Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

  • Michihito Sasaki1Email author,

    Affiliated with

    • Agus Setiyono3Email author,

      Affiliated with

      • Ekowati Handharyani3Email author,

        Affiliated with

        • Ibenu Rahmadani4,

          Affiliated with

          • Siswatiana Taha5,

            Affiliated with

            • Sri Adiani6,

              Affiliated with

              • Mawar Subangkit3,

                Affiliated with

                • Hirofumi Sawa1,

                  Affiliated with

                  • Ichiro Nakamura2 and

                    Affiliated with

                    • Takashi Kimura1Email author

                      Affiliated with

                      Virology Journal20129:240

                      DOI: 10.1186/1743-422X-9-240

                      Received: 3 May 2012

                      Accepted: 17 October 2012

                      Published: 19 October 2012

                      Abstract

                      Background

                      Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia.

                      Methods

                      RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes.

                      Results

                      Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus.

                      Conclusions

                      This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia.

                      Background

                      The genus Henipavirus in the subfamily Paramyxovirinae, family Paramyxoviridae, contains two highly pathogenic viruses, i.e., Hendra virus and Nipah virus. Hendra virus causes fatal pneumonia and encephalitis in horses and humans. The first case was identified in 1994 and Hendra virus disease still continues to arise sporadically in Australia [1, 2]. Nipah virus also causes acute encephalitis and respiratory symptoms in animals and humans, with a high mortality rate. Outbreaks of Nipah virus have occurred in Malaysia, Singapore, Bangladesh, and India [1, 2]. Henipaviruses have been isolated from fruit bats including Pteropus vampyrus[3], Pteropus hypomelanus[4], Pteropus lylei[5], Pteropus poliocephalus, and Pteropus alecto[6], which are considered to be their natural reservoirs. Epidemiological studies demonstrate that Hendra and/or Nipah virus-seropositive fruit bats are widely distributed throughout Asian countries [711]. No human cases of henipavirus infection have been reported in Indonesia, although Pteropus vampyrus that are seropositive for both Nipah virus and Hendra virus are distributed nationwide [12, 13]. These findings indicate the presence of henipavirus or henipa-like viruses in Indonesian fruit bats, suggesting the need for further epidemiological investigations.

                      Menangle virus, belonging to the genus Rubulavirus of the Paramyxoviridae family, has been identified in pteropus bats from Australia [14]. Menangle virus is a zoonotic paramyxovirus that causes febrile illness with rash in humans [15]. Tioman virus, belonging to the genus Rubulavirus, has also been isolated from Pteropus hypomelanus on the island of Tioman, Malaysia [16]. Although Tioman virus showed antigenic cross-reactivity to Menangle virus, the pathogenicity of Tioman virus remains unclear. There have been no reports of rubulavirus infections in the Indonesian fruit bat population.

                      The current study used molecular sequencing and phylogenetic analyses to identify RNA sequence from potential paramyxoviruses in fruit bats from Indonesia.

                      Results

                      A total of 110 fruit bats belonging to four different species were sampled from four locations in Indonesia (Figure  1). Pteropus vampyrus was captured in Panjalu District (n = 26) and Lima Puluh Kota District (n = 20). Other pteropus bats captured in Popayato District (n = 4) and Paguyaman District (n = 25) were considered to be closely related to Pteropus hypomelanus, based on the shared nucleotide sequence identity of their 16S rRNA (96%) and cytochrome b (cyt b) (95%) with corresponding sequences from Pteropus hypomelanus (GenBank/EMBL/DDBJ entry AF069537 and AB062472). Acerodon celebensis was captured in Paguyaman District (n = 18). Dobsonia bats that were captured in Paguyaman District (n = 17) had high sequence similarity with 16S rRNA (96%) and cyt b (94%) from Dobsonia moluccensis (JN398196 and FJ218484). Information on the samples is summarized in Table  1.
                      http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-9-240/MediaObjects/12985_2012_1884_Fig1_HTML.jpg
                      Figure 1

                      Map of the sampling locations in Indonesia.

