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Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean
Virus Research 170 (2012) 159–163
Contents lists available at SciVerse ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean David A. Wilkinson a,b,c,∗ , Sarah Temmam a,d,1 , Camille Lebarbenchon a,b,1 , Erwan Lagadec a,d,e,1 , Julien Chotte a,c,1 , Julia Guillebaud a,c,1 , Beza Ramasindrazana g , Jean-Michel Héraud i , Xavier de Lamballerie f , Steven M. Goodman g,h , Koussay Dellagi a,c , Hervé Pascalis a,c,∗ a
Centre de Recherche et de Veille sur les Maladies émergentes dans l’Océan Indien (CRVOI), Plateforme de Recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde, Reunion Université de La Réunion, 97715 Saint-Denis, Reunion c Institut de Recherche pour le Développement (IRD), IRD – BP 50172, 97492 Sainte-Clotilde, Reunion d CNRS, UMR 5557 Ecologie Microbienne, Université Claude Bernard Lyon I 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, Lyon, France e CIRAD, Station de La Bretagne BP 20, 97408 Saint-Denis, Reunion f UMR190, Aix-Marseille Univ – IRD, Marseille, France g Association Vahatra, B.P. 3972, Antananarivo 101, Madagascar h Field Museum of Natural History, 1400 S. Lake Shore Dr., Chicago, IL 60605-2496, USA i Institut Pasteur de Madagascar, B.P. 1274 – Ambatofotsikely, Antananarivo 101, Madagascar b
a r t i c l e
i n f o
Article history: Received 18 May 2012 Received in revised form 20 August 2012 Accepted 31 August 2012 Available online 7 September 2012 Keywords: Bats viruses Paramyxovirus Madagascar Union of Comoros Mauritius Indian Ocean
a b s t r a c t Bats are reservoirs for many emerging zoonotic viruses. In this study, we screened 197 animals from 15 different bat species of the Southwest Indian Ocean for paramyxovirus infection and identified paramyxoviruses in five insectivorous bat-species from the Union of the Comoros (3/66), Mauritius (1/55) and Madagascar (4/76). Viral isolation was possible via cell culture and phylogenetic analysis revealed these viruses clustered in a Morbillivirus-related lineage, with relatively high nucleotide sequence similarity to other recently discovered insectivorous-bat paramyxoviruses but distinct from those known to circulate in frugivorous bats. © 2012 Elsevier B.V. All rights reserved.
Paramyxoviruses have been described in a large diversity of mammalian hosts. Recent studies have suggested that bats may represent an important and unique reservoir for several emerging viruses (Wang et al., 2011), some of which have been incriminated in human epidemics and mortality (Field, 2009; Wong et al., 2007). For instance, fruit bat-species (Pteropodidae) may represent the natural reservoir for Hendra and Nipah viruses (Field et al., 2011; Luby et al., 2009). Such viruses are recognized as a threat to human and veterinary health, and recent studies have shown that bats and rodents worldwide host a broad spectrum of paramyxoviruses (Drexler et al., 2012; Kurth et al., 2012) includ-
Abbreviations: SWIO, Southwestern Indian Ocean; RMH, Respirovirus/ Morbillivirus and Henipavirus; PMV, Paramyxovirinae. ∗ Corresponding authors at: CRVOI, Plateforme de Recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde, Reunion. Tel.: +262 262 93 88 20. E-mail addresses: [email protected], [email protected] (D.A. Wilkinson), [email protected] (H. Pascalis). 1 These authors contributed equally to this article. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.08.022
ing Henipah-related, Respiratory and Morbillivirus-related viruses. Fifty-seven species of bat, including 50 endemic species, have been described in the Southwestern Indian Ocean (SWIO) region, and earlier studies have detected antibodies to paramyxoviruses in Pteropodidae of the region (Iehle et al., 2007). Here, we investigated the circulation of paramyxoviruses in 15 bat-species in the islands of the SWIO, which are part of hotspots of biodiversity neighboring the countries of East Africa, known for several emerging pathogens (Jones et al., 2008; Myers et al., 2000; Tortosa et al., 2012). As part of a program aiming to establish an inventory of infectious agents circulating in wild fauna of the SWIO area, bats were definitively captured at four locations: Anjouan and Grande Comore Islands (Union of Comoros), Mauritius and Madagascar using mist nets or harp traps. The appropriate national research permits were obtained for each location. Individual bats were identified by external and cranio-dental characteristics based on voucher specimens, and details of age/sex were recorded. Tissue samples were collected within 30 min of each individual being dispatched, frozen in liquid nitrogen and stored at −80 ◦ C until testing.
