Genomic and serological detection of bat coronavirus from bats in the Philippines

Abstract

Bat 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 collected 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-dependent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others belonged 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.

Fig. 1

Phylogenetic analysis of isolated BtCoVs. A phylogenetic tree was constructed by the maximum-likelihood method, using a conserved 372-nt sequence from the RdRp genes of isolates of BtCoV. Virus lineages detected in this study are marked with black circles. The percentage of replicate trees in which the associated taxa clustered in the bootstrap test (1000 replicates) is shown next to the branches. Phylogenetic analysis was performed using MEGA5 [17]. This model was selected by a modelfit using MEGA5 [17]. Coronaviral sequences used for comparison and their GenBank accession numbers were as follows: human coronavirus 229E (HCoV 229E; NC_002645), human coronavirus NL63 (HCoV NL63; NC_005831), human coronavirus OC43 (NC_005147), human coronavirus HKU1 (NC_006577), SARS coronavirus (NC_004718), canine coronavirus (AF_124986), feline infectious peritonitis virus (FIPV; AY_994055), transmissible gastroenteritis virus (TGEV; NC_002306), porcine epidemic diarrhea virus (PEDV; NC_003436), porcine hemagglutinating encephalomyelitis virus (PHEV; NC_007732), bovine coronavirus (NC_003045), murine hepatitis virus (MHV; NC_001846), avian infectious bronchitis virus (IBV; NC_001451), turkey coronavirus isolate MG10 (EU095850), BtCoV HKU2 (DQ249235), BtCoV HKU6 (DQ_249224), BtCoV HKU7 (DQ_249226), BtCoV HKU8 (NC_010438), BtCoV HKU9-1 (EF_065513), BtCoV HKU9-3 (EF_065515), bat SARS-CoV Rf1 (DQ_412042), BtCoV/A970/2005 (DQ_648854), BtCoV/A515/2005 (DQ_648822), BtKY22/Chaerephon sp./Kenya/2006 (HQ728486), BtCoV/Philippines/Dilliman1552G1/2008 (AB_539080) and BtCoV/Philippines/Dilliman/1525G2/2008 (AB_539081)

Fig. 2

Expression and western blotting of the recombinant BtCoV N protein. (a) CBB staining of the recombinant N protein. BL21 (DE3) cells expressing recombinant N protein were suspended in PBS containing 1 % Triton X-100, followed by sonication and centrifugation. Supernatants were collected (lane 1), and the pellet was resuspended in 0.5 % Triton X-100/PBS. After another round of centrifugation, the secondary supernatant was collected (lane 2). Two supernatant samples and the pellet (lane 3) were analyzed by SDS-PAGE. The gel was stained with CBB. (b) Western blotting of the recombinant N protein. Secondary supernatant (lane 2 in a) was separated by 8 % SDS-PAGE and detected by serum from a CoV-negative bat (lane 1 diluted 1:1000), serum from a BtCoV-infected bat (lane 2, diluted 1:1000) [14], and HRP-conjugated anti-6x His antibody (lane 3, diluted 1:5000). Strips incubated with bat sera were subsequently detected using rabbit anti-bat IgG antibody (diluted 1:1000) as the secondary antibody, and HRP-conjugated anti-rabbit IgG antibody (diluted 1:2000) as the tertiary antibody. (c) WB analysis of bat sera. Recombinant BtCoV N protein was detected by bat serum specimens as described in Fig. 2b