Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

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 [7][8][9][10][11]. 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 * Correspondence: kimura@czc.hokudai.ac.jp † Equal contributors 1 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. 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).  (Table 1).
BLAST search showed that all six amplicons shared less than 65% nucleotide identity with homologous fragments of paramyxovirus sequences previously deposited in Gen-Bank. 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.  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. 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.
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. 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).
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 batassociated 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.

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 Super-Script 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 neighborjoining 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.