JGV Papers in Press. Published February 20, 2015 as doi:10.1099/vir.0.000099
Journal of General Virology Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution. --Manuscript Draft-Manuscript Number:
JGV-D-14-00287R1
Full Title:
Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution.
Short Title:
Novel paramyxoviruses in Australian flying-foxes
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Miranda Emily Vidgen, PhD University of the Sunshine Coast Sippy Downs, Queensland AUSTRALIA
First Author:
Miranda E. Vidgen, PhD
Order of Authors:
Miranda E. Vidgen, PhD Carol de Jong Karrie Rose Jane Hall Hume E. Field Craig S. Smith
Abstract:
Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses in Australian flying-fox species (Pteropus alecto, P. scapulatus, P. poliocephalus and P. conspicillatus) and in the Christmas Island species P. melanotus natalis. Using a degenerative RT-PCR specific for the L gene of known species of Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously unknown paramyxoviruses (UPV), with a representative 31 UPVs clustering in eight distinct groups (100 UPV/495 samples). No new henipaviruses were identified. The findings are consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.
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Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution. Running title: Novel paramyxoviruses in Australian flying-foxes Contents Category: Animal, negative-strand RNA viruses Miranda E. Vidgen1,5, Carol de Jong1, Karrie Rose2,4, Jane Hall2, Hume E. Field1, 3, and Craig S. Smith1 1
Queensland Centre of Emerging Infectious Diseases, Biosecurity Queensland, Department
of Agriculture, Fisheries and Forestry, 39 Kessels Road, Coopers Plains Queensland, 4108, Australia 2
Australian Registry of Wildlife Health, Taronga Conservation Society Australia, PO Box
20, Mosman NSW, 2088, Australia 3 4
EcoHealth Alliance, 460 West 34th Street, New York, NY 10001, USA School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook
University, Townsville Queensland, 4811, Australia 5
School of Health and Sports Science, University of Sunshine Coast, 90 Sippy Down Drive,
Sippy Downs Queensland, 4556, Australia
Corresponding Author: Dr. Miranda Vidgen, School of Health and Sport Science, University of the Sunshine Coast, Sippy Downs, Queensland, 4556, Australia; Phone: +61 (7) 5456 3436; Fax: none available; Email: [email protected]
Word Count- Summary/Abstract: 149 words Word Count- Main text: 2497 words
Unpublished References: There are two unpublished references cited in this manuscript; Goldspink, et al. and Phalen et al. Permission has been obtained from the authors to cite the unpublished work.
The GenBank accession number for the partial L gene sequence are KF871289 to KF871319.
1
Abstract (Summary)
2
Understanding the diversity of henipaviruses and related viruses is important in determining
3
the viral ecology within flying-fox populations and assessing the potential threat posed by
4
these agents. This study sought to identify the abundance and diversity of previously
5
unknown paramyxoviruses in Australian flying-fox species (Pteropus alecto, P. scapulatus,
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P. poliocephalus and P. conspicillatus) and in the Christmas Island species P. melanotus
7
natalis. Using a degenerative RT-PCR specific for the L gene of known species of
8
Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus,
9
we identified an abundance and diversity of previously unknown paramyxoviruses (UPV),
10
with a representative 31 UPVs clustering in eight distinct groups (100 UPV/495 samples). No
11
new henipaviruses were identified. The findings are consistent with a hypothesis of co-
12
evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-
13
speciation between host and virus (beyond the scope of this study) would strengthen this
14
hypothesis.
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Main Text
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Hendra (HeV) and Nipah (NiV) virus (genus Henipavirus, family Paramyxoviridae) cause
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fatal encephalitic and respiratory diseases in animals and humans (Field et al., 2010; Playford
18
et al., 2010). A third henipavirus, Cedar Virus (CeV), (Marsh et al., 2012), and an
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undescribed henipavirus in Africa (Baker et al., 2013a; Drexler et al., 2009) have recently
20
been identified. The natural reservoir hosts of these viruses in Australia and Southern Asia
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are bats of the genus Pteropus (order Chiroptera), commonly known as flying-foxes (Johara
22
et al., 2001; Young et al., 1996).
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It is recognised that bats have played an important role in the evolution of paramyxoviruses,
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both as historical and current reservoir hosts (Drexler et al., 2012). Multiple studies of the
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family Pteropodidae have reported broad diversity of unknown paramyxoviruses (UPV)
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(Anthony et al., 2013; Baker et al., 2013a; Barr et al., 2014; Drexler et al., 2009; Sasaki et
28
al., 2012). Phylogenetic analyses suggest that some of these UPV are either members of the
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genus Henipavirus (Drexler et al., 2009) or belong to novel genera closely related to
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henipaviruses (Baker et al., 2012; Baker et al., 2013a; Sasaki et al., 2012). This study
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investigates the diversity of UPVs, with an emphasis on henipaviruses, in flying-fox
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populations endemic to Australia and Christmas Island to inform emerging disease risk
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prediction and to gain insight into the evolutionary biology of bat paramyxoviruses.
