Title Detection of novel gammaherpesviruses from fruit bats in Indonesia Wada, Yuji; Sasaki, Michihito; Setiyono, Agus; Handharyani, Ekowati; Rahmadani, Ibenu; Taha, Siswatiana; Adiani, Author(s) Sri; Latief, Munira; Kholilullah, Zainal Abidin; Subangkit, Mawar; Kobayashi, Shintaro; Nakamura, Ichiro; Kimura, Takashi; Orba, Yasuko; Sawa, Hirofumi Journal of medical microbiology, 67(3), 415-422 Citation https://doi.org/10.1099/jmm.0.000689 Issue Date 2018-03 Doc URL http://hdl.handle.net/2115/74394 Type article (author version) File Information manuscript (revice).pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP 1 Detection of novel gammaherpesviruses from fruit bats in 2 Indonesia 3 4 Yuji Wada1, Michihito Sasaki1, Agus Setiyono2, Ekowati Handharyani2, Ibenu 5 Rahmadani3, Siswatiana Taha4, Sri Adiani5, Munira Latief6, Zainal Abidin 6 Kholilullah7, Mawar Subangkit2, Shintaro Kobayashi1,#a, Ichiro Nakamura8, 7 Takashi Kimura1,#b, Yasuko Orba1 and Hirofumi Sawa1,9,10 8 9 1 Division of Molecular Pathobiology, Research Center for Zoonosis Control, 10 Hokkaido University, Sapporo, Hokkaido, Japan 11 2 Laboratory of Veterinary Pathology, Faculty of Veterinary Medicine, Bogor 12 Agricultural University, Bogor, Indonesia 13 3 Veterinary Investigation and Diagnostic Center, Bukittinggi, Indonesia 14 4 Faculty of Agriculture, Gorontalo State University, Gorontalo, Indonesia 15 5 Faculty of Animal Husbandry, Sam Ratulangi University, Manado, Indonesia 16 6 Office of Animal Husbandry and Fisheries, Soppeng, Indonesia 17 7 Veterinary Medicine Study Program, Faculty of Medicine, Hasanudin University, 18 Makassar, Indonesia 19 8 Unit of International Cooperation, Research Center for Zoonosis Control, 20 Hokkaido University, Sapporo, Hokkaido Japan 21 9 Global Institution for Collaborative Research and Education (GI-CoRE), 22 Hokkaido University, Sapporo, Hokkaido, Japan 23 10 Global Virus Network, Baltimore, MD 21201, USA 1 24 25 Current addresses: 26 #a Laboratory of Public Health, Department of Preventive Veterinary Medicine, 27 Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 28 Japan. 29 #b Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, 30 Hokkaido University, Sapporo, Hokkaido, Japan 31 32 Corresponding author: 33 Hirofumi Sawa ([email protected]) 34 35 Key words: 36 Gammaherpesvirus, Fruit bat, PCR screening, Long-distance PCR, Phylogenetic 37 analysis 38 39 Word count for abstract: 124 40 Word count for main text: 2150 41 42 Database depository: 43 The DDBJ accession numbers for the MgBHV and MgGHV DPOL, gB and 44 DPOL-gB sequences are LC268906-LC268990 2 45 Abstract 46 Bats are an important natural reservoir of zoonotic viral pathogens. We 47 previously isolated an alphaherpesvirus in fruit bats in Indonesia, and here 48 establish the presence of viruses belonging to other taxa of the family 49 Herpesviridae. We screened the same fruit bat population with pan-herpesvirus 50 PCR and discovered 68 sequences of novel gammaherpesvirus, designated 51 'megabat gammaherpesvirus' (MgGHV). A phylogenetic analysis of approximately 52 3.4 kbp of continuous MgGHV sequences encompassing the glycoprotein B gene 53 and DNA polymerase gene revealed that the MgGHV sequences are distinct from 54 those of other reported gammaherpesviruses. Further analysis suggested the 55 existence of coinfections of herpesviruses in Indonesian fruit bats. Our findings 56 extend our understanding of the infectious cycles of herpesviruses in bats in 57 Indonesia and the phylogenetic diversity of the gammaherpesviruses. 58 3 59 Text 60 Outbreaks of emerging and re-emerging zoonoses have compromised public 61 health and damaged the global economy in recent years. Bats are known to be 62 carriers of lyssaviruses, henipaviruses, severe acute respiratory syndrome-like 63 coronaviruses, and filoviruses, so they are regarded as an important natural 64 reservoirs of zoonotic viral pathogens [1-5]. 65 Herpesviruses are enveloped, double-stranded DNA viruses belonging to the 66 family Herpesviridae. These viruses include three genera: Alphaherpesvirinae, 67 Betaherpesvirinae, and Gammaherpesvirinae. Gammaherpesvirinae contains 68 viruses that are oncogenic to humans, including Human gammaherpesvirus 4 69 (also known as Epstein-Barr virus) and Human gammaherpesvirus 8 (also known 70 as Kaposi's sarcoma-associated herpesvirus). Therefore, the viruses of the genus 71 Gammaherpesvirinae are important in human health. 72 Many gammaherpesvirus sequences have recently been detected in a broad 73 range of bat species worldwide [6-9]. Myotis gammaherpesvirus 8 was isolated 74 from a tumor cell line derived from Myotis velifer incautus (mouse-eared bat), and 75 this virus has been shown to cause cytopathic effects in Vero cells and can 76 replicate in some human cell lines [10, 11]. 