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D.A. Wilkinson et al. / Virus Research 170 (2012) 159–163
Table 1 Paramyxoviruses in bat species on different islands of the southwest Indian Ocean (SWIO) and their typical day roost sites. Results are presented for RT-PCR based screening using Respirovirus/Morbillivirus/Henipahvirus (RMH) and Paramyxovirinae (PMV) detection systems. *: island endemic and +: SWOI regional endemic. For roosting site information: C, cave; S, synanthropic; V, vegetation. All species feed predominantly on insects, with the exception of the frugivorous Rousettus obliviosus. In two cases (indicated by #), individual Miniopterus griveaudi obtained in caves in the Union of the Comoros had co-infections of paramyxovirus – one of these on Grande Comore and the other on Anjouan. In both cases, these caves only had day roost sites of Miniopterus griveaudi, but elsewhere in the archipelago, this species co-occurs in the same caves as Rousettus obliviosus. Location
Bat species
Bat family
Typical roosting sites
Detection System
Total tested
Total positive
RMH
PMV
The Union of the Comoros
+Miniopterus griveaudi# *Rousettus obliviosus +Chaerephon pusillus
Miniopteridae Pteropodidae Molossidae
C C S
3 0 0
2 0 0
20 26 20
3 0 0
Mauritius
*Mormopterus acetabulosus
Molossidae
C
1
1
55
1
Madagascar
*Miniopterus gleni *Miniopterus mahafaliensis *Miniopterus griffithsi *Miniopterus sororculus *Mormopterus jugularis *Otomops madagascariensis *Chaerephon atsinanana *Myotis goudoti *Pipistrellus raceyi *Triaenops menamena *Triaenops furculus
Miniopteridae Miniopteridae Miniopteridae Miniopteridae Molossidae Molossidae Molossidae Vespertilionidae Vespertilionidae Hipposideridae Hipposideridae
C C C C S C S C V, S C C
1 0 0 1 0 0 0 0 0 2 0
1 0 0 0 0 0 0 0 0 0 0
7 11 2 1 13 8 20 2 1 10 1
1 0 0 1 0 0 0 0 0 2 0
Pteropodidae Miniopteridae Molossidae Vespertilionidae Hipposideridae
– – – – –
0 5 1 0 2
0 3 1 0 0
26 41 116 3 11
0 5 1 0 2
–
8
4
197
8
Family totals
Grand total
Approximately 1 mm3 of lung, kidney and spleen collected from the same animal were dissected on ice from the frozen samples, pooled in DMEM medium, and homogenized in a TissueLyser (QIAGEN) for 2 min at 25 Hz using 3 mm tungsten beads. Total nucleic acids were extracted from the mixture supernatant using the viral mini kit v2.0 and an EZ1 BioRobot (QIAGEN). cDNA was generated via reverse transcription (Promega cDNA kit). The presence of paramyxoviruses was detected via semi-nested PCR using primers targeting the L-gene polymerase locus, for the detection of Respirovirus/Morbillivirus and Henipavirus (RMH) and Paramyxovirinae (PMV) subfamilies (Tong et al., 2008). PCR products (400–600 bp in length) were cloned into the pGEMt-Easy vector (Promega) and at least 2 positive clones were sequenced for each individual. To identify the genetic relatedness of the detected viruses, phylogenetic analyses were performed with published Paramyxoviridae sequences. Primer sequences were removed and leading Ns were added to place every sequence in the same reading frame. Partial paramyxovirus L-gene sequences and full-genomes were collected and filtered by hand from the results of several BLAST nucleotide searches as well as direct references from available and up-to-date literature. Representative sequences were chosen from all of the major classified and unclassified genera of the Paramyxoviridae family. A total of 218 partial and full L-gene nucleotide sequences were then aligned using the ClustalW translation alignment tool in MEGA (Ver. 5.05). Free end gaps were trimmed but internal gaps were permitted, resulting in a final alignment length of 474 bps. The appropriate substitution model (GTR + I + ) was selected using jModeltest (Guindon and Gascuel, 2003) and a Bayesian analysis was performed using the MrBayes plugin (Huelsenbeck and Ronquist, 2001) for Geneious® Pro (Kearse et al., 2012). A total of 5,000,000 iterations were run for the phylogenetic analysis, sampling results every 5000 iterations. The first 10% trees were discarded as burn-in and an effective sample size superior to 200 was obtained for each of the estimated model parameters to ensure adequate sample size.