34 35
Pooled urine samples were collected from underneath ten roosting sites of P. alecto, P.
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poliocephalus, P. conspicillatus and P. scapulatus during 16 sampling events between May
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2012 and July 2013 (Table 1). Samples were collected as described by Field et al (2011).
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Individual urine samples were collected from 28 P. melanotus natalis between July and
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August 2010, as described by Hall et al (2014). The samples were aliquoted into lysis buffer
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(Life Technologies, Australia) before being transported at 4oC and then stored at -80oC. RNA
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extractions were performed using the MagMAX Viral RNA Isolation Kit (Life Technologies,
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Australia).
43 44
The RNA samples were screened for the presence of respiroviruses, morbilliviruses and
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henipaviruses (RMH) using a previously described semi-nested PCR (Tong et al., 2008).
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Phylogenetic analysis of the nucleotide sequences were carried out with Maximum
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Likelihood analysis using the General Time Reversible (GTR) model with Gamma
2
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distribution, and Bootstrapping (n=1000). Representative UPVs were submitted to GenBank
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(KF871289 to KF871319).
50 51
A total of 495 samples from Australia (n = 467) and Christmas Island (n = 28) were tested
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using the RMH PCR. Of these, 100 samples (Australia n = 97, Christmas Island n = 3)
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yielded UPVs from 15 of the 17 sampling events (Table 1). Phylogenetic analysis (Fig. 1)
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and comparative analysis of the P-distances of the UPVs found no molecular evidence of
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henipaviruses in Christmas Island flying-foxes (P. melanotus natalis) or of any new
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henipaviruses in Australian flying-foxes. CeV RNA was detected in a P. poliocephalus roost
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site in Sydney (n=2), and in a mixed roost site of P. alecto and P. poliocephalus in Boonah
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(n=1) (Fig. 2). This is the first published finding of CeV since it was characterised (Marsh et
59
al., 2012), and the lowest latitude (33.900oS) at which any henipavirus has been identified.
60 61
We have avoided using taxonomic terminology as it denotes a level of comparative analysis
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not applicable with partial sequences from unidentified viruses. To aid the discussion of
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phylogenetic analysis informal definitions have been applied. When a monophyletic clade
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contained UPVs with P-distances less than the difference between NiV variants from
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Malaysia and Bangladesh (4.7%), a single representative sequence was selected and termed
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‘representative UPV’. A clade of UPVs is considered a UPV Group when the P-distance
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between members of the cluster is less than the sequence identity of the two most distant
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species of the Henipavirus genus (CeV and NiV-Malaysia: 33.0%) and they are contained on
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a monophyletic clade that is well supported by bootstrapping (≥75%) at the common
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ancestral node.
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Of the 100 UPVs, a total of 31 representative UPVs were identified. These representative
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UPVs formed eight distinct groups (Fig. 1). None had sufficient sequence similarity to be
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defined as members of a known genera of the family Paramyxoviridae (Fig. 1). This level of
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UPV group diversity is consistent with the variety reported in Asia and Africa (Anthony et
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al., 2013; Baker et al., 2013a; Drexler et al., 2009; Sasaki et al., 2012), and supports the
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existence of a diverse range of unidentified viruses that are not associated with known
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diseases (Anthony et al., 2013). It should be noted that diversity may be higher in the flying-
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fox populations studied as the primers used would not have identified all paramyxoviruses.
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We propose that these UPV groups represent new genera within the family. Isolation and
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characterisation of representative strains and detailed comparative analyses are needed to
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confirm this.
83 84
Comparative analysis identified the same UPV groups in flying-foxes from both Australia
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and Christmas Island (Groups 1 and 5), despite the populations being geographically isolated
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from each other. It also revealed the same UPV groups in Indonesian bats (Sasaki et al.,
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2012), and in flying-foxes from Australia (Groups 1, 3 & 8) and Christmas Island (Group 1).
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This wide distribution of related UPV groups suggests a regional connectivity of flying-foxes
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between Australia, Christmas Island and Indonesia. However, there is no evidence supporting
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regional connectivity of individual UPVs, with an absence of the same UPV in flying-foxes
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from different countries. This pattern of distribution of UPVs is consistent with the regional
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distribution of henipaviruses throughout Australasia, i.e. HeV and CeV are localised to
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Australia, and NiV to Southern Asia (Eaton et al., 2006; Marsh et al., 2012). Further, the
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pattern of distribution of UPV groups and representatives is suggestive of an ancient
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association of these viruses with flying-foxes that has evolved either through periods of
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geographical isolation or localised regional association in an evolutionary timescale.
