Virus Genes (2015) 51:136–139 DOI 10.1007/s11262-015-1197-6
Detection of a novel herpesvirus from bats in the Philippines Kaori Sano1 • Sachiko Okazaki1 • Satoshi Taniguchi2 • Joseph S. Masangkay3 • Roberto Puentespina Jr.4 • Eduardo Eres5 • Edison Cosico5 • Nin˜a Quibod5 • Taisuke Kondo6 • Hiroshi Shimoda7 • Yuuki Hatta1 • Shumpei Mitomo1 • Mami Oba1 • Yukie Katayama1 • Yukiko Sassa8 • Tetsuya Furuya9 • Makoto Nagai8 • Yumi Une10 • Ken Maeda7 • Shigeru Kyuwa6 • Yasuhiro Yoshikawa11 Hiroomi Akashi12 • Tsutomu Omatsu1 • Tetsuya Mizutani1
•
Received: 27 November 2014 / Accepted: 9 April 2015 / Published online: 9 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel batborne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences
of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.
Edited by Keizo Tomonaga. & Shigeru Kyuwa [email protected] & Tetsuya Mizutani [email protected] 1
2
Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan Special Pathogens Laboratory, Department of Virology I, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan
3
College of Veterinary Medicine, University of the Philippines Los Ban˜os, 4031 Los Ban˜os, Laguna, Philippines
4
College of Science, University of the Philippines Mindanao, Bago-Oshiro, Davao, Philippines
5
Museum of Natural History, University of the Philippines Los Ban˜os, 4031 Los Ban˜os, Laguna, Philippines
6
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
123
7
Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
8
Laboratory of Epizootiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
9
Laboratory of Microbiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
10
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan
11
Department of Animal Risk Management, Faculty of Risk and Crisis Management, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan
12
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Virus Genes (2015) 51:136–139
Keywords Bats The Philippines Herpesvirus Next generation sequencing The interest in bats, of the order Chiroptera, has increased since they were identified as a natural host of many emerging viruses such as Nipah virus and Ebola virus [1]. In fact, because of the large number of viruses identified in bats, it is likely that new bat-borne infectious diseases may emerge in the near future [2], thus highlighting the importance of monitoring viruses infecting bats. This information may also be useful to identify natural hosts in an outbreak of a novel infectious disease of bat origin. In this study, next generation sequencing techniques and conventional PCR methods were used to analyze bat samples collected from three locations in the Philippines to investigate which virus species infect bat populations in the region. A total of 70 bats belonging to four megabat species (19 Cynopterus brachyotis, 4 Macroglossus minimus, 7 Ptenochirus jagori, 40 Rousettus amplexicaudatus) and 1 microbat species (Rhinolophus rufus) were collected from three different locations in Davao region, in the Philippines in 2012. Written permission from the regional director of the Department of Environment and Natural Resources (DENR) was obtained to capture bats. The bats were euthanized, and various tissue samples were obtained. DNA was extracted from 70 intestine, 69 lung, and 52 blood clot samples using High Pure viral nucleic acid kits (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. DNA extracts were divided into sample pools (intestine: seven pools, lung: seven pools, blood clot: six pools). DNA libraries of each sample pool were prepared using the TruSeq DNA Sample Prep Kit v2—Set A (Illumina, San Diego, USA) for sequencing using the Genome Analyzer IIx (Illumina, San Diego, USA). A total of 2,000,000–12,000,000 raw reads were obtained from each sample pool. More than 50 contigs were assembled from the reads obtained for each sample pool using CLC Genomics Workbench version 6. All reads had a minimum length of 500 nucleotides (nt). Contigs were mapped to viral genome sequence data downloaded from the NCBI database; they showed similarity to sequences of different viral species belonging to the Retroviridae and Herpesviridae family (E value, \1E-20). As the available sequence data did not allow differentiation of endogenous and exogenous retroviruses, the study reported here is focused on further characterization and detection of the herpesvirus identified. To confirm the presence of herpesvirus in the samples, nested PCR was conducted using consensus primers targeting herpesvirus genes. The PCR conditions were similar to those described previously [3], except for using 1.0 U/50 ll reaction mixture of KOD FX DNA polymerase
137
