key: cord-0961811-s8ajikp0
authors: Tsuda, Shumpei; Watanabe, Shumpei; Masangkay, Joseph S.; Mizutani, Tetsuya; Alviola, Phillip; Ueda, Naoya; Iha, Koichiro; Taniguchi, Satoshi; Fujii, Hikaru; Kato, Kentaro; Horimoto, Taisuke; Kyuwa, Shigeru; Yoshikawa, Yasuhiro; Akashi, Hiroomi
title: Genomic and serological detection of bat coronavirus from bats in the Philippines
date: 2012-07-26
journal: Arch Virol
DOI: 10.1007/s00705-012-1410-z
sha: 88ab3976824ae7fff5ef24eb63ccf6a830951ac4
doc_id: 961811
cord_uid: s8ajikp0

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.

SARS was caused by a previously unknown coronavirus [2] . From 2002 to 2003, the SARS epidemic affected mainly China, Hong Kong and Taiwan, afflicting about 8000 people and killing over 600 [3] . Investigations of bats revealed that horseshoe bats (Rhinolophus) in southern China were natural reservoirs of a coronavirus related to SARS-CoV [4, 5] .

This prompted surveys of bat coronaviruses (BtCoVs) worldwide, and novel viruses were detected in North and South America, Africa, Europe and Asia [6] [7] [8] [9] [10] [11] [12] , revealing a broad diversity of BtCoVs. Molecular phylogenetic analysis suggested that all mammalian coronaviruses including SARS-CoV were likely to have evolved from BtCoVs [13] .

We previously reported that two groups of BtCoVs were detected in the Philippines: one belonging to the genus Alphacoronavirus and the other belonging to the genus Betacoronavirus [14] . To explore the diversity of BtCoVs in the Philippines, we carried out more surveys in this study. We investigated 179 intestinal or fecal samples to detect coronaviral RNA and performed phylogenetic analysis on isolated coronavirus sequences. We also detected virus-specific antibodies, indicating how widely distributed BtCoVs are in the Philippines. Each sampling was performed after receiving permission from the Government of the Philippines. For anesthesia, 15 mg/kg tiletamine and zolazepam (Virbac, Carros, France) were injected intraperitoneally into captured bats, and the anesthetized bats were sacrificed by cardiac exsanguination. Liver, lienal, renal, lung, fecal and intestinal specimens were collected from each captured bat and were shared with the coworkers. All of the experiments using animals were conducted under the supervision of, and with approval from the Animal Care and Use Committee of the Faculty of Agriculture, the University of Tokyo.

To determine the presence of viral RNA in bat samples, reverse transcriptase polymerase chain reaction (RT-PCR) was performed with a pair of consensus primers targeted to a highly conserved region of the RNA-dependent RNA polymerase (RdRp) gene [10] . Viral RNA was extracted from feces or intestinal tissue of the bats using a Total RNA Isolation Mini Kit (Agilent Technologies, Santa Clara, CA, USA). Complementary DNA was synthesized using a SuperScript III Kit (Invitrogen, Carlsbad, CA, USA). PCR was performed using GoTaq PCR Master Mix (Promega, Madison, WI, USA) and 0.2 lM primers (primer 1, 5 0 -GGTTGGGACTATCCTAAGTGTGA-3 0 ; primer 2, 5 0 -CCATCATCAGATAGAATCATCATA-3 0 ). PCR products were analyzed by agarose gel electrophoresis. Amplicons (440 nucleotides [nt] long) were gel-purified 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 blot (WB) analysis was performed. Recombinant BtCoV nucleocapsid (N) protein produced by prokaryotic expression was used as an antigen. To amplify the full-length BtCoV N protein-coding region (1389 nt; AB543561), PCR was performed using KOD-Plus (Toyobo, Osaka, Japan) and 0.3 lM primers (forward primer, 5' -GAACAGATTGG AGGTATGTCTGGACGGAATAAGC-3'; reverse primer, 5'-TGCTCGAGTGCGGCCTTAGGATCTCTCATTAGC A-3'). The amplified 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 induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside. 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 blotting, nitrocellulose membranes were cut into strips and incubated for 1 h with bat serum samples (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 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 total of 179 bats at five 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 collected in 2010 and 2011 were examined. As shown in Table 1 , the overall prevalence of BtCoV was 53/179 (29.6 %). Amplified 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 (BtCoV/Philippines/Diliman1525G2/ 2008). These sequences were[98 % similar to one another and were obtained from lesser dog-faced fruit bats (Cynopterus brachyotis) and greater musky fruit bats (Ptenochirus 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 were [98 % identical were also amplified from Philippine sheath-tailed bats (Emballonura alecto), large rufous horseshoe bats (Rhinolophus rufus), Geoffroy's rousette bats (Rousettus amplexicaudatus), and lesser long-tongued fruit bats (Macroglossus minimus). A novel sequence was detected in a diadem roundleaf bat (Hipposideros diadema), which had 82 % similarity to BtCoV/A977/2005, found in China [16] , and 75 % to the BtCoV we reported previously (BtCoV/Philippines/Diliman1552G1/2008) [14] . Phylogenetic reconstruction from isolated sequences was performed using MEGA5 software [17] (Fig. 1) . This suggested that one of the detected sequences (from the diadem roundleaf bat) was related to members of the genus Alphacoronavirus (BtCoV2231/Philippines/2010), while most of the BtCoVs belonged to the genus Betacoronavirus (BtCoV/Philippines/Diliman1525G2/2008 and BtCoV22 65/Philippines/2010). The two sequences (BtCoV2265/ Philippines/2010 and BtCoV2231/Philippines/2010) were deposited into the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) with the accession numbers AB683970 and AB683971, respectively.