4
97 98
The UPVs of the Christmas Island P. melanotus natalis population are suggestive of viral
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evolution due to geographical isolation that diverged from the UPV Group after their
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ancestors colonised the island. This is consistent with a recent phylogenetic analysis which
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recommends P. melanotus natalis be considered a separate flying-fox species due to
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allopatric speciation (Phalen et al., in prep). The suggestion that UPV groups have ancient
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associations with flying-foxes is further supported by comparison of Australasian and African
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UPV groups. Most UPVs identified in Eidolon helvum (Baker et al., 2012; Drexler et al.,
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2009) remained in discrete groups that were inter-dispersed within the UPVs identified within
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Australia and Christmas Island (this study), and Indonesia (Sasaki et al., 2012). The
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clustering of Groups 3 and 4 with UPVs from E. helvum is not well supported by
108
bootstrapping and has nucleotide diversity greater than 33%. This indicates that E. helvum
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UPVs and Australian Pteropus UPVs are more distantly related to one another than UPVs
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from within their respective regions. E. helvum is both genetically and geographically a
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distant relative of Australasian Pteropus (Juste et al., 1999). A previous analysis of bat-borne
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viruses has suggested an ancient evolutionary history (Badrane & Tordo, 2001; Gouilh et al.,
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2011), culminating in unique immunological associations between bats and their viruses
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(Baker et al., 2013b).
115 116
It is widely accepted in phylogeography studies that Pteropodidae originated in Asia (Chan et
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al., 2011; O'Brien et al., 2009; Phalen et al., in prep) and migrated to Africa through overland
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routes when forest corridors existed between Africa and Asia approximately 15 to 25 million
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years ago (MYA) (Juste et al., 1999), and colonised Indian Ocean islands using oceanic
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routes that occurred between 0.37 to 2.58 MYA (Almeida et al., 2014; Bastian et al., 2002).
121
The replacement of forest corridors by deserts and tectonic movements have since isolated
5
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pteropid populations to their respective regions, and presumably also their viral assemblages.
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Thus we postulate that bat paramyxoviruses originated with ancestral bats in Asia and
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accompanied their hosts’ dispersal to Africa. This hypothesis is in direct contrast to the
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proposed origins of henipaviruses in Africa and subsequent global dispersal (Drexler et al.,
126
2012). To further elucidate the origins of bat paramyxoviruses, and whether these
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relationships represent co-speciation and host-virus specificity, additional research into co-
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phylogeny and virus diversity is needed.
129 130
There is a tendency to view related species of bats (i.e. Pteropodidae, Pteropus genus) as a
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single host population (Breed et al., 2011; Breed et al., 2013; Plowright et al., 2008), with
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viruses moving easily between species, rather than considering species or closely related
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groups of species as alternative hosts that require a host-switch event for viral replication and
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excretion to occur. In this study there is evidence of UPV specificity to a single species,
135
despite overlapping geographical ranges for the four Australian species (Hall & Richards,
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2000). None of the eight UPVs found in P. scapulatus were identified in any other of the
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three Australian species (Fig. 1). In contrast, there were examples of UPVs detected in
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multiple roosts with different resident Pteropus species: AU-15 (Group 7) was identified
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from a P. poliocephalus roost and P. alecto roost; and AU-12 (Group 2) was identified in P.
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conspicillatus roost and in a mixed roost of P. alecto and P. poliocephalus. During the
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evolutionary history of P. scapulatus this species is believed to have undergone an extended
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period of allopatric speciation (Bastian et al., 2002; Phalen et al., in prep) and colonised
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Australia much earlier than the ancestor of P. alecto and P. conspicillatus, and the ancestor of
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P. poliocephalus (Almeida et al., 2014). Taken together this is suggestive of ancient
145
association of UPVs with flying-foxes that have evolved through periods of geographical
146
isolation.
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147 148
This association is further supported by research into natural HeV infection dynamics in
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Australian flying-foxes. Despite a substantial seroprevalence in the Australian species (Breed
150
et al., 2011; Field, 2005; Plowright et al., 2008), it is suggested that P. alecto and
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P. conspicillatus are the predominant natural host with other species playing a limited role in
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the maintenance of the infection or spillover of HeV (Goldspink et al., Submitted; Smith et
153
al., 2014). Based on the UPV specificity observed in P. scapulatus in this study, and
154
mounting evidence of species-specificity in relation to HeV infection, we suggest that flying-
155
foxes have developed a unique infection dynamic with individual paramyxoviruses that has
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resulted in species-specific or group-specific relationships similar to those reported between
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coronaviruses and bats (Gouilh et al., 2011). Exposure of a flying-fox species to an
158
unfamiliar paramyxovirus may result in no or limited infection, characterised by a lack of
159
virus replication and excretion, but simply an immunological response to the antigen. If this
160
is so, the use of seroprevalence and serosurveillance as proxy of infection dynamics as a
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means of risk assessment associated with bat borne paramyxoviruses should be used with
162
caution (Gilbert et al., 2013).