(TOYOBO Co., Ltd., Osaka, Japan). PCR products were visualized using 1.8 % agarose gels, and amplification of target-sized DNA (210-bp) was observed in one intestine, four lung, and one blood clot samples. Target DNA was purified using the Fast Gene Gel/PCR Extraction Kit (NIPPON Genetics Co. Ltd., Tokyo, Japan) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, New York, USA) and the same set of inner primers for the nested PCR described above. Sequence analysis was performed on the ABI 3130xl Genetic Analyzer (Applied Biosystems, USA). A 166-bp nucleotide sequence was obtained from three lung samples, and the three sequences were 100 % identical (data not shown). The sequences obtained were analyzed using the blastn and tblastx algorithms of the NCBI database. The nucleotide sequences were most identical to a DNA polymerase gene of herpesviruses, Pteropus giganteus herpesvirus-1 (PgHV1) and Pteropus giganteus herpesvirus-3 (PgHV-3) detected from Pteropus giganteus [4], and Elephant gammaherpesvirus 5B [6] with nucleotide identities of 72, 67, and 71 %, respectively. Amino acid sequences were most identical to a DNA polymerase gene of PgHV-1, PgHV-3, and a gammaherpesvirus detected from Rousettus aegyptiacus [5]. In this case, the identities were 81 % for all three nucleotide sequences. A phylogenetic analysis performed using MEGA 6 (Fig. 1) identified that the obtained sequence belonged to the Gammaherpesvirinae cluster. Therefore, the obtained nucleotide sequence was identified as a novel bat herpesvirus gene and deposited in GenBank as Bat herpesvirus pol gene for DNA polymerase, partial cds, isolate: Pja-414 (accession number LC008326). To evaluate the infection rate of this novel herpesvirus among all the bats captured for this study, a set of specific primers were designed from the inner region of the obtained nucleotide sequence as follows: forward primer, 50 TTATTACCGTGCGTGCGC-30 and reverse primer, 50 AAAATTAGCGTGTTCGTCG-30 . The expected amplicon size was 150-bp. PCR reactions were conducted using all intestine, lung, and blood clot samples. The reaction mixture (25 ll) contained 12.5 ll of 29 GoTaq Master Mix (Promega, Madison, WI, USA), 1 ll of DNA template, 0.25 lM of forward and reverse primers each, and nuclease-free water. Thermal cycling conditions were as follows: 95 °C for 2 min, 40 cycles of 95 °C for 1 min, 55 °C for 30 s, and 72 °C for 20 s, followed by a final extension period of 72 °C for 5 min. The PCR products were confirmed to be positive by sequencing as described above. The novel herpesvirus was detected from 20 % of the intestine samples (14/70) and 10 % of the lung (7/69) and blood clot samples (5/52). All three tissues of one animal, P. jagori, were positive for the herpesvirus by PCR. In
123
138
Virus Genes (2015) 51:136–139 Psittacid herpesvirus 1(AY372243.1) 61 Gallid herpesvirus 2(AF147806.2) Alphaherpesvirinae
Human herpesvirus 1(AB070847)
21 33
Equid herpesvirus 8(JQ343919.1) Human herpesvirus 6B(AB021506)
86
Human herpesvirus 7(NC001716)
Betaherpesvirinae 99
Human herpesvirus 5(AB329634) 69
Rhesus cytomegalovirus(AF033184)
Elephant gammaherpesvirus 5B(JX268522.1)
49
Ptenochirus jagori Ptenochirus jagori
herpesvirus
Kaposis sarcoma-associated herpesvirus(AF005477.2)
85
Gammaherpesvirinae
Equid herpesvirus 5(JX125459.1)
86 91
Equid herpesvirus 2(HQ247775.1)
0.1
Fig. 1 Herpesvirus phylogenetic tree based on a 166-bp partial DNAdependent DNA polymerase gene. The virus detected in this study is highlighted in gray. The tree was constructed using the maximum likelihood method with 1000 bootstrap repetitions. The tree with the
highest log likelihood (-735.5090) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches
total, 30 % (21/71) of the bats analyzed were positive for the herpesvirus sequence that was detected either in intestine, lung, or blood clot samples. This infection rate changed with the sampling site and ranged between 28 and 33 %. The infection rate for each bat species was 16 % for C. brachyotis (3/19), 25 % for M. minimus (1/4), 71 % for P. jagori (5/7), 28 % for R. amplexicaudatus (11/40), and 100 % for R. rufus (1/1). These results indicated that this novel herpesvirus may be widely distributed in the region where bat sampling was conducted. In this study, we present the partial genome of a novel herpesvirus detected from bats captured in Davao region using next generation sequencing techniques and PCR. This virus may be widely infecting bat populations in the region because 30 % of the bats captured were positive for the herpesvirus. Phylogenetic analysis indicated that this virus belongs to the subfamily Gammaherpesvirinae. Although each virus in the family Herpesviridae has a restricted host range, interspecies transmission of herpesviruses leading to severe clinical symptoms has been reported [7, 8]. Gammaherpesviruses are known to cause diseases including cancer in many animal species, and there are reports indicating interspecies transmission of gammaherpesviruses [9–11]. Thus, it is possible that this herpesvirus may emerge as a novel disease if a spillover event to other animal species occurs. Therefore, further
studies to verify the pathogenicity of this novel virus are recommended.