A total of 197 bats were collected: 86.8% from insectivorous species and 13.2% from frugivorous species. Of these, 8 individuals belonging to 5 SWOI-endemic bat-species and from 3 bat-families (Miniopteridae, Molossidae, and Vespertilionidae) tested positive for paramyxoviruses (Table 1). All PMV-positive samples were also detected using the RMH system. Additionally, phylogenetic analysis of sequences derived from the PMV system provided no additional information (data not shown), and therefore genetic analyses are only presented for sequences derived using the RMH system. The average nucleotide sequence similarity was 74.2%, suggesting a high level of genetic diversity between viruses. Two individuals of the species Miniopterus griveaudi from Anjouan and Grande Comore, genetically close bat-populations of Malagasy origin with proved dispersal exchange (Weyeneth et al., 2011), had evidence of co-infection by two genetically distinct paramyxoviruses. The observed genetic identities between pairs of co-infecting viruses were 70.8% and 74.7%. Phylogenetic analysis supported that all 10 of the obtained sequences were new members of the recently identified “Morbillivirus-related” branch of the Paramyxoviridae family, comprising both Rodentian paramyxoviruses and bat paramyxoviruses from many locations across the globe (Fig. 1) (McCarthy and Goodman, 2010). Broadly speaking, all viruses belonging to the Morbillvirus-related group of paramyxoviruses likely to share a common ancestor with the rodentian viruses J. paramyxovirus, Beilong and Tailam viruses (Li et al., 2006; Woo et al., 2011) as well as the more distant Nariva virus, Mossman virus and Tupaia virus, again of rodent origin (Lambeth et al., 2009; Miller et al., 2003) (Fig. 1b). Although the phylogeny could not be completely resolved due to the high level of sequence variation, a certain degree of species specificity was observed between different strongly supported lineages with separate clusters being either rodent-specific of bat-specific (Fig. 1b). The majority of identified sequences were grouped by geographical location and host-species. Eight of the 10 sequences obtained from Madagascar, Mauritius and the Union
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Fig. 1. Phylogeny of the Paramyxoviridae. (a) A global phylogeny of 218 partial L-gene sequences calculated in 5,000,000 iterations in MrBayes with the GTR + I + evolutionary model and a 10% burn-in. Main virus subfamilies and genera were annotated according to Drexler et al., 2012. The dashed-line rectangle highlights the position of the partial phylogenetic tree detailed in (b) which contains all of the sequences obtained during this study. (b) Tips of sequences obtained during this study are highlighted in bold. Scientific names of paramyxoviruses are abbreviated as follows: Tupaia virus, TpV; Mossman virus, MosV; Nariva virus, NarV; Rodent paramyxovirus, RodPV; Bat paramyxovirus, BatPV; J. paramyxovirus, JV; Tailam virus, TaiV; Beilong virus, BeiV. Co-infected individual bats are identified with symbols + and *. Viral strains that were successfully isolated by cell culture techniques are indicated by the symbol #. Nomenclature code description and scale bars are also presented.
of Comoros grouped with other paramyxoviruses detected in insectivorous bats of the Hipposideridae family and one Coleura afra, bats known to inhabit in large areas of Africa and Northern Madagascar (Goodman et al., 2008), sharing similar roost sites and diets to those of the studied bat species. Interestingly, one M. griveaudi was co-infected with viruses belonging to two separate sub-branches of this main phylogenetic group. This suggests that the sequence variation within this group is representative of separate viral strains, and that there is active circulation of these viruses in separate regions and between different species. This also infers that, as previously suggested (Drexler et al., 2012), a large number of different viral genera are represented within “Morbillivirus-related subfamily”. The remaining two sequences, both from M. griveaudi in the Union of Comoros, grouped with other insectivorous bat species originating from Europe. Interestingly one of the obtained sequences in this subgroup originated from a co-infection in M. griveaudi suggestive of active viral transmission across large geographical boundaries and between species. The relatively large amount of currently available genetic data derived from bat paramyxoviruses suggests that differences may exist between viruses circulating in insectivorous and frugivorous bat-species. The recently published sequences from the frugivorous Eidolon helvum (Pteropodidae) collected in Ghana (Baker et al., 2012; Drexler et al., 2009, 2012) formed genetic lineages that were unrelated to the Morbilliviruses and more closely related to the Henipaviruses. In addition, none of the 36 individuals of Rousettus oblivious (Pteropodidae; frugivorous) that we collected tested positive, despite an insectivorous species (M. griveaudi) from the same location being paramyxovirus positive. Most of the analyzed bats from the SWOI are cave dwelling, with single cave systems holding from one to 10 different species roosting in syntopy (Goodman, 2011). Hence, there is considerable potential for horizontal transfer. However, these results suggest that host-related adaptation may exist between insectivorous and frugivorous bats, which may potentially limit viral gene flow between bat-species. In an attempt to isolate the detected viruses, clarified tissue supernatant from all paramyxovirus-positive animals was diluted in DMEM to 1/5 and applied to a 12.5 cm2 tissue-layer of Vero cells