163 164
An alternative explanation of bat and paramyxoviruses interactions is localised regional
165
association in an evolutionary timescale. In this study, UPV representatives in Group 2 and 7
166
were detected in multiple single species roost with different resident Pteropus species (Fig.
167
1). In a localised regional association scenario, the assumption is that the origins of a viral
168
group or host switch event occurred in a location where multiple species were regularly
169
cohabitating, and then evolved in multiple distantly related species. We do not suggest that
170
the proposed evolutionary scenarios are mutually exclusive, but rather that they may co-occur
171
depending on prevailing host dynamics.
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172 173
NiV could be an example of co-occurring evolutionary scenarios. The host of NiV-
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Bangladesh, P. giganteus (Yadav et al., 2012), is proposed to have diverged approximately
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0.42 MYA (Almeida et al., 2014) from ancestors of the hosts of NiV-Malaysia, P. lylei or P.
176
vampyrus (Rahman et al., 2010; Wacharapluesadee et al., 2005). Conversely, P. lylei and P.
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vampyrus ancestors diverged approximately 0.5 to 1.18 MYA but both remained on the
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Southeast Asian mainland (Almeida et al., 2014). This suggests NiV-Malaysia evolved in
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multiple species through localised regional association in an evolutionary timescale. In our
180
study an Australian UPV from Group 3 was closely related to a UPV identified from P.
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vampyrus (Sasaki et al., 2012) (P-distance of 7.4%). This is more variation than is observed
182
between the NiV-Malaysia and NiV-Bangladesh (6.4%) but less than observed between NiV
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and HeV (21.4 - 21.6%). If the rate of evolution in bat paramyxoviruses is consistent, the
184
divergence between these two UPVs would be equivalent to those observed in NiV-
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Bangladesh and NiV-Malaysia (> 0.5 MYA). This timeframe is consistent with the
186
introduction of P. alecto ancestor to Australia from Indonesia/Papua New Guinea.
187 188
A number of studies postulate an ancient association between Pteropodidae and their
189
associated viruses, including paramyxoviruses and particularly henipaviruses (Badrane &
190
Tordo, 2001; Drexler et al., 2012; Gouilh et al., 2011). Here we suggest that ancient
191
associations with their host or a closely related group of hosts may have resulted in host-
192
specificity. If this is the case, there are limitations to the ability of these viruses to produce an
193
infection in other species of flying-foxes. An earlier quantitative risk assessment (Breed,
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2012) identified that the probability of NiV becoming endemic in Australian flying-foxes was
195
very low. Our study suggests the improbability of NiV establishing infection in Australian
8
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mainland species regardless of any movement of flying-foxes between Australia, Indonesia
197
and Papua New Guinea (Breed et al., 2010).
198 199
In recent years there has been an increased effort to understand virus diversity beyond the
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scope of known pathogens in flying-foxes. In this study we found numerous and diverse UPV
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RNA in Australian and Christmas Island flying-foxes, but no novel henipaviruses. The
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patterns of regional distribution and host-specificity observed have led us to propose an
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ancient association with flying-foxes and their paramyxoviruses due to species-specificity or
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host-group specificity, owing either to periods of geographical isolation or localised regional
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association in an evolutionary timescale. Together, this hypothesis presents a plausible
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scenario for the co-evolution of paramyxoviruses and their hosts as they originated in Asia.
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However, further research into bat/virus relationships is needed, as it has implications for the
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interpretation of seroprevalence studies and biosecurity risk management.
209 210
Acknowledgements
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We acknowledge and thank: Alice Broos for laboratory assistance and field assistance; Dan
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Edson, Lauren Goldspink, Joanne Kristofferson, Debra Melville, Amanda McLaughlin, and
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Lee McMichael for field assistance with Australian collections; Christmas Island National
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Park for funding investigations into P. melanotus natalis; and Biosecurity Queensland,
215
Department of Agriculture, Fisheries and Forestry for logistic and base funding. This research
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was partly supported by the State of Queensland, the State of New South Wales and the
217
Commonwealth of Australia under the National Hendra Virus Research Program.