123
Acknowledgments This work was supported by a grant from the Ministry of Education, Science, Sports, and Culture, Japan (JSPS KAKENHI Grant Number 25304043).
References 1. C.H. Calisher, J.E. Childs, H.E. Field, K.V. Holmes, T. Schountz, Clin. Microbiol. Rev. 19, 531 (2006) 2. S. Wong, S. Lau, P. Woo, K.Y. Yuen, Rev. Med. Virol. 17, 67 (2007) 3. D.R. VanDevanter, P. Warrener, L. Bennett, E.R. Schultz, S. Coulter, R.L. Garber, T.M. Rose, J. Clin. Microbiol. 34, 1666 (1996) 4. S.J. Anthony, J.H. Epstein, K.A. Murray, I. Navarrete-Macias, C.M. Zambrana-Torrelio, A. Solovyov, R. Ojeda-Flores, N.C. Arrigo, A. Islam, S. Ali Khan, P. Hosseini, T.L. Bogich, K.J. Olival, M.D. Sanchez-Leon, W.B. Karesh, T. Goldstein, S.P. Luby, S.S. Morse, J.A.K. Mazet, P. Daszak, W.I. Lipkin, MBio. 4, e00598 (2013) 5. M. Ja´noska, M. Vidovszky, V. Molna´r, M. Liptovszky, B. Harrach, M. Benko, Vet. J. 189, 118 (2011) 6. E. Latimer, J. Zong, S. Heaggans, L. Richman, G. Hayward, Vet. Microbiol. 147, 28 (2011) 7. A. Abdelgawad, W. Azab, A.M. Damiani, K. Baumgartner, H. Will, N. Osterrieder, A.D. Greenwood, Vet. Microbiol. 169, 102 (2014)
Virus Genes (2015) 51:136–139 8. M.D. Schrenzel, T.A. Tucker, T.A. Donovan, M.D.M. Busch, A.G. Wise, R.K. Maes, M. Kiupel, Emerg. Infect. Dis. 14, 1616 (2008) 9. B. Ehlers, G. Dural, N. Yasmum, T. Lembo, B. de Thoisy, M.-P. RyserDegiorgis, R.G. Ulrich, D.J. McGeoch, J. Virol. 82, 3509 (2008) 10. B. Ehlers, K. Spiess, F. Leendertz, M. Peeters, C. Boesch, D. Gatherer, D.J. McGeoch, J. Gen. Virol. 91, 630 (2010)
139 11. R.M. Troyer, J.A. Beatty, K.R. Stutzman-Rodriguez, S. Carver, C.C. Lozano, J.S. Lee, M.R. Lappin, S.P.D. Riley, L.E.K. Serieys, K.A. Logan, L.L. Sweanor, W.M. Boyce, T.W. Vickers, R. McBride, K.R. Crooks, J.S. Lewis, M.W. Cunningham, J. Rovnak, S.L. Quackenbush, S. VandeWoude, J. Virol. 88, 3914 (2014)
123
Detection of a novel herpesvirus from bats in the Philippines Kaori Sano1 • Sachiko Okazaki1 • Satoshi Taniguchi2 • Joseph S. Masangkay3 • Roberto Puentespina Jr.4 • Eduardo Eres5 • Edison Cosico5 • Nin˜a Quibod5 • Taisuke Kondo6 • Hiroshi Shimoda7 • Yuuki Hatta1 • Shumpei Mitomo1 • Mami Oba1 • Yukie Katayama1 • Yukiko Sassa8 • Tetsuya Furuya9 • Makoto Nagai8 • Yumi Une10 • Ken Maeda7 • Shigeru Kyuwa6 • Yasuhiro Yoshikawa11 Hiroomi Akashi12 • Tsutomu Omatsu1 • Tetsuya Mizutani1
•
Received: 27 November 2014 / Accepted: 9 April 2015 / Published online: 9 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel batborne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences
of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.
