Received: 12 March 2012/Accepted: 29 May 2012/Published online: 26 July 2012 ? Springer-Verlag 2012 AbstractBat coronavirus (BtCoV) is assumed to be a progenitor of severe acute respiratory syndrome (SARS)- related coronaviruses. To explore the distribution of BtCoVs in the Philippines, we collecte
3、d 179 bats and detected viral RNA from intestinal or fecal samples by RT- PCR. The overall prevalence of BtCoVs among bats was 29.6 %. Phylogenetic analysis of the partial RNA-depen- dent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others bel
4、onged to the genus Betacoronavirus. Western blotting revealed that 66.5 % of bat sera had antibodies to BtCoV. These surveys suggested the endemic presence of BtCoVs in the Philippines. KeywordsBat coronavirus ? Coronavirus ? Bats ? Antibody ? Viruses ? SARS ? Severe acute respiratory syndrome ? Zoo
5、nosis Abbreviations BtCoVBat coronavirus CoVCoronavirus SARSSevere acute respiratory syndrome RT-PCRReverse transcriptase polymerase chain reaction RdRpRNAdependent RNA polymerase ntNucleotide NNucleocapsid PBSPhosphate-buffered saline WBWestern blotting SDS-PAGESodium dodecyl sulfate polyacrylamide
6、 gel electrophoresis CBBCoomassie brilliant blue HRPHorseradish peroxidase Recently, bats have been recognized as a major reservoir of emerging viral infections that cause serious diseases in humans and other mammals 1. Several viruses that cause severe diseases in humans have been detected in bats,
7、 such as lyssaviruses related to rabies virus, Hendra virus, Nipah virus, Ebola virus, Marburg virus and severe acute respi- ratory syndrome (SARS) coronavirus 1. Hence, there is a growing focus on bats as zoonotic hosts. S. Tsuda ? S. Watanabe ? H. Fujii ? K. Kato ? T. Horimoto ? H. Akashi ( primer
8、 2,50-CCATCATCAGATAGAATCATCATA-30).PCR products were analyzed by agarose gel electrophoresis. Amplicons (440 nucleotides nt long) were gelpurifi ed and directly sequenced with an ABI 3130 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). To detect anti-BtCoV antibodies in bat sera, western
9、 blot (WB) analysis was performed. Recombinant BtCoV nucle- ocapsid (N) protein produced by prokaryotic expression was used as an antigen. To amplify the full-length BtCoV N proteincoding region (1389 nt; AB543561), PCR was performed using KODPlus (Toyobo, Osaka, Japan) and 0.3 lM primers (forward p
10、rimer, 5-GAACAGATTGG AGGTATGTCTGGACGGAATAAGC-3; reverse primer, 5-TGCTCGAGTGCGGCCTTAGGATCTCTCATTAGC A-3). The amplifi ed fragment was cloned into the pE- SUMOstar vector (LifeSensors, Malvern, PA, USA). E. coli strain BL21 (DE3) was transformed with the recombinant plasmid. N protein expression was
11、induced by the addition of 1 mM isopropylbDthiogalactopyrano- side. The purity of proteins was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), Coomassie brilliant blue (CBB) staining and WB analysis. Recombinant BtCoV N protein was separated by 8 % SDS-PAGE. After b
12、lotting, nitrocellulose membranes were cut into strips and incubated for 1 h with bat serum sam- ples (diluted 1:1000) or horseradish peroxidase (HRP)- conjugated anti-6x His tag antibody (Abcam, Cambridge, UK) at a dilution of 1:5000. The strips were rinsed and further incubated for 1 h with rabbit
13、 anti-bat IgG antibody (diluted 1:1000) 15 and subsequently incubated for 1 h with HRP-conjugated donkey anti-rabbit IgG (diluted 1:2000; GE Healthcare, Milwaukee, WI, USA). Signals were detected using an Enhanced Chemiluminescence (ECL) Detection Kit (GE Healthcare). From 2009 to 2011, we sampled a
14、 total of 179 bats at fi ve sites in the Philippines. To detect viral RNA, RT-PCR was performed using a pair of primers for a conserved 440-nt region within the coronavirus RdRp gene. Intestinal samples containing feces obtained in 2009 and feces col- lected in 2010 and 2011 were examined. As shown
15、in Table 1, the overall prevalence of BtCoV was 53/179 (29.6 %). Amplifi ed fragments were sequenced directly for characterization. A BLAST search (http:/blast.ncbi.nlm. nih.gov/Blast) suggested that most of the sequences obtained in this study were similar to the sequence we reported previously (Bt
16、CoV/Philippines/Diliman1525G2/ 2008). These sequences were98 % similar to one another and were obtained from lesser dog-faced fruit bats (Cyn- opterus brachyotis) and greater musky fruit bats (Pteno- chirus jagori). Sequences from greater musky fruit bats (5/16 specimens positive by RT-PCR) that were similar to BtCoV/Philippines/Diliman1525G2/2008 were also found (87 % identical at the nucleotide level and 95 % identical in their translated amino acid sequences). Sequences that were98 % identic