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Eaton, B. T., Broder, C. C., Middleton, D. & Wang, L. F. (2006). Hendra and Nipah viruses: different and dangerous. Nature Reviews Microbiology 4, 23-35. Field, H. (2005). The ecology of Hendra virus and Australian bat lyssavirus. PhD thesis, The University of Queensland, Brisbane. Field, H., de Jong, C., Melville, D., Smith, C., Smith, I., Broos, A., Kung, Y. H., McLaughlin, A. & Zeddeman, A. (2011). Hendra Virus Infection Dynamics in Australian Fruit Bats. PLoS ONE 6, e28678. Field, H., Schaaf, K., Kung, N., Simon, C., Waltisbuhl, D., Hobert, H., Moore, F., Middleton, D., Crook, A. & other authors (2010). Hendra Virus Outbreak with Novel Clinical Features, Australia. Emerging Infectious Diseases 16, 338-340. Gilbert, A. T., Fooks, A. R., Hayman, D. T., Horton, D. L., Muller, T., Plowright, R., Peel, A. J., Bowen, R., Wood, J. L. & other authors (2013). Deciphering serology to understand the ecology of infectious diseases in wildlife. EcoHealth 10, 298-313. Goldspink, L. K., Edson, D. W., Vidgen, M. E., Bingham, J., Field , H. E. & Smith, C. S. (Submitted). Natural Hendra virus infection in flying-foxes - new insights into infection dynamics. PLoS One. Gouilh, M. A., Puechmaille, S. J., Gonzalez, J.-P., Teeling, E., Kittayapong, P. & Manuguerra, J.-C. (2011). SARS-Coronavirus ancestor's foot-prints in South-East Asian bat colonies and the refuge theory. Infection Genetics and Evolution 11, 16901702. Hall, L. & Richards, G. (2000). Flying foxes: fruit and blossom bats of Australia. UNSW Press. Johara, M. Y., Field, H., Rashdi, A. M., Morrissy, C., van der Heide, B., Rota, P., bin Adzhar, A., White, J., Daniels, P. W. & other authors (2001). Nipah Virus Infection in Bats (Order Chiroptera) in Peninsular Malaysia. Emerging Infectious Diseases 7, 439-441. Juste, J., Alvarez, Y., Tabares, E., Garrido-Pertierra, A., Ibanez, C. & Bautista, J. M. (1999). Phylogeography of African fruitbats (Megachiroptera). Molecular Phylogenetics and Evolution 13, 596-604. Marsh, G. A., de Jong, C., Barr, J. A., Tachedjian, M., Smith, C., Middleton, D., Yu, M., Todd, S., Foord, A. J. & other authors (2012). Cedar Virus: A Novel Henipavirus Isolated from Australian Bats. PLoS Pathogens 8. O'Brien, J., Mariani, C., Olson, L., Russell, A. L., Say, L., Yoder, A. D. & Hayden, T. J. (2009). Multiple colonisations of the western Indian Ocean by Pteropus fruit bats (Megachiroptera: Pteropodidae): The furthest islands were colonised first. Molecular Phylogenetics and Evolution 51, 294-303. Phalen, D., Hall, J., Ganesh, G., Hartigan, A., Smith, C., De Jong, C., Field, F. & Rose, K. (in prep). The genetic diversity and phylogenetic relationship of the Christmas Island Flying Fox (Pteropus melanotus natalis) to other members of the Genus Pteropus. Playford, E. G., McCall, B., Smith, G., Slinko, V., Allen, G., Smith, I., Moore, F., Taylor, C., Kung, Y.-H. & other authors (2010). Human Hendra Virus Encephalitis Associated with Equine Outbreak, Australia, 2008. Emerging Infectious Diseases 16, 219-223. Plowright, R., Field, H., Smith, C., Divljan, A., Palmer, C., Tabor, G., Daszak, P. & Foley, J. (2008). Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proceedings of the Royal Society B-Biological Sciences 275, 861 - 869. Rahman, S., Hassan, S., Olival, K., Mohamed, M., Chang, L.-Y., Hassan, L., Suri, A., Saad, N., Shohaimi, S. & other authors (2010). Characterization of Nipah Virus 11
318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341
from Naturally Infected Pteropus vampyrus Bats, Malaysia. Emerging Infectious Disease 16, 1990 - 1993. Sasaki, M., Setiyono, A., Handharyani, E., Rahmadani, I., Taha, S., Adiani, S., Subangkit, M., Sawa, H., Nakamura, I. & other authors (2012). Molecular detection of a novel paramyxovirus in fruit bats from Indonesia. Virology Journal 9. Smith, C., Skelly, C., Kung, N., Roberts, B. & Field, H. (2014). Flying-Fox Species Density - A Spatial Risk Factor for Hendra Virus Infection in Horses in Eastern Australia. PLoS ONE 9, e99965. Tong, S., Chern, S.-W. W., Li, Y., Pallansch, M. A. & Anderson, L. J. (2008). Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel Paramyxoviruses. Journal of Clinical Microbiology 46, 2652-2658. Wacharapluesadee, S., Lumlertdacha, B., Boongird, K., Wanghongsa, S., Chanhome, L., Rollin, P., Stockton, P., Rupprecht, C. E., Ksiazek, T. G. & other authors (2005). Bat Nipah virus, Thailand. Emerging Infectious Diseases 11, 1949-1951. Yadav, P. D., Raut, C. G., Shete, A. M., Mishra, A. C., Towner, J. S., Nichol, S. T. & Mourya, D. T. (2012). Short Report: Detection of Nipah Virus RNA in Fruit Bat (Pteropus giganteus) from India. American Journal of Tropical Medicine and Hygiene 87, 576-578. Young, P. L., Halpin, K., Selleck, P. W., Field, H., Gravel, J. L., Kelly, M. A. & Mackenzie, J. S. (1996). Serologic Evidence for the presence in Pteropus Bats of a Paramyxovirus Related to Equine Morbillivirus. Emerging Infectious Diseases 2, 239240.