Edited by Keizo Tomonaga. & Shigeru Kyuwa [email protected] & Tetsuya Mizutani [email protected] 1
2
Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan Special Pathogens Laboratory, Department of Virology I, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan
3
College of Veterinary Medicine, University of the Philippines Los Ban˜os, 4031 Los Ban˜os, Laguna, Philippines
4
College of Science, University of the Philippines Mindanao, Bago-Oshiro, Davao, Philippines
5
Museum of Natural History, University of the Philippines Los Ban˜os, 4031 Los Ban˜os, Laguna, Philippines
6
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
123
7
Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
8
Laboratory of Epizootiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
9
Laboratory of Microbiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
10
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan
11
Department of Animal Risk Management, Faculty of Risk and Crisis Management, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan
12
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Virus Genes (2015) 51:136–139
Keywords Bats The Philippines Herpesvirus Next generation sequencing The interest in bats, of the order Chiroptera, has increased since they were identified as a natural host of many emerging viruses such as Nipah virus and Ebola virus [1]. In fact, because of the large number of viruses identified in bats, it is likely that new bat-borne infectious diseases may emerge in the near future [2], thus highlighting the importance of monitoring viruses infecting bats. This information may also be useful to identify natural hosts in an outbreak of a novel infectious disease of bat origin. In this study, next generation sequencing techniques and conventional PCR methods were used to analyze bat samples collected from three locations in the Philippines to investigate which virus species infect bat populations in the region. A total of 70 bats belonging to four megabat species (19 Cynopterus brachyotis, 4 Macroglossus minimus, 7 Ptenochirus jagori, 40 Rousettus amplexicaudatus) and 1 microbat species (Rhinolophus rufus) were collected from three different locations in Davao region, in the Philippines in 2012. Written permission from the regional director of the Department of Environment and Natural Resources (DENR) was obtained to capture bats. The bats were euthanized, and various tissue samples were obtained. DNA was extracted from 70 intestine, 69 lung, and 52 blood clot samples using High Pure viral nucleic acid kits (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. DNA extracts were divided into sample pools (intestine: seven pools, lung: seven pools, blood clot: six pools). DNA libraries of each sample pool were prepared using the TruSeq DNA Sample Prep Kit v2—Set A (Illumina, San Diego, USA) for sequencing using the Genome Analyzer IIx (Illumina, San Diego, USA). A total of 2,000,000–12,000,000 raw reads were obtained from each sample pool. More than 50 contigs were assembled from the reads obtained for each sample pool using CLC Genomics Workbench version 6. All reads had a minimum length of 500 nucleotides (nt). Contigs were mapped to viral genome sequence data downloaded from the NCBI database; they showed similarity to sequences of different viral species belonging to the Retroviridae and Herpesviridae family (E value, \1E-20). As the available sequence data did not allow differentiation of endogenous and exogenous retroviruses, the study reported here is focused on further characterization and detection of the herpesvirus identified. To confirm the presence of herpesvirus in the samples, nested PCR was conducted using consensus primers targeting herpesvirus genes. The PCR conditions were similar to those described previously [3], except for using 1.0 U/50 ll reaction mixture of KOD FX DNA polymerase
137