                      Table 1

                      Sample information and result of the semi-nested RT-PCR

                      Species

                      Location collected

                      Year collected

                      Nested RT-PCR

                         

                      Number of analyzed

                      Number of positive

                      Pteropus vampyrus

                      Panjalu district

                      2010

                      26

                      1

                       

                      Lima Puluh Kota district

                      2011

                      20

                      0

                      Pteropus sp.*

                      Popayato district

                      2011

                      4

                      0

                       

                      Paguyaman district

                      2011

                      23

                      4

                        

                      2012

                      2

                      0

                      Acerodon celebensis

                      Paguyaman district

                      2012

                      18

                      1

                      Dobsonia sp.**

                      Paguyaman district

                      2012

                      17

                      0

                      *Genetically closely related to Pteropus hypomelanus.

                      **Genetically closely related to Dobsonia moluccensis.

                      RNA samples from each fruit bat spleen were screened using semi-nested broad spectrum reverse transcription PCR (RT-PCR), as described previously [17]. The primers were designed based on a conserved sequence within the RNA polymerase large (L) gene of the Paramyxovirinae subfamily, which includes Avulavirus, Rubulavirus, Respirovirus, Morbillivirus, and Henipavirus[17]. Semi-nested RT-PCR was positive for 1/26 (4%) Pteropus vampyrus specimens captured in Panjalu District. The size of PCR product detected in the positive sample (sample number IFBPV01/2010) was 584 bp, and the amplified viral sequence excluding the primer-derived sequences (530 bp) was deposited in GenBank (accession number AB691542). Positive results with amplification of the 530 bp viral sequence (excluding the primer-derived sequences) were also obtained for 4/25 (16%) Pteropus sp. captured in Paguyaman District, i.e., IFBPV25/2011 (AB691543), IFBPV32/2011 (AB691544), IFBPV39/2011 (AB691545), and IFBPV46/2011 (AB691546), and for 1/18 (6%) Acerodon celebensis specimens captured in Paguyaman District, i.e., IFBPV32/2012 (AB710472). No positive results were obtained for the 20 Pteropus vampyrus captured in Lima Puluh Kota District, the four Pteropus sp. captured in Popayato District, or the 17 Dobsonia sp. captured in Paguyaman District (Table  1).

                      BLAST search showed that all six amplicons shared less than 65% nucleotide identity with homologous fragments of paramyxovirus sequences previously deposited in GenBank. Deduced pairwise amino acid identities were then calculated to compare the homologous region with known paramyxovirus L proteins (Table  2). IFBPV32/2011 shared 98% nucleotide identity and 100% amino acid identity with IFBPV39/2011, suggesting that they belonged to the same strain. IFBPV01/2010, IFBPV32/2011, IFBPV39/2011, and IFBPV46/2011 were most closely related to Nipah virus of all the known paramyxoviruses. IFBPV25/2011 shared 72% amino acid sequence identity with Tuhoko virus 2, which was isolated from Rousettus leschenaulti in China [18]. IFBPV32/2012 shared 78% amino acid sequence identity with Tioman virus.
                      Table 2

                      Pairwise amino acid identities of predicted L gene products compared with known paramyxoviruses

                      Genus

                      Species

                      Percentage of amino acid sequence identity

                        

                      IFBPV01/2010

                      IFBPV25/2011

                      IFBPV32/2011, IFBPV39/2011

                      IFBPV46/2011

                      IFBPV32/2012

                       

                      IFBPV01/2010

                       

                      37

                      90

                      66

                      35

                       

                      IFBPV25/2011

                      37

                       

                      36

                      38

                      70

                       

                      IFBPV32/2011, IFBPV39/2011

                      90

                      36

                       

                      65

                      36

                       

                      IFBPV46/2011

                      66

                      38

                      65

                       

                      38

                       

                      IFBPV32/2012

                      35

                      70

                      36

                      38

                       

                      Avulavirus

                      Avian paramyxovirus 3

                      29

                      36

                      30

                      34

                      35

                       

                      Newcastle disease virus

                      34

                      37

                      34

                      36

                      40

                      Rubulavirus

                      Mapuera virus

                      41

                      65

                      40

                      40

                      64

                       