12
342
Table 1. Sampling information and results from Respirovirus-Morbillivirus-Henipavirus
343
nested PCR.
Species P. poliocephalus P. poliocephalus P. poliocephalus
Sydney (Centennial Park), NSW Sydney (Centennial Park), NSW Sydney (Centennial Park), NSW
Nested RMH-PCR No. No. positive tested (percentage)
June 2012
30
4 (13.3%)
July 2012
30
7 (23.3%)
July 2012
30
4 (13.3%)
P. alecto
Tannum Sands, Qld
June 2012
36
2 (5.5%)
P. alecto
Tannum Sands, Qld
Oct 2012
28
7 (25%)
P. alecto
Pine Creek, NT
May 2012
30
2 (6.6%)
P. scapulatus
Duaringa, Qld
June 2012
50
28 (56%)
P. scapulatus
Duaringa, Qld
Oct 2012
29
14 (48.3%)
P. conspicillatus
Cairns, Qld
May 2012
29
1 (3.4%)
P. conspicillatus
Cairns, Qld
June 2012
30
0
P. conspicillatus
Yungaburra, Qld
May 2012
29
0
P. melanotus natalis*
Christmas Island
Aug 2010
28
3 (10.7%)
Boonah, QLD
June 2013
17
9 (52.9%)
Boonah, QLD
July 2013
16
4 (25.0%)
Redcliffe, Qld
July 2013
30
8 (26.6%)
Nambucca Heads, NSW
June 2012
23
6 (26.1%)
Byron Bay, NSW
June 2012
30
1 (3.3%)
495
100 (20.2%)
P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto
344
Location
Date collected
*
Collections that were of individual urine samples
345
13
346
Fig. 1. Maximum Likelihood analysis of the sub-family Paramyxovirinae based partial L
347
gene nucleotide sequence. Groups of unknown paramyxoviruses (UPVs) identified in this
348
study are in grey boxes. UPV or bat paramyxoviruses (BatPV) that were identified in other
349
studies but have not been formally classified are marked by an asterisk (*). The number of
350
viral sequences that correspond to the representative sequence is provided in brackets and
351
shows the number found at each location. Location abbreviations: Christmas Island = CI,
352
Pine Creek= NT, Cairns= Cai, Tannum Sands = TS, Duaringa = Dua, Redcliffe = Red,
353
Boonah = Boo, Byron Bay = Byr, Nambucca Heads = NH, Sydney = CP. Flying-fox roost
354
composition are represented by: ( ) P. alecto & P. poliocephalus mixed roost, ( ) P. alecto,
355
( ) P. poliocephalus, ( ) P. scapulatus, ( ) P. conspiculatus, and ( ) P. melanotus natalis.
356 357
Fig. 2. Collection locations for this study ( ) and a previous Indonesian study ( ) (Sasaki et
358
al., 2012).
359
14
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Journal of General Virology Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution. --Manuscript Draft-Manuscript Number:
JGV-D-14-00287R1
Full Title:
Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution.
Short Title:
Novel paramyxoviruses in Australian flying-foxes
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Miranda Emily Vidgen, PhD University of the Sunshine Coast Sippy Downs, Queensland AUSTRALIA
First Author:
Miranda E. Vidgen, PhD
Order of Authors:
Miranda E. Vidgen, PhD Carol de Jong Karrie Rose Jane Hall Hume E. Field Craig S. Smith
Abstract:
Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses in Australian flying-fox species (Pteropus alecto, P. scapulatus, P. poliocephalus and P. conspicillatus) and in the Christmas Island species P. melanotus natalis. Using a degenerative RT-PCR specific for the L gene of known species of Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously unknown paramyxoviruses (UPV), with a representative 31 UPVs clustering in eight distinct groups (100 UPV/495 samples). No new henipaviruses were identified. The findings are consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.
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Novel paramyxoviruses in Australian flying-fox populations support host-virus coevolution. Running title: Novel paramyxoviruses in Australian flying-foxes Contents Category: Animal, negative-strand RNA viruses Miranda E. Vidgen1,5, Carol de Jong1, Karrie Rose2,4, Jane Hall2, Hume E. Field1, 3, and Craig S. Smith1 1
Queensland Centre of Emerging Infectious Diseases, Biosecurity Queensland, Department
of Agriculture, Fisheries and Forestry, 39 Kessels Road, Coopers Plains Queensland, 4108, Australia 2
Australian Registry of Wildlife Health, Taronga Conservation Society Australia, PO Box
20, Mosman NSW, 2088, Australia 3 4
EcoHealth Alliance, 460 West 34th Street, New York, NY 10001, USA School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook
University, Townsville Queensland, 4811, Australia 5
School of Health and Sports Science, University of Sunshine Coast, 90 Sippy Down Drive,
Sippy Downs Queensland, 4556, Australia
Corresponding Author: Dr. Miranda Vidgen, School of Health and Sport Science, University of the Sunshine Coast, Sippy Downs, Queensland, 4556, Australia; Phone: +61 (7) 5456 3436; Fax: none available; Email: [email protected]
Word Count- Summary/Abstract: 149 words Word Count- Main text: 2497 words
Unpublished References: There are two unpublished references cited in this manuscript; Goldspink, et al. and Phalen et al. Permission has been obtained from the authors to cite the unpublished work.