(TOYOBO Co., Ltd., Osaka, Japan). PCR products were visualized using 1.8 % agarose gels, and amplification of target-sized DNA (210-bp) was observed in one intestine, four lung, and one blood clot samples. Target DNA was purified using the Fast Gene Gel/PCR Extraction Kit (NIPPON Genetics Co. Ltd., Tokyo, Japan) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, New York, USA) and the same set of inner primers for the nested PCR described above. Sequence analysis was performed on the ABI 3130xl Genetic Analyzer (Applied Biosystems, USA). A 166-bp nucleotide sequence was obtained from three lung samples, and the three sequences were 100 % identical (data not shown). The sequences obtained were analyzed using the blastn and tblastx algorithms of the NCBI database. The nucleotide sequences were most identical to a DNA polymerase gene of herpesviruses, Pteropus giganteus herpesvirus-1 (PgHV1) and Pteropus giganteus herpesvirus-3 (PgHV-3) detected from Pteropus giganteus [4], and Elephant gammaherpesvirus 5B [6] with nucleotide identities of 72, 67, and 71 %, respectively. Amino acid sequences were most identical to a DNA polymerase gene of PgHV-1, PgHV-3, and a gammaherpesvirus detected from Rousettus aegyptiacus [5]. In this case, the identities were 81 % for all three nucleotide sequences. A phylogenetic analysis performed using MEGA 6 (Fig. 1) identified that the obtained sequence belonged to the Gammaherpesvirinae cluster. Therefore, the obtained nucleotide sequence was identified as a novel bat herpesvirus gene and deposited in GenBank as Bat herpesvirus pol gene for DNA polymerase, partial cds, isolate: Pja-414 (accession number LC008326). To evaluate the infection rate of this novel herpesvirus among all the bats captured for this study, a set of specific primers were designed from the inner region of the obtained nucleotide sequence as follows: forward primer, 50 TTATTACCGTGCGTGCGC-30 and reverse primer, 50 AAAATTAGCGTGTTCGTCG-30 . The expected amplicon size was 150-bp. PCR reactions were conducted using all intestine, lung, and blood clot samples. The reaction mixture (25 ll) contained 12.5 ll of 29 GoTaq Master Mix (Promega, Madison, WI, USA), 1 ll of DNA template, 0.25 lM of forward and reverse primers each, and nuclease-free water. Thermal cycling conditions were as follows: 95 °C for 2 min, 40 cycles of 95 °C for 1 min, 55 °C for 30 s, and 72 °C for 20 s, followed by a final extension period of 72 °C for 5 min. The PCR products were confirmed to be positive by sequencing as described above. The novel herpesvirus was detected from 20 % of the intestine samples (14/70) and 10 % of the lung (7/69) and blood clot samples (5/52). All three tissues of one animal, P. jagori, were positive for the herpesvirus by PCR. In
123
138
Virus Genes (2015) 51:136–139 Psittacid herpesvirus 1(AY372243.1) 61 Gallid herpesvirus 2(AF147806.2) Alphaherpesvirinae
Human herpesvirus 1(AB070847)
21 33
Equid herpesvirus 8(JQ343919.1) Human herpesvirus 6B(AB021506)
86
Human herpesvirus 7(NC001716)
Betaherpesvirinae 99
Human herpesvirus 5(AB329634) 69
Rhesus cytomegalovirus(AF033184)
Elephant gammaherpesvirus 5B(JX268522.1)
49
Ptenochirus jagori Ptenochirus jagori
herpesvirus
Kaposis sarcoma-associated herpesvirus(AF005477.2)
85
Gammaherpesvirinae
Equid herpesvirus 5(JX125459.1)
86 91
Equid herpesvirus 2(HQ247775.1)
0.1
Fig. 1 Herpesvirus phylogenetic tree based on a 166-bp partial DNAdependent DNA polymerase gene. The virus detected in this study is highlighted in gray. The tree was constructed using the maximum likelihood method with 1000 bootstrap repetitions. The tree with the
highest log likelihood (-735.5090) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches
total, 30 % (21/71) of the bats analyzed were positive for the herpesvirus sequence that was detected either in intestine, lung, or blood clot samples. This infection rate changed with the sampling site and ranged between 28 and 33 %. The infection rate for each bat species was 16 % for C. brachyotis (3/19), 25 % for M. minimus (1/4), 71 % for P. jagori (5/7), 28 % for R. amplexicaudatus (11/40), and 100 % for R. rufus (1/1). These results indicated that this novel herpesvirus may be widely distributed in the region where bat sampling was conducted. In this study, we present the partial genome of a novel herpesvirus detected from bats captured in Davao region using next generation sequencing techniques and PCR. This virus may be widely infecting bat populations in the region because 30 % of the bats captured were positive for the herpesvirus. Phylogenetic analysis indicated that this virus belongs to the subfamily Gammaherpesvirinae. Although each virus in the family Herpesviridae has a restricted host range, interspecies transmission of herpesviruses leading to severe clinical symptoms has been reported [7, 8]. Gammaherpesviruses are known to cause diseases including cancer in many animal species, and there are reports indicating interspecies transmission of gammaherpesviruses [9–11]. Thus, it is possible that this herpesvirus may emerge as a novel disease if a spillover event to other animal species occurs. Therefore, further
studies to verify the pathogenicity of this novel virus are recommended.
