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Bat Coronavirus of Pteropus alecto from Gorontalo Province, Indonesia

Bats have been known as natural reservoirs for potential emerging infectious viruses, such as Lyssaviruses, Coronaviruses, Ebola viruses, Nipah virus, and many others. Because of their abudance in population, wide distribution and mobility, bats have a greater risk as source for zoonotic transmission than other animals. Despite the facts of their role as reservoirs for many pathogens, not until an epidemic of Severe Acute Respiratory Coronavirus (SARS-CoV) in 2003 and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012, that people pay much attention about coronavirus in bats. SARS-like virus also found in bats with a higher prevalence rate. This study aims to detect the coronavirus of bats in Gorontalo province Indonesia, characterization at the molecular level of the coronavirus genome and determining the level of kinship (through trees filogenetic). This study was conducted as part of bigger PREDICT Indonesia project, in particular to examine coronavirus in bats from Gorontalo province, Indonesia.  As many as  95 rectal swab samples collected from flying foxes (Pteropus alecto) were analyzed in the laboratory using Consensus Polymerase Chain Reaction (PCR) technique to amplify the target sequence from RNA-dependent RNA Polymerase (RdRp) gene with 434 basepair product, resulted 24 samples determined as presumptive positive. Eight out of 24 presumptive positive samples by PCR were analyzed further by nucleotide sequencing and confirmed coronavirus positive. Phylogenetic tree analyses to the eight coronavirus confirmed-sequences were constructed with MEGA-6.0 . The conclusion was 24 out of 95 samples suggested as presumptive positive to Bat CoV. Eight out of 24 samples were analyzed further by nucleotide sequencing and have similarities in the kinship. Three samples had the 98% nucleotide identity to BatCoV from Indonesia and five samples were 85-88% nucleotide identity to BatCoV from Thailand.


Background
Indonesia has a megabiodiversity, both flora and fauna, and it has about 162 million hectares of forest area with a variety of ecosystems, including the habitat of bats. Sulawesi Island, which most of their area has included in the Wallace line ecological zone, become the second largest home to flora or fauna next to Brazil in terms of biodiversity (Achmaliadi et al., 2011). About 62 species of bats in the world are found in Sulawesi (Heinrichs et al., 1997). Bats are known as potential reservoir for emerging pathogens. Because of their abundance, distribution, and mobility, bats give high risk for transmission to other animals (Calisher et al., 2006). Human interaction with wildlife is mediated by multiple triggers (drivers): forest clearing, mining industry, eco-tourism, transportation, wildlife hunting and illegal trading, as well as consumption of wildlife meat. Sulawesi an community (Minahasa) known as consumer of domestic animals or wildlife. Bats are one of the most favorite meat, even it becomes popular extreme culinary (Whitten et al., 1987) Previously, the infection of coronavirus (CoV) in human received less attention until a new type of coronavirus causing epidemics in humans was reported; This Severe Acute Respiratory Syndrome Coronavirus was discovered in 2003 and the Middle-East Respiratory Syndrome Coronavirus (Mers-CoV) in 2012 which infected more than 8000 people in the world and caused 774 deaths (Poon et al.,2004). Common symptoms of SARS include a high fever over 38°C of body temperature, followed by symptoms of pneumonia (CDC, 2015;Mahon et al.,2014). In Southeast Asia, it was documented that bats are known to carry coronavirus in several countries, including the Philippines (Watanabe et al.,2010) and Thailand (Gouilh et al.,2011).
Surveillance of virus in bats indicates the type of virus which is classified as pathogens that causing emerging infectious diseases and potentially zoonotic. The interaction between bats and humans (human-animal interface) especially in North Sulawesi region could increase the risk of transmission of pathogen against zoonotic diseases. Only a few studies focused on coronavirus of bats were conducted in Indonesia. Therefore, it is necessary to conduct research on identification and characterization of CoV in bats from their natural habitats in Indonesia, for example Gorontalo Province.

Materials and Methods
This project was conducted under research grant from PREDICT Indonesia in the EPT-1 Program.

Sample collection
We obtained 95 bats of one species: black flying fox (Pteropus alecto) during 2013 field sampling from Olibuu mangroves, Gorontalo province, after receiving permission from the goverment. All bats captured from mangrove forests Olibuu using relevant equipment, conditioned on a certain place and then taxonomically classified on the basis of morphology. The specimens were sampled from urine or uro-genital swab, blood, fecal or rectal swab, throat swab, and skin biopsy when necessary for the host genetic identification. All specimens in lysis buffer or VTM (Viral Transport Medium) were collected in accordance with the Guidelines for the Institutional Animal Care and USE Committee at the PRC-IPB with protocol number approval PRC-16-D001 and stored in a cold chain in order to keep the low temperature. Recommendations to carry out the study was issued by Indonesian Institute of Science (LIPI) approval number: 1689/IPH.1/HK.04.04/VII/2013. Approval for shipping of specimens from Olibuu mangrove forests to Bogor was issued by the Directorate General of Conservation of Natural Resources and Ecosystem, Ministry of Forestry, number 02/SATS/BKSDA-28/2013