that had been grown to 70–80% confluence. Infections were left for 90 min at 37 ◦ C before removal of the inoculants, washing in minimal medium and the addition of 5 ml of growth medium. Cells were observed microscopically over the course of 10 days, and paramyxovirus infections were followed using RMH and PMV PCR systems. Two of the eight positive samples produced visible cytopathic effects, which were evident after 5 days of cellular growth and had cells that tested PCR-positive for paramyxovirus infection (Fig. 2). To establish the viral culture, sequential passages were performed, first with a mixture of cells and supernatant and then with clarified supernatant. Viral isolation was confirmed via electron microscopy by the presence of paramyxovirus-like particles in the growth medium of these cultures (Fig. 2). One of the isolated viruses originated from a M. griveaudi, identified as co-infected with 2 separate paramyxoviruses, thus sequencing was used to identify which of the viral strains had been isolated (indicated in Fig. 1b). Repeated attempts to isolate viruses from the remaining 6 animals were unsuccessful. The successful isolation of two different viral strains suggests that Vero cell lines may be suitable for the isolation of the rapidly increasing number of novel Morbillivirus-related paramyxoviruses (Baker et al., 2012; Drexler et al., 2012; Kurth et al., 2012). Both paramyxovirus strains isolated by this method were genetically closely related, suggesting that there may exist differences between the strains within this large and broadly defined genus that will likely affect the optimal methodology for viral isolation. Importantly, the isolation of similar viruses has recently been shown to be possible from urine samples (Baker et al., 2012), suggesting that non-invasive methods of viral isolation may be used for the study of paramyxoviruses infecting endemic bats that are often protected species.Conclusions In this study, we identified new members of Paramyxoviridae circulating in insectivorous bat-species of the SWIO and isolated 2 of these viruses by cell culture techniques. Our findings suggest that a large diversity of paramyxoviruses is circulating in bat-reservoirs with likely important differences between viruses from frugivorous and insectivorous species. Additionally, differences between viral strains co-infecting bats from the Union of Comoros suggest active geographical circulation of these viruses, however the mechanism
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Fig. 2. Viral isolation of paramyxovirus strains in Vero cell cultures. (a) Representative photos of Vero cell cultures at 20× magnification: (i) negative control at day 1 after infection, (ii) negative control at day 5 after infection, (iii) Paramyxovirus strain 1 (corresponding to Genbank accession number JQ886100) and (iv) Paramyxovirus strain 2 (corresponding to Genbank accession number JQ886104) at day 5 after infection in a second consecutive passage. Infected cell-lines demonstrate substantial cellular lysis and vacuolization after 5 days of infection. (b) Representative electron micrograph of a paramyxovirus-like particle imaged from the culture depicted in ((a)(iii)). Scale bar represents 100 nm. This displayed viral particle has a diameter of approximately 165 nm.
for this circulation remains to be characterized. Whereas the potential for bat to human species-transgression has been described for fruit bat-species, less is known about the potential of viral emergence from insectivorous bats, although many insectivorous bat species can be synantropic and may enter in direct contact with human populations. Further investigation will be required to assess the pathogenicity of the new group of Morbillivirus-related paramyxoviruses, as little is known as to whether these viruses have any impact on animal or human health. Acknowledgements The authors wish to thank the Direction du Système des Aires Protégées, Direction Générale de l’Environnement et des Forêts (Madagascar), the National Parks and Conservation Service and the
Ministry of Agriculture, Food Technology and Natural Resources (Mauritius), and the Center National de Documentation et de Recherche Scientifique (Union of the Comoros) which delivered permits for this research. We also thank Yahaya Ibrahim and Ishaka Saïd for their help for collecting bats and administrative aspects in the Union of the Comoros, and Odile Py, from UMR190, AixMarseille Univ – IRD – EHESP French School of Public Health, Marseille, France, for her technical assistance. This work was supported by funding from CRVOI and ERDF/French Government/Regional Council of La Réunion, FEDER POCT (2007–2013) Pathogènes associés à la Faune Sauvage Océan Indien #31189. D.A. Wilkinson and C. Lebarbenchon post-doctoral fellowships were funded by “RUN-Emerge: European project funded by European Commission under FP7 program”.