WB analysis was performed to detect antibodies to the CoV N protein. The 6x His-SUMO-fused full-length N protein was expressed in BL21 (DE3) cells and used as antigen. The expression of the *63-kDa recombinant protein was confirmed by CBB staining and WB using an anti-6x His antibody (Fig. 2) . As the recombinant N protein was present in the pellet after centrifugation, this pellet was resuspended in phosphate-buffered saline (PBS) containing 0.5 % Triton X-100. It was confirmed that the recombinant protein was pure and showed a single band in WB using an anti-6x His antibody (Fig. 2b) . The secondary supernatant (isolated after two rounds of centrifugation of the E. coli cells) was used as antigen to detect antibodies to BtCoV. Bat serum samples that detected a 63-kDa band at a dilution of 1:1000 were considered positive (Fig. 2c) . Sera from 10 Leschnault rousette bats (Rousettus leschnaulti), which were obtained from a zoo in Japan, were used as negative controls; none showed bands at 63 kDa. As a positive control, serum from coronavirus infected Leschnault rousette bat was used [14] . Table 1 Our surveys revealed that diverse populations of BtCoVs are continuously circulating in bats in the Philippines. Interestingly, CoVs in two different lineages were detected in greater musky fruit bats (BtCoV/Philippines/ Diliman1525G2/2008 and BtCoV2265/Philippines/2010). Because this bat species is endemic to the Philippines [18] , BtCoVs may have adapted to the local bat species and developed a unique ecology. While the bat-CoV/Philippines/Diliman1525G2/2008 lineage was only present in lesser dog-faced fruit bats and greater musky fruit bats, the BtCoV2265/Philippines/2010 lineage was detected in various bat species, including both insectivorous and 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; [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 Detection of bat coronavirus from bats in the Philippines 2353 frugivorous bats. These observations imply that interspecies jumping has occurred with BtCoVs in the Philippines. Given the wide host range of mammalian coronaviruses, it would not be surprising if BtCoVs crossed species barriers between frugivorous and insectivorous bats as well as barriers between different frugivorous bats. Besides SARS-CoV, cross-species transmission events have been implicated in the emergence of novel CoVs [19] . Recombination commonly occurs among CoVs [20] and also seems to happen in BtCoVs [21] . These two features could enable novel BtCoVs to emerge, leading to diversity and complexity among BtCoVs. While numerous surveys to detect BtCoV RNA have been conducted, serological studies to find anti-CoV antibodies in bats are limited. It is possible that this lack of information has made our understanding of BtCoVs obscure. Only a few studies have investigated antibodies to SARS-related coronavirus in bats [4, 22, 23] . Our survey revealed that antibodies to BtCoV N protein were highly prevalent in bat species (66.5 %). This observation also supports the idea of continuous circulation of BtCoVs in the Philippines and implies that repetitive infection enables BtCoVs to circulate among bat colonies.

We previously reported two lineages of BtCoVs in the Philippines. One was an alphacoronavirus and the other was a betacoronavirus. In this study, we found two more distinct lineages: a betacoronavirus related to another previously described virus [14] and a novel alphacoronavirus. Our genetic and serological surveys suggest the widespread distribution and diversity of BtCoVs in the Philippines.

Bats prove to be rich reservoirs for emerging viruses

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Cumulative number of reported probable cases of Severe Acute Respiratory Syndrome (SARS)

Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats

Bats are natural reservoirs of SARS-like coronaviruses

Detection of novel SARS-like and other coronaviruses in bats from Kenya

Detection and phylogenetic analysis of group 1 coronaviruses in South American bats

Detection and prevalence patterns of group I coronaviruses in bats

Detection of group 1 coronaviruses in bats in North America

Identification of a novel coronavirus in bats

Metagenomic analysis of the viromes of three North American bat species: viral diversity among different bat species that share a common habitat

Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses

Evolutionary insights into the ecology of coronaviruses

Molecular evolution inferred from immunological cross-reactivity of immunoglobulin G among Chiroptera and closely related species

Prevalence and genetic diversity of coronaviruses in bats from China

MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods

Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event

The molecular biology of coronaviruses

Ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related Rhinolophus bat coronavirus in China reveal bats as a reservoir for acute, self-limiting infection that allows recombination events

Coronavirus antibodies in African bat species

Coexistence of different Genotypes in the same bat and sCharacterization of rousettus bat coronavirus HKU9 belonging to a novel betacoronavirus subgroup

Acknowledgments We greatly thank Mr. Edison Cosico and Mr. Eduardo Eres for the arrangements for bat collection. This study was supported by a grant from a Ministry of Education, Culture, Sports, Science and Technology, Japan.