                      Menangle virus

                      34

                      65

                      33

                      37

                      77

                       

                      Mumps virus

                      38

                      65

                      36

                      40

                      68

                       

                      Tioman virus

                      34

                      69

                      34

                      37

                      78

                       

                      Tuhoko virus 2

                      38

                      72

                      36

                      38

                      72

                      Respirovirus

                      Human parainfluenza virus 1

                      51

                      35

                      49

                      52

                      34

                       

                      Human parainfluenza virus 3

                      51

                      37

                      51

                      52

                      35

                       

                      Sendai virus

                      51

                      35

                      49

                      51

                      34

                      Morbillivirus

                      Canine distemper virus

                      57

                      37

                      56

                      55

                      34

                       

                      Measles virus

                      58

                      37

                      57

                      56

                      37

                       

                      Rinderpest virus

                      57

                      36

                      57

                      56

                      38

                      Henipavirus

                      Hendra Virus

                      67

                      36

                      66

                      69

                      35

                       

                      Nipah Virus

                      70

                      36

                      69

                      70

                      35

                      Unclassified

                      J-virus

                      61

                      38

                      62

                      60

                      38

                      A phylogenetic analysis was performed based on the deduced amino acid sequences (176 amino acids) from the six nucleotide sequences obtained (Figure  2). The phylogenetic tree showed that IFBPV01/2010, IFBPV32/2011, IFBPV39/2011, and IFBPV46/2011 formed three distinct branches that were closely related to the genus Henipavirus. IFBPV25/2011 and IFBPV32/2012 were most closely related to the genus Rubulavirus.
                      http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-9-240/MediaObjects/12985_2012_1884_Fig2_HTML.jpg
                      Figure 2

                      Phylogenetic analysis of amino acid sequences derived from partial L gene fragments. The phylogenetic tree was constructed based on the 176 amino acid sequences deduced from the partial L gene fragments identified in the present study (gray shade) and homologous sequences from known paramyxoviruses. The bootstrap values obtained after 1000 replicates are indicated at each branch. Scale bars indicate amino acid substitutions per site.

                      We also amplified other regions of L gene by using different degenerate primer sets for Morbillivirus Respirovirus Henipavirus subgroup or Rubulavirus Avulavirus subgroup [17]. The partial viral sequences measuring 439 bp (excluding the primer-derived sequences) was obtained from four henipavirus-like RNA-positive samples, i.e., IFBPV01/2010 (AB748559), IFBPV32/2011 (AB748560), IFBPV39/2011 (AB748560), and IFBPV46/2011 (AB748561). A phylogenetic analysis on the deduced amino acid sequences showed that IFBPV01/2010, IFBPV32/2011, IFBPV39/2011 and IFBPV46/2011 were related to the genus Henipavirus (Additional file 1). The viral sequence measuring 169 bp (excluding the primer-derived sequences) was obtained from two rubulavirus-like RNA-positive samples, i.e., IFBPV25/2011 (AB691543) and IFBPV32/2012 (AB710472). Phylogenetic analysis performed on the deduced amino acid sequences showed that IFBPV25/2011 and IFBPV32/2012 were related to the genus Rubulavirus (Additional file 2).

                      The amino acid sequence GDNQ is highly conserved in the viral RNA polymerase of non-segmented negative-stranded RNA viruses and it is responsible for polymerase activity [19, 20]. However, this motif is replaced by GDNE in the L protein of Henipavirus[20, 21]. The region encoding the GDNQ/GDNE motif was amplified by RT-PCR to determine whether the putative henipavirus-like nucleotide sequences contained the characteristic GDNE motif in the L protein. The deduced amino acid sequence comparison showed that only IFBPV46/2011 encoded the GDNE motif, among the six samples obtained (Figure  3).
                      http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-9-240/MediaObjects/12985_2012_1884_Fig3_HTML.jpg
                      Figure 3

                      Amino acid alignment of the GDNQ/GDNE motif. An amino acid alignment of the GDNQ/GDNE motif was constructed based on the sequences of nonsegmented negative-stranded RNA virus RNA polymerase, including paramyxovirus L protein. The conserved GDNQ/GDNE sequences are boxed while GDNE is also shaded in gray.