The GenBank accession number for the partial L gene sequence are KF871289 to KF871319.
1
Abstract (Summary)
2
Understanding the diversity of henipaviruses and related viruses is important in determining
3
the viral ecology within flying-fox populations and assessing the potential threat posed by
4
these agents. This study sought to identify the abundance and diversity of previously
5
unknown paramyxoviruses in Australian flying-fox species (Pteropus alecto, P. scapulatus,
6
P. poliocephalus and P. conspicillatus) and in the Christmas Island species P. melanotus
7
natalis. Using a degenerative RT-PCR specific for the L gene of known species of
8
Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus,
9
we identified an abundance and diversity of previously unknown paramyxoviruses (UPV),
10
with a representative 31 UPVs clustering in eight distinct groups (100 UPV/495 samples). No
11
new henipaviruses were identified. The findings are consistent with a hypothesis of co-
12
evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-
13
speciation between host and virus (beyond the scope of this study) would strengthen this
14
hypothesis.
15
Main Text
16
Hendra (HeV) and Nipah (NiV) virus (genus Henipavirus, family Paramyxoviridae) cause
17
fatal encephalitic and respiratory diseases in animals and humans (Field et al., 2010; Playford
18
et al., 2010). A third henipavirus, Cedar Virus (CeV), (Marsh et al., 2012), and an
19
undescribed henipavirus in Africa (Baker et al., 2013a; Drexler et al., 2009) have recently
20
been identified. The natural reservoir hosts of these viruses in Australia and Southern Asia
21
are bats of the genus Pteropus (order Chiroptera), commonly known as flying-foxes (Johara
22
et al., 2001; Young et al., 1996).
23
1
24
It is recognised that bats have played an important role in the evolution of paramyxoviruses,
25
both as historical and current reservoir hosts (Drexler et al., 2012). Multiple studies of the
26
family Pteropodidae have reported broad diversity of unknown paramyxoviruses (UPV)
27
(Anthony et al., 2013; Baker et al., 2013a; Barr et al., 2014; Drexler et al., 2009; Sasaki et
28
al., 2012). Phylogenetic analyses suggest that some of these UPV are either members of the
29
genus Henipavirus (Drexler et al., 2009) or belong to novel genera closely related to
30
henipaviruses (Baker et al., 2012; Baker et al., 2013a; Sasaki et al., 2012). This study
31
investigates the diversity of UPVs, with an emphasis on henipaviruses, in flying-fox
32
populations endemic to Australia and Christmas Island to inform emerging disease risk
33
prediction and to gain insight into the evolutionary biology of bat paramyxoviruses.
34 35
Pooled urine samples were collected from underneath ten roosting sites of P. alecto, P.
36
poliocephalus, P. conspicillatus and P. scapulatus during 16 sampling events between May
37
2012 and July 2013 (Table 1). Samples were collected as described by Field et al (2011).
38
Individual urine samples were collected from 28 P. melanotus natalis between July and
39
August 2010, as described by Hall et al (2014). The samples were aliquoted into lysis buffer
40
(Life Technologies, Australia) before being transported at 4oC and then stored at -80oC. RNA
41
extractions were performed using the MagMAX Viral RNA Isolation Kit (Life Technologies,
42
Australia).
43 44
The RNA samples were screened for the presence of respiroviruses, morbilliviruses and
45
henipaviruses (RMH) using a previously described semi-nested PCR (Tong et al., 2008).
46
Phylogenetic analysis of the nucleotide sequences were carried out with Maximum
47
Likelihood analysis using the General Time Reversible (GTR) model with Gamma
2
48
distribution, and Bootstrapping (n=1000). Representative UPVs were submitted to GenBank
49
(KF871289 to KF871319).
50 51
A total of 495 samples from Australia (n = 467) and Christmas Island (n = 28) were tested
52
using the RMH PCR. Of these, 100 samples (Australia n = 97, Christmas Island n = 3)
53
yielded UPVs from 15 of the 17 sampling events (Table 1). Phylogenetic analysis (Fig. 1)
54
and comparative analysis of the P-distances of the UPVs found no molecular evidence of
55
henipaviruses in Christmas Island flying-foxes (P. melanotus natalis) or of any new
56
henipaviruses in Australian flying-foxes. CeV RNA was detected in a P. poliocephalus roost
57
site in Sydney (n=2), and in a mixed roost site of P. alecto and P. poliocephalus in Boonah
58
(n=1) (Fig. 2). This is the first published finding of CeV since it was characterised (Marsh et
59
al., 2012), and the lowest latitude (33.900oS) at which any henipavirus has been identified.