123
Acknowledgments This work was supported by a grant from the Ministry of Education, Science, Sports, and Culture, Japan (JSPS KAKENHI Grant Number 25304043).
References 1. C.H. Calisher, J.E. Childs, H.E. Field, K.V. Holmes, T. Schountz, Clin. Microbiol. Rev. 19, 531 (2006) 2. S. Wong, S. Lau, P. Woo, K.Y. Yuen, Rev. Med. Virol. 17, 67 (2007) 3. D.R. VanDevanter, P. Warrener, L. Bennett, E.R. Schultz, S. Coulter, R.L. Garber, T.M. Rose, J. Clin. Microbiol. 34, 1666 (1996) 4. S.J. Anthony, J.H. Epstein, K.A. Murray, I. Navarrete-Macias, C.M. Zambrana-Torrelio, A. Solovyov, R. Ojeda-Flores, N.C. Arrigo, A. Islam, S. Ali Khan, P. Hosseini, T.L. Bogich, K.J. Olival, M.D. Sanchez-Leon, W.B. Karesh, T. Goldstein, S.P. Luby, S.S. Morse, J.A.K. Mazet, P. Daszak, W.I. Lipkin, MBio. 4, e00598 (2013) 5. M. Ja´noska, M. Vidovszky, V. Molna´r, M. Liptovszky, B. Harrach, M. Benko, Vet. J. 189, 118 (2011) 6. E. Latimer, J. Zong, S. Heaggans, L. Richman, G. Hayward, Vet. Microbiol. 147, 28 (2011) 7. A. Abdelgawad, W. Azab, A.M. Damiani, K. Baumgartner, H. Will, N. Osterrieder, A.D. Greenwood, Vet. Microbiol. 169, 102 (2014)
Virus Genes (2015) 51:136–139 8. M.D. Schrenzel, T.A. Tucker, T.A. Donovan, M.D.M. Busch, A.G. Wise, R.K. Maes, M. Kiupel, Emerg. Infect. Dis. 14, 1616 (2008) 9. B. Ehlers, G. Dural, N. Yasmum, T. Lembo, B. de Thoisy, M.-P. RyserDegiorgis, R.G. Ulrich, D.J. McGeoch, J. Virol. 82, 3509 (2008) 10. B. Ehlers, K. Spiess, F. Leendertz, M. Peeters, C. Boesch, D. Gatherer, D.J. McGeoch, J. Gen. Virol. 91, 630 (2010)
139 11. R.M. Troyer, J.A. Beatty, K.R. Stutzman-Rodriguez, S. Carver, C.C. Lozano, J.S. Lee, M.R. Lappin, S.P.D. Riley, L.E.K. Serieys, K.A. Logan, L.L. Sweanor, W.M. Boyce, T.W. Vickers, R. McBride, K.R. Crooks, J.S. Lewis, M.W. Cunningham, J. Rovnak, S.L. Quackenbush, S. VandeWoude, J. Virol. 88, 3914 (2014)
123