                      Virus isolation was attempted using African green monkey kidney (Vero) and rabbit kidney (RK13) cells because these cell lines are used for the isolation or propagation of various paramyxoviruses [3, 4, 6, 16, 22]. After serial passages, an RT-PCR assay detected no paramyxovirus RNA in the culture supernatants (data not shown).

                      Discussion

                      Four henipavirus-like and two rubulavirus-like nucleic acid sequences were detected in fruit bats from Indonesia. The phylogenetic analysis showed that these novel viral sequences possessed considerable sequence divergence, suggesting that a variety of paramyxoviruses are circulating in the Indonesian fruit bat population.

                      In addition to fruit bats of the genus Pteropus, partial paramyxovirus sequences were identified from fruit bats of the genus Eidolon, Rousettus and Epomophorus[18, 23, 24]. To our knowledge, this is the first study that has detected paramyxovirus RNA from fruit bats of the genus Acerodon. This finding broadens the number of megabat genera which are associated with paramyxoviruses.

                      Tuhoko virus, Tioman virus, Menangle virus, and Mapuera virus have been identified as fruit bat-associated rubulaviruses. Menangle virus causes central nervous system degeneration in pigs and it also infects humans [14, 15]. Therefore, it would be useful to investigate infections of humans or domestic animals with the novel rubula-like viruses detected in this study.

                      Conclusions

                      This study identified unique paramyxovirus sequence from three species of fruit bats (Pteropus vampyrus, Pteropus hypomelanus and Acerodon celebensis), potentially representing three new henipaviruses and two new rubulaviruses. To the best of our knowledge, this is the first study to identify viral genome sequence from potential paramyxoviruses in the tissues of fruit bats from Indonesia. Local people consume bat meat in Indonesia, so further epidemiological and experimental studies are needed to determine the risk of fruit bat-associated paramyxovirus infection of humans in Indonesia.

                      Methods

                      Animal samples and RNA extraction

                      Fruit bats were captured in: Panjalu District, West Java Province during February, 2010 (n = 26); Lima Puluh Kota District, West Sumatra Province during February, 2011 (n = 20); Popayato District, Gorontalo Province during February, 2011 (n = 4); and Paguyaman District, Gorontalo Province during February, 2011 and February, 2012 (n = 23 and n = 37, respectively). All animal research was performed in accordance with the ethical guidelines of the Animal Care and Use Committee of Veterinary Teaching Hospital, Bogor Agricultural University. All of the spleen tissues from fruit bats were divided into two samples. Samples for RNA extraction were stored in RNAlater (Life Technologies, Carlsbad, CA), followed by RNA extraction with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Samples for virus isolation were frozen at −80°C. These samples were exported with the permission of the Directorate General of Livestock and Animal Health Services, Ministry of Agriculture, Republic of Indonesia.

                      Species identification

                      Fruit bats were identified based on morphological characters and nucleotide sequence analysis of their mitochondrial 16S rRNA using the primer set 12l-f (5-AGAGGAGAYAAGTCGTAMCAAG-3) [25] and 16q-r (5-GTTTGCCGAGTTCCTTTTAC-3) [25], and mitochondrial cyt b using the primer set Ptecytb-26 (5-TTGTATTTCAACTACARGAAC-3) designed in this study and H15149p (5-CTGCAGCCCCTCAGAATGATATTTGTCCTC-3) [26].

                      RT-PCR

                      RNA samples were screened for paramyxoviruses using semi-nested RT-PCR, as described previously [17]. The primer annealing temperature of the PCR programs was modified to 48°C. The degenerate primers used for amplification of the L gene of the Paramyxovirinae subfamily were as follows: for one-step RT-PCR, PAR-F1 and PAR-R; for semi-nested PCR, PAR-F2 and PAR-R [17]. The upstream region of L gene of the Respirovirus-Morbillivirus Henipavirus subgroup or Avulavirus Rubulavirus subgroup were amplified by using the following degenerate primer sets: RES-MOR-HEN F1, RES-MOR-HEN F2 and RES-MOR-HEN R, or AVU-RUB F1, AVU-RUB F2 and AVU-RUB R, respectively [17]. PCR products were electrophoresed on 1.6% agarose gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Direct cycle sequence reactions were performed in both directions using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and analyzed with an ABI Prism 3130 genetic analyzer (Life Technologies).