77 In a previous study, we reported a high prevalence of fruit bat 78 alphaherpesvirus 1 (FBAHV1) in fruit bats in Indonesia [12]. Here, to extend our 79 understanding of the herpesviruses carried by fruit bats, we used PCR to screen 80 for a broad spectrum of herpesviruses, and followed by phylogenetically analyzed 81 the sequences detected. 4 82 In this study, 183 bat tissue samples were analyzed: 69 from Pteropus 83 vampyrus (large flying fox), 61 from unassigned Pteropus bats (Pteropus sp.), 17 84 from Dobsonia moluccensis (Moluccan bare-backed fruit bat), and 36 from 85 Acerodon celebensis (Sulawesi fruit bat). The samples were collected in the Lima 86 Puluh Kota, Magelang, Panjalu, Paguyaman, Popayato, Sidrap, Soppeng, and 87 Surabaya regions of Indonesia in 2010-2014, as shown in Fig. 1 and Table 1. 88 Several healthy bats were captured from flocks at each location and euthanized 89 with an overdose of ketamine and xylazine, with the permission of the Directorate 90 General of Livestock and Animal Health Services, Ministry of Agriculture, Republic 91 of Indonesia. All procedures were approved by the Animal Care and Use 92 Committee of the Veterinary Teaching Hospital, Bogor Agricultural University 93 (permit number 05-2010 RSHP-IPB). The samples were stored at -80°C and have 94 been used previously to screen for FBAHV1 and other viruses [12-15]. The bat 95 species were identified according to their morphological characteristics, and a 96 nucleotide sequence analysis of their mitochondrial 16S rRNA and cytochrome b 97 genes, as previously described [12, 15]. The mitochondrial nucleotide sequences 98 of Pteropus sp. showed greatest identity (96% 16S rRNA and 95% cytochrome b 99 genes) to the corresponding sequences of P. hypomelanus (small flying fox; 100 accession numbers AF069537 and AB062472). Therefore, we concluded that 101 these bats were closely related to P. hypomelanus, but should be classified as a 102 different species. 103 Total DNA was extracted from spleen tissue samples with DNAzol (Molecular 104 Research Center) or the QIAamp DNA Mini Kit (Qiagen). We screened for 5 105 herpesviruses with semi-nested PCR using TaKaRa Ex Taq Hot Start Version 106 (Takara Bio) and degenerate primers targeting the DNA polymerase (DPOL) gene 107 of the herpesviruses [16]. This primer set has been used to detect herpesviruses 108 in a broad range of wild animals [17-19]. The PCR products, of approximately 250 109 bp, were purified from the gel after electrophoresis and sequenced directly with 110 the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The 111 sequences were analyzed with BLASTN, and 71 of the 183 tested samples were 112 positive for herpesvirus sequences. The BLASTN results suggested that the 67 of 113 these 71 sequences (172 bp in length) originated from gammaherpesviruses and 114 that the remaining four sequences (181 bp in length) originated from 115 betaherpesviruses, as summarized in Table S1 (available in the online 116 Supplementary Material). Therefore, the origins of these herpesvirus sequences 117 were designated 'megabat gammaherpesvirus' (MgGHV) and 'megabat 118 betaherpesvirus' (MgBHV), respectively. The DPOL sequences determined were 119 deposited in GenBank under accession numbers LC268906-LC268972. The 120 sequences of MgGHV were detected in the bat samples collected at every 121 location that we sampled, suggesting that MgGHVs are geographically distributed 122 throughout Indonesia, as summarized in Figure 1 and Table 1. 123 Because the majority of the sequences belonged to MgGHV, distributed 124 geographically throughout Indonesia, we focused our investigation on MgGHV. A 125 multiple-sequence alignment by CLUSTAL W and a subsequent Maximum 126 likelihood phylogenetic analysis of the partial MgGHV DPOL sequences were 127 performed by the MEGA 7 program [20]. The MgGHVs were phylogenetically 6 128 divided into seven groups (MgGHV Group-1 to -7), as shown in Fig. 2(a). The 129 MgGHV sequences clustered according to the bat species in which they were 130 detected, except MgGHV Group-1, which contained sequences detected in 131 Pteropus sp. and Acerodon celebensis. All of the MgGHV groups were 132 phylogenetically related to previously described gammaherpesviruses detected in 133 Pteropus species. MgGHV Group-1, -2 and -3 formed a cluster with Pteropus 134 giganteus gammaherpesvirus isolate-3 (PgHV-3); MgGHV Group-4 and -5 formed 135 a cluster with PgHV-2; and MgGHV Group-6 was closely related to PgHV-1. 136 These PgHVs were detected in P. giganteus in Bangladesh [21]. MgGHV Group-7 137 contained a single sequence that was identical to that of