D.A. Wilkinson et al. / Virus Research 170 (2012) 159–163
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Contents lists available at SciVerse ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean David A. Wilkinson a,b,c,∗ , Sarah Temmam a,d,1 , Camille Lebarbenchon a,b,1 , Erwan Lagadec a,d,e,1 , Julien Chotte a,c,1 , Julia Guillebaud a,c,1 , Beza Ramasindrazana g , Jean-Michel Héraud i , Xavier de Lamballerie f , Steven M. Goodman g,h , Koussay Dellagi a,c , Hervé Pascalis a,c,∗ a
Centre de Recherche et de Veille sur les Maladies émergentes dans l’Océan Indien (CRVOI), Plateforme de Recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde, Reunion Université de La Réunion, 97715 Saint-Denis, Reunion c Institut de Recherche pour le Développement (IRD), IRD – BP 50172, 97492 Sainte-Clotilde, Reunion d CNRS, UMR 5557 Ecologie Microbienne, Université Claude Bernard Lyon I 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, Lyon, France e CIRAD, Station de La Bretagne BP 20, 97408 Saint-Denis, Reunion f UMR190, Aix-Marseille Univ – IRD, Marseille, France g Association Vahatra, B.P. 3972, Antananarivo 101, Madagascar h Field Museum of Natural History, 1400 S. Lake Shore Dr., Chicago, IL 60605-2496, USA i Institut Pasteur de Madagascar, B.P. 1274 – Ambatofotsikely, Antananarivo 101, Madagascar b
a r t i c l e
i n f o
Article history: Received 18 May 2012 Received in revised form 20 August 2012 Accepted 31 August 2012 Available online 7 September 2012 Keywords: Bats viruses Paramyxovirus Madagascar Union of Comoros Mauritius Indian Ocean
a b s t r a c t Bats are reservoirs for many emerging zoonotic viruses. In this study, we screened 197 animals from 15 different bat species of the Southwest Indian Ocean for paramyxovirus infection and identified paramyxoviruses in five insectivorous bat-species from the Union of the Comoros (3/66), Mauritius (1/55) and Madagascar (4/76). Viral isolation was possible via cell culture and phylogenetic analysis revealed these viruses clustered in a Morbillivirus-related lineage, with relatively high nucleotide sequence similarity to other recently discovered insectivorous-bat paramyxoviruses but distinct from those known to circulate in frugivorous bats. © 2012 Elsevier B.V. All rights reserved.
Paramyxoviruses have been described in a large diversity of mammalian hosts. Recent studies have suggested that bats may represent an important and unique reservoir for several emerging viruses (Wang et al., 2011), some of which have been incriminated in human epidemics and mortality (Field, 2009; Wong et al., 2007). For instance, fruit bat-species (Pteropodidae) may represent the natural reservoir for Hendra and Nipah viruses (Field et al., 2011; Luby et al., 2009). Such viruses are recognized as a threat to human and veterinary health, and recent studies have shown that bats and rodents worldwide host a broad spectrum of paramyxoviruses (Drexler et al., 2012; Kurth et al., 2012) includ-
Abbreviations: SWIO, Southwestern Indian Ocean; RMH, Respirovirus/ Morbillivirus and Henipavirus; PMV, Paramyxovirinae. ∗ Corresponding authors at: CRVOI, Plateforme de Recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde, Reunion. Tel.: +262 262 93 88 20. E-mail addresses: [email protected], [email protected] (D.A. Wilkinson), [email protected] (H. Pascalis). 1 These authors contributed equally to this article. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.08.022
ing Henipah-related, Respiratory and Morbillivirus-related viruses. Fifty-seven species of bat, including 50 endemic species, have been described in the Southwestern Indian Ocean (SWIO) region, and earlier studies have detected antibodies to paramyxoviruses in Pteropodidae of the region (Iehle et al., 2007). Here, we investigated the circulation of paramyxoviruses in 15 bat-species in the islands of the SWIO, which are part of hotspots of biodiversity neighboring the countries of East Africa, known for several emerging pathogens (Jones et al., 2008; Myers et al., 2000; Tortosa et al., 2012). As part of a program aiming to establish an inventory of infectious agents circulating in wild fauna of the SWIO area, bats were definitively captured at four locations: Anjouan and Grande Comore Islands (Union of Comoros), Mauritius and Madagascar using mist nets or harp traps. The appropriate national research permits were obtained for each location. Individual bats were identified by external and cranio-dental characteristics based on voucher specimens, and details of age/sex were recorded. Tissue samples were collected within 30 min of each individual being dispatched, frozen in liquid nitrogen and stored at −80 ◦ C until testing.