                      The nucleotide sequence encoding the GDNQ/GDNE motif from each sample was amplified using a SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Life Technologies). The inner primer PAR-F2 used in the semi-nested PCR was designed for a nucleotide sequence encoding this GDNQ/GDNE motif, so one-step RT-PCR was performed with the outer forward primer PAR-F1 and reverse primers specifically for each sample.

                      Phylogenetic analysis

                      The obtained nucleotide sequences and the deduced amino acid sequences were compared with those of known paramyxoviruses. Nucleotide and amino acid identity values were calculated using GENETYX software ver. 10 (GENETYX, Tokyo, Japan). Multiple sequence alignments were constructed based on the amino acid sequences deduced from the six nucleotide sequences obtained using the MEGA5 program [27]. Phylogenetic analysis was performed using the neighbor-joining method with 1000 bootstrap replicates [28, 29].

                      Viral isolation

                      Virus isolation was attempted in the biosafety level (BSL)-3 facility at the Research Center for Zoonosis Control, Hokkaido University. Frozen spleen tissues were homogenized (10%, wt/vol) in MEM containing penicillin (100 units/ml), streptomycin (100 μg/ml), and fungizone (2.5 μg/ml) (all obtained from Life Technologies). The homogenates were clarified by centrifugation at 1000 × g for 5 min and inoculated onto Vero cells and RK13 cells for 2 h at 37°C. Cells were washed with phosphate-buffered saline (−) and cultured with MEM containing 2% fatal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) and fungizone (2.5 μg/ml). All cells were subcultured every 5 or 6 days. After three serial passages, RNA samples were prepared from each culture supernatant using a High Pure Viral RNA Kit (Roche Diagnostics, Mannheim, Germany) and analyzed by semi-nested RT-PCR, as described above.

                      Accession number

                      The amplified nucleotide sequences reported in this study have been deposited in the GenBank nucleotide database under accession numbers AB691542 to AB691546, AB748559 to AB748561 and AB710472. AB691543 and AB710472 are composed of three overlapping sequences that were amplified using three primer sets; PAR-F2 and PAR-R, AVU-RUB F2 and AVU-RUB R, and the primers for the amplification of GDNQ/GDNE motif.

                      The GenBank/EMBL/DDBJ accession numbers of the amino acid sequences used in this study were ACB46872 (Avian paramyxovirus 3), NP_071471 (Newcastle disease virus), NP_054714 (Mumps virus), YP_001249278 (Mapuera virus), YP_415514 (Menangle virus), NP_665871 (Tioman virus), ADI80722 (Tuhoko virus 2), NP_056879 (Sendai virus), NP_604442 (Human parainfluenza virus 1), NP_067153 (Human parainfluenza virus 3), AAA75501 (Measles virus), YP_087126 (Rinderpest virus), NP_047207 (Canine distemper virus), NP_047113 (Hendra virus), NP_112028 (Nipah virus), and AAX86035 (J-virus), NP_056797 (Rabies virus), NP_478343 (Australian bat lyssavirus), NP_066251 (Ebola virus), and ACT79225 (Marburg virus).

                      Declarations

                      Acknowledgments

                      This study was supported by the Japan Initiative for Global Research Network of Infectious Diseases (J-GRID) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan.

                      Authors’ Affiliations

                      (1)
                      Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University
                      (2)
                      Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University
                      (3)
                      Laboratory of Veterinary Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University
                      (4)
                      Veterinary Investigation and Diagnostic Center
                      (5)
                      Faculty of Agriculture, Gorontalo State University
                      (6)
                      Faculty of Animal Husbandry, Sam Ratulangi University

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                      Copyright

                      © Sasaki et al.; licensee BioMed Central Ltd. 2012

                      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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