60 61
We have avoided using taxonomic terminology as it denotes a level of comparative analysis
62
not applicable with partial sequences from unidentified viruses. To aid the discussion of
63
phylogenetic analysis informal definitions have been applied. When a monophyletic clade
64
contained UPVs with P-distances less than the difference between NiV variants from
65
Malaysia and Bangladesh (4.7%), a single representative sequence was selected and termed
66
‘representative UPV’. A clade of UPVs is considered a UPV Group when the P-distance
67
between members of the cluster is less than the sequence identity of the two most distant
68
species of the Henipavirus genus (CeV and NiV-Malaysia: 33.0%) and they are contained on
69
a monophyletic clade that is well supported by bootstrapping (≥75%) at the common
70
ancestral node.
71
3
72
Of the 100 UPVs, a total of 31 representative UPVs were identified. These representative
73
UPVs formed eight distinct groups (Fig. 1). None had sufficient sequence similarity to be
74
defined as members of a known genera of the family Paramyxoviridae (Fig. 1). This level of
75
UPV group diversity is consistent with the variety reported in Asia and Africa (Anthony et
76
al., 2013; Baker et al., 2013a; Drexler et al., 2009; Sasaki et al., 2012), and supports the
77
existence of a diverse range of unidentified viruses that are not associated with known
78
diseases (Anthony et al., 2013). It should be noted that diversity may be higher in the flying-
79
fox populations studied as the primers used would not have identified all paramyxoviruses.
80
We propose that these UPV groups represent new genera within the family. Isolation and
81
characterisation of representative strains and detailed comparative analyses are needed to
82
confirm this.
83 84
Comparative analysis identified the same UPV groups in flying-foxes from both Australia
85
and Christmas Island (Groups 1 and 5), despite the populations being geographically isolated
86
from each other. It also revealed the same UPV groups in Indonesian bats (Sasaki et al.,
87
2012), and in flying-foxes from Australia (Groups 1, 3 & 8) and Christmas Island (Group 1).
88
This wide distribution of related UPV groups suggests a regional connectivity of flying-foxes
89
between Australia, Christmas Island and Indonesia. However, there is no evidence supporting
90
regional connectivity of individual UPVs, with an absence of the same UPV in flying-foxes
91
from different countries. This pattern of distribution of UPVs is consistent with the regional
92
distribution of henipaviruses throughout Australasia, i.e. HeV and CeV are localised to
93
Australia, and NiV to Southern Asia (Eaton et al., 2006; Marsh et al., 2012). Further, the
94
pattern of distribution of UPV groups and representatives is suggestive of an ancient
95
association of these viruses with flying-foxes that has evolved either through periods of
96
geographical isolation or localised regional association in an evolutionary timescale.
4
97 98
The UPVs of the Christmas Island P. melanotus natalis population are suggestive of viral
99
evolution due to geographical isolation that diverged from the UPV Group after their
100
ancestors colonised the island. This is consistent with a recent phylogenetic analysis which
101
recommends P. melanotus natalis be considered a separate flying-fox species due to
102
allopatric speciation (Phalen et al., in prep). The suggestion that UPV groups have ancient
103
associations with flying-foxes is further supported by comparison of Australasian and African
104
UPV groups. Most UPVs identified in Eidolon helvum (Baker et al., 2012; Drexler et al.,
105
2009) remained in discrete groups that were inter-dispersed within the UPVs identified within
106
Australia and Christmas Island (this study), and Indonesia (Sasaki et al., 2012). The
107
clustering of Groups 3 and 4 with UPVs from E. helvum is not well supported by
108
bootstrapping and has nucleotide diversity greater than 33%. This indicates that E. helvum
109
UPVs and Australian Pteropus UPVs are more distantly related to one another than UPVs
110
from within their respective regions. E. helvum is both genetically and geographically a
111
distant relative of Australasian Pteropus (Juste et al., 1999). A previous analysis of bat-borne
112
viruses has suggested an ancient evolutionary history (Badrane & Tordo, 2001; Gouilh et al.,
113
2011), culminating in unique immunological associations between bats and their viruses
114
(Baker et al., 2013b).
115 116
It is widely accepted in phylogeography studies that Pteropodidae originated in Asia (Chan et
117
al., 2011; O'Brien et al., 2009; Phalen et al., in prep) and migrated to Africa through overland
118
routes when forest corridors existed between Africa and Asia approximately 15 to 25 million
119
years ago (MYA) (Juste et al., 1999), and colonised Indian Ocean islands using oceanic
120
routes that occurred between 0.37 