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Table 1 Paramyxoviruses in bat species on different islands of the southwest Indian Ocean (SWIO) and their typical day roost sites. Results are presented for RT-PCR based screening using Respirovirus/Morbillivirus/Henipahvirus (RMH) and Paramyxovirinae (PMV) detection systems. *: island endemic and +: SWOI regional endemic. For roosting site information: C, cave; S, synanthropic; V, vegetation. All species feed predominantly on insects, with the exception of the frugivorous Rousettus obliviosus. In two cases (indicated by #), individual Miniopterus griveaudi obtained in caves in the Union of the Comoros had co-infections of paramyxovirus – one of these on Grande Comore and the other on Anjouan. In both cases, these caves only had day roost sites of Miniopterus griveaudi, but elsewhere in the archipelago, this species co-occurs in the same caves as Rousettus obliviosus. Location
Bat species
Bat family
Typical roosting sites
Detection System
Total tested
Total positive
RMH
PMV
The Union of the Comoros
+Miniopterus griveaudi# *Rousettus obliviosus +Chaerephon pusillus
Miniopteridae Pteropodidae Molossidae
C C S
3 0 0
2 0 0
20 26 20
3 0 0
Mauritius
*Mormopterus acetabulosus
Molossidae
C
1
1
55
1
Madagascar
*Miniopterus gleni *Miniopterus mahafaliensis *Miniopterus griffithsi *Miniopterus sororculus *Mormopterus jugularis *Otomops madagascariensis *Chaerephon atsinanana *Myotis goudoti *Pipistrellus raceyi *Triaenops menamena *Triaenops furculus
Miniopteridae Miniopteridae Miniopteridae Miniopteridae Molossidae Molossidae Molossidae Vespertilionidae Vespertilionidae Hipposideridae Hipposideridae
C C C C S C S C V, S C C
1 0 0 1 0 0 0 0 0 2 0
1 0 0 0 0 0 0 0 0 0 0
7 11 2 1 13 8 20 2 1 10 1
1 0 0 1 0 0 0 0 0 2 0
Pteropodidae Miniopteridae Molossidae Vespertilionidae Hipposideridae
– – – – –
0 5 1 0 2
0 3 1 0 0
26 41 116 3 11
0 5 1 0 2
–
8
4
197
8
Family totals
Grand total
Approximately 1 mm3 of lung, kidney and spleen collected from the same animal were dissected on ice from the frozen samples, pooled in DMEM medium, and homogenized in a TissueLyser (QIAGEN) for 2 min at 25 Hz using 3 mm tungsten beads. Total nucleic acids were extracted from the mixture supernatant using the viral mini kit v2.0 and an EZ1 BioRobot (QIAGEN). cDNA was generated via reverse transcription (Promega cDNA kit). The presence of paramyxoviruses was detected via semi-nested PCR using primers targeting the L-gene polymerase locus, for the detection of Respirovirus/Morbillivirus and Henipavirus (RMH) and Paramyxovirinae (PMV) subfamilies (Tong et al., 2008). PCR products (400–600 bp in length) were cloned into the pGEMt-Easy vector (Promega) and at least 2 positive clones were sequenced for each individual. To identify the genetic relatedness of the detected viruses, phylogenetic analyses were performed with published Paramyxoviridae sequences. Primer sequences were removed and leading Ns were added to place every sequence in the same reading frame. Partial paramyxovirus L-gene sequences and full-genomes were collected and filtered by hand from the results of several BLAST nucleotide searches as well as direct references from available and up-to-date literature. Representative sequences were chosen from all of the major classified and unclassified genera of the Paramyxoviridae family. A total of 218 partial and full L-gene nucleotide sequences were then aligned using the ClustalW translation alignment tool in MEGA (Ver. 5.05). Free end gaps were trimmed but internal gaps were permitted, resulting in a final alignment length of 474 bps. The appropriate substitution model (GTR + I + ) was selected using jModeltest (Guindon and Gascuel, 2003) and a Bayesian analysis was performed using the MrBayes plugin (Huelsenbeck and Ronquist, 2001) for Geneious® Pro (Kearse et al., 2012). A total of 5,000,000 iterations were run for the phylogenetic analysis, sampling results every 5000 iterations. The first 10% trees were discarded as burn-in and an effective sample size superior to 200 was obtained for each of the estimated model parameters to ensure adequate sample size.