RNA Extraction and cDNA synthetis
Viral RNA was extracted from rectal swab specimens using QIAamp viral RNA minikit (QIAGEN, Hilden, Germany) according to the manufacture's instructions. These RNA then used as the template for reverse transcription using Superscript ™ III First-Strand cDNA Synthesis System for RT-PCR (Life Technologies, CA, USA). Briefly, 10 µL of extracted RNA containing 1 µL of 10 mM dNTPs, 1 µL of 50 pmol/µL random hexamer was incubated at 65 o C for 5 minutes. The mixture then added to 10 µL of a reverse transcriptase enzyme mix containing 10x RT buffer, 25mM MgCl2, mM MgSO4, 0.1 M DTT, RNAs out, and 1 µL (200U) of reverse transcriptase enzyme. Samples were subjected to an initial cycle of 50°C for 50 min and 85°C for 5 min. One microliter of RNaseH 2U/ µL was added and incubated at 37 o C for 20 min

PCR and DNA sequencing
All cDNA samples obtained from Olibuu mangroves were tested using conventional nested PCR. We used two sets of consensus primers amplifying highly conserved region of the RNA-dependent RNA polymerase (RdRp) gene, modified of Watanabe et al., 2010. As much as 2.5 µL of cDNA was added to a 22.5 µL reaction mixture of 2x KAPA Taq Hotstart ready mix (containing 1U KAPA Taq DNA polymerase, KAPA Taq buffer, 0.2 mM dNTPs, 1.5mM MgCl2) (KAPA Biosystem, Boston, USA), 1 µL of each primer (10 pmol/ µL) and nuclease free water. The primer for first round PCR were CoV-Forward-1: GGTTGGGAYTAYCCHAA-RTGTGA and CoV-Reverse-1: CCATCA-TCASWYRAATCATCATA; and for the second round were CoV-forward-2 as forward primer: GAYTAYCCHAARTGT-GAUMGWGC and the same CoV-Reverse-1 primer as used in first round. PCR amplification was undertaken in Thermal Cycler (Veriti Applied Biosystem) at 94 o C for 2 minutes, 35 cycles at 94 o C for 20 sesonds, 50 o C for 30 seconds and 72 o C for 30 seconds and a final extension of 72 o C for 5 minutes. This protocols are the same for second round PCR. The amplified PCR product then analyzed by 1.8% agarose gel electrophoresis in tris-acetic EDTA (TAE buffer) and visualized in GelDoc machine (BioRad) using Quantity One program

Sequences and Data Analysis
The presumptive PCR positive samples were results then nucleotide sequenced in commercial sequencing provider. The results then analyzed by BioEdit program and further analyzed by alignment method using the Basic Local Alignment Search Tool (BLAST) program from NCBI. Sequences were aligned with previously published CoV sequences from GenBank by using ClustalW program. The GenBank accession numbers of all sequences used are noted in the taxon names in Figures 2. The partial RdRp gene sequences were trimmed to equal length about 390 bp. Phylogenetic trees were constructed to see the kinship of the virus using the Molecular Evolutionary Genetics Analysis 6.0 (MEGA 6.0) with neighborjoining (NJ) method. Bootstrapping was perfomed to asses the robustness of tree topologies by using 1000 replicate (Tamura et al.,2013).

bp
Wenti Dwi Febriani, Uus Saepuloh, Ellis Dwi Ayuningsih, R. Suryo Saputro, Azhari Purbatrapsila, Meis Jacinta Nangoy, Tiltje Andretha Ransaleleh, Indyah Wahyuni, Safriyanto Dako, Rachmitasari Noviana, Diah Iskandriati, Ligaya ITA Tumbelaka, Joko Pamungkas (2018) Int. J. Trop. Vet. Biomed. Res. 3(2):36-42 39 from Thailand's Scotophilus heathii [12] showed in Table 1. Based on Table 1 shows that Coronavirus (CoV) found in samples of bat originating from Olibuu Gorontalo province has similarities in nucleotide sequences with virus samples that have been found in Indonesia before by Anindita, 2015. She mentioned the coronavirus found in Dobsonia moluccensis bat species obtained from 3 regions in Indonesia, Paguyaman, Gorontalo, Surabaya and Jogjakarta. whereas the similarity of the sequences of the next nucleotides is 85-88% found in Thailand in the bat species Scotophilus heathii (Wacharapluesadee, 2015). Three samples had the 98% nucleotide identity to BatCoV from Indonesia and five samples were 85-88% nucleotide identity to BatCoV from Thailand Phylogenetic analysis of nucleotide sequences of 390 bp of RdRp gene showed that 8 samples were clustered as Betacoronavirus genus and branched to two clustered groups. The first group consist of three specimens (INDSWBT101, IND-  In this study, we found that 23.3% presumptive positive to bat coronavirus were detected from Pteropus alecto rectal swab specimens. Meanwhile, only 8 presumptive positive out of 97 samples (8.3%) were detected from throat swab specimens of those samples (unpublish data). This finding suggests that bat CoV circulation is found more around the digestive tract. This means to detect the genetic material of bats CoV is more likely found in rectal swab than throat swab, although most of CoV are transmitted by both and by aerosols of respiratory secretions. These data are consistent with the result of bat CoV detection from fecal sample or rectal swab or intestine tissue that previously reported. (Watanabe et al.,2010;Anindita et al.,2015;Wacharapluesadee et al.,2015). The species of bats captured from Olibuu Mangrove was identified as Pteropus alecto (black flying fox), meanwhile the CoV genetic material were detected closely related to the bat CoV found in Indonesian Dobsonia moluccensis (Moluccan nakedbacked fruit bat) and Thailand's Scotophilus heathii (greater Asiatic yellow bat) (Anindita et al.,2015;Wacharapluesadee et al.,2015). Therefore, bat CoV with similar genetic material were found in different bats species and not solely correlated with one host species. This data also supported the previous study about the diversity of CoV detected in different bats species (Wacharapluesadee et al.,2015). The RdRp gene is the one most commonly used for PCR amplification in CoV surveillance, since it contains the most region that are conserved in all CoV (Xu et al.,2003). The primers designed in this research were nested consensus primer that amplified the RdRp partial genes resulted about 434 bp PCR product modified of Watanabe et al 2010(Anthony et al.,2013. The gene target position was a different region slightly more upstream. On the Human CoV genome (Strain 229E) it targets roughly nucleotides at the position 14,370-14,750 of CoV whole genome. The primers developed by modified some degenerate sequences to increase the ability of the assay to detect widely variant CoV in family level. A hemi-nested PCR step has also been conducted to increase the sensitivity. This step can be performed using a forward primer that is optimized for bat CoV, or other coronaviruses, depending on the sample being investigated (Anthony et al.,2013) The BLAST and the phylogenetic tree result to the 8 bats CoV positive samples indicated there were two group bats CoV detected from Olibuu Pteropus alecto. Three samples (INDSWBT101, INDSWBT 102, and INDSWBT103) clustered with the known bat CoV that previously reported from Paguyaman, Indonesia with 98% nucleotide identity. Olibuu and Paguyaman are the same province region of Gorontalo Indonesia. Five specimens (INDSWBT180,  INDSWBT195, INDSWBT192, INDSWB-T110, and INDSWBT198) possibly assumed as new bats CoV due to low similarity (85%) to others bat CoV in Genbank, although still clustered with bat CoV from Thailand. Further characterization to the whole RdRp gene or the other gene region of these bat CoV is needed to prove this assumption. In general, CoV in bats that taken from Olibuu mangroves, Gorontalo province were genetically still has a kinship with the others CoV found in some areas of the world (Kenya, China, Thailand, Philippine, Middle East, and Madagascar).
This study shows that fruit bats from Indonesia is a reservoir for viruses that have potentially zoonotic, because the fruit bats are also consumed in some areas in Indonesia. Although CoV does not cause illness in bats and the kinship of the findings of phylogenetic in this study were far distance with SARS and MERS-CoV, but this studies finding need more attention about the impact of the CoV to human health and could serve as an early warning. The information could serve as additional data to support global monitoring system for the emergence of bats CoV which potentially become dangerous pathogens to human health, thus further and assist the authorities to develop relevant policies. The threat of emerging pandemic diseases is facilitated by the interaction of wildlife and humans at human-animal interfaces. In Indonesia, as in other countries, increasing contact between wildlife and humans leads to greater risk of human exposure to pathogens, both well-known and new. Habitat disturbance, such as forest clearing for agriculture or development purposes; transportation; handling of wildlife; and wildlife consumption, among other humanmediated practices, promotes direct opportunities for pathogens to move from wildlife to people. Development of early warning systems, including expanded surveillance and diagnostic capacity for potential disease threats to human beings, is urgently needed by government agencies, research, and academic institutions in order to better serve and protect the public

Conclusion
Twenty four out of 95 samples suggested as presumptive positive to Bat CoV. Eight out of 24 samples were analyzed further by nucleotide sequencing and confirmed as Bat CoV. Three samples had the 98% nucleotide identity to BatCoV from Indonesia and five samples were 85-88% nucleotide identity to BatCoV from Thailand.