A total of 197 bats were collected: 86.8% from insectivorous species and 13.2% from frugivorous species. Of these, 8 individuals belonging to 5 SWOI-endemic bat-species and from 3 bat-families (Miniopteridae, Molossidae, and Vespertilionidae) tested positive for paramyxoviruses (Table 1). All PMV-positive samples were also detected using the RMH system. Additionally, phylogenetic analysis of sequences derived from the PMV system provided no additional information (data not shown), and therefore genetic analyses are only presented for sequences derived using the RMH system. The average nucleotide sequence similarity was 74.2%, suggesting a high level of genetic diversity between viruses. Two individuals of the species Miniopterus griveaudi from Anjouan and Grande Comore, genetically close bat-populations of Malagasy origin with proved dispersal exchange (Weyeneth et al., 2011), had evidence of co-infection by two genetically distinct paramyxoviruses. The observed genetic identities between pairs of co-infecting viruses were 70.8% and 74.7%. Phylogenetic analysis supported that all 10 of the obtained sequences were new members of the recently identified “Morbillivirus-related” branch of the Paramyxoviridae family, comprising both Rodentian paramyxoviruses and bat paramyxoviruses from many locations across the globe (Fig. 1) (McCarthy and Goodman, 2010). Broadly speaking, all viruses belonging to the Morbillvirus-related group of paramyxoviruses likely to share a common ancestor with the rodentian viruses J. paramyxovirus, Beilong and Tailam viruses (Li et al., 2006; Woo et al., 2011) as well as the more distant Nariva virus, Mossman virus and Tupaia virus, again of rodent origin (Lambeth et al., 2009; Miller et al., 2003) (Fig. 1b). Although the phylogeny could not be completely resolved due to the high level of sequence variation, a certain degree of species specificity was observed between different strongly supported lineages with separate clusters being either rodent-specific of bat-specific (Fig. 1b). The majority of identified sequences were grouped by geographical location and host-species. Eight of the 10 sequences obtained from Madagascar, Mauritius and the Union
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Fig. 1. Phylogeny of the Paramyxoviridae. (a) A global phylogeny of 218 partial L-gene sequences calculated in 5,000,000 iterations in MrBayes with the GTR + I + evolutionary model and a 10% burn-in. Main virus subfamilies and genera were annotated according to Drexler et al., 2012. The dashed-line rectangle highlights the position of the partial phylogenetic tree detailed in (b) which contains all of the sequences obtained during this study. (b) Tips of sequences obtained during this study are highlighted in bold. Scientific names of paramyxoviruses are abbreviated as follows: Tupaia virus, TpV; Mossman virus, MosV; Nariva virus, NarV; Rodent paramyxovirus, RodPV; Bat paramyxovirus, BatPV; J. paramyxovirus, JV; Tailam virus, TaiV; Beilong virus, BeiV. Co-infected individual bats are identified with symbols + and *. Viral strains that were successfully isolated by cell culture techniques are indicated by the symbol #. Nomenclature code description and scale bars are also presented.
of Comoros grouped with other paramyxoviruses detected in insectivorous bats of the Hipposideridae family and one Coleura afra, bats known to inhabit in large areas of Africa and Northern Madagascar (Goodman et al., 2008), sharing similar roost sites and diets to those of the studied bat species. Interestingly, one M. griveaudi was co-infected with viruses belonging to two separate sub-branches of this main phylogenetic group. This suggests that the sequence variation within this group is representative of separate viral strains, and that there is active circulation of these viruses in separate regions and between different species. This also infers that, as previously suggested (Drexler et al., 2012), a large number of different viral genera are represented within “Morbillivirus-related subfamily”. The remaining two sequences, both from M. griveaudi in the Union of Comoros, grouped with other insectivorous bat species originating from Europe. Interestingly one of the obtained sequences in this subgroup originated from a co-infection in M. griveaudi suggestive of active viral transmission across large geographical boundaries and between species. The relatively large amount of currently available genetic data derived from bat paramyxoviruses suggests that differences may exist between viruses circulating in insectivorous and frugivorous bat-species. The recently published sequences from the frugivorous Eidolon helvum (Pteropodidae) collected in Ghana (Baker et al., 2012; Drexler et al., 2009, 2012) formed genetic lineages that were unrelated to the Morbilliviruses and more closely related to the Henipaviruses. In addition, none of the 36 individuals of Rousettus oblivious (Pteropodidae; frugivorous) that we collected tested positive, despite an insectivorous species (M. griveaudi) from the same location being paramyxovirus positive. Most of the analyzed bats from the SWOI are cave dwelling, with single cave systems holding from one to 10 different species roosting in syntopy (Goodman, 2011). Hence, there is considerable potential for horizontal transfer. However, these results suggest that host-related adaptation may exist between insectivorous and frugivorous bats, which may potentially limit viral gene flow between bat-species. In an attempt to isolate the detected viruses, clarified tissue supernatant from all paramyxovirus-positive animals was diluted in DMEM to 1/5 and applied to a 12.5 cm2 tissue-layer of Vero cells
that had been grown to 70–80% confluence. Infections were left for 90 min at 37 ◦ C before removal of the inoculants, washing in minimal medium and the addition of 5 ml of growth medium. Cells were observed microscopically over the course of 10 days, and paramyxovirus infections were followed using RMH and PMV PCR systems. Two of the eight positive samples produced visible cytopathic effects, which were evident after 5 days of cellular growth and had cells that tested PCR-positive for paramyxovirus infection (Fig. 2). To establish the viral culture, sequential passages were performed, first with a mixture of cells and supernatant and then with clarified supernatant. Viral isolation was confirmed via electron microscopy by the presence of paramyxovirus-like particles in the growth medium of these cultures (Fig. 2). One of the isolated viruses originated from a M. griveaudi, identified as co-infected with 2 separate paramyxoviruses, thus sequencing was used to identify which of the viral strains had been isolated (indicated in Fig. 1b). Repeated attempts to isolate viruses from the remaining 6 animals were unsuccessful. The successful isolation of two different viral strains suggests that Vero cell lines may be suitable for the isolation of the rapidly increasing number of novel Morbillivirus-related paramyxoviruses (Baker et al., 2012; Drexler et al., 2012; Kurth et al., 2012). Both paramyxovirus strains isolated by this method were genetically closely related, suggesting that there may exist differences between the strains within this large and broadly defined genus that will likely affect the optimal methodology for viral isolation. Importantly, the isolation of similar viruses has recently been shown to be possible from urine samples (Baker et al., 2012), suggesting that non-invasive methods of viral isolation may be used for the study of paramyxoviruses infecting endemic bats that are often protected species.Conclusions In this study, we identified new members of Paramyxoviridae circulating in insectivorous bat-species of the SWIO and isolated 2 of these viruses by cell culture techniques. Our findings suggest that a large diversity of paramyxoviruses is circulating in bat-reservoirs with likely important differences between viruses from frugivorous and insectivorous species. Additionally, differences between viral strains co-infecting bats from the Union of Comoros suggest active geographical circulation of these viruses, however the mechanism
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Fig. 2. Viral isolation of paramyxovirus strains in Vero cell cultures. (a) Representative photos of Vero cell cultures at 20× magnification: (i) negative control at day 1 after infection, (ii) negative control at day 5 after infection, (iii) Paramyxovirus strain 1 (corresponding to Genbank accession number JQ886100) and (iv) Paramyxovirus strain 2 (corresponding to Genbank accession number JQ886104) at day 5 after infection in a second consecutive passage. Infected cell-lines demonstrate substantial cellular lysis and vacuolization after 5 days of infection. (b) Representative electron micrograph of a paramyxovirus-like particle imaged from the culture depicted in ((a)(iii)). Scale bar represents 100 nm. This displayed viral particle has a diameter of approximately 165 nm.
for this circulation remains to be characterized. Whereas the potential for bat to human species-transgression has been described for fruit bat-species, less is known about the potential of viral emergence from insectivorous bats, although many insectivorous bat species can be synantropic and may enter in direct contact with human populations. Further investigation will be required to assess the pathogenicity of the new group of Morbillivirus-related paramyxoviruses, as little is known as to whether these viruses have any impact on animal or human health. Acknowledgements The authors wish to thank the Direction du Système des Aires Protégées, Direction Générale de l’Environnement et des Forêts (Madagascar), the National Parks and Conservation Service and the
Ministry of Agriculture, Food Technology and Natural Resources (Mauritius), and the Center National de Documentation et de Recherche Scientifique (Union of the Comoros) which delivered permits for this research. We also thank Yahaya Ibrahim and Ishaka Saïd for their help for collecting bats and administrative aspects in the Union of the Comoros, and Odile Py, from UMR190, AixMarseille Univ – IRD – EHESP French School of Public Health, Marseille, France, for her technical assistance. This work was supported by funding from CRVOI and ERDF/French Government/Regional Council of La Réunion, FEDER POCT (2007–2013) Pathogènes associés à la Faune Sauvage Océan Indien #31189. D.A. Wilkinson and C. Lebarbenchon post-doctoral fellowships were funded by “RUN-Emerge: European project funded by European Commission under FP7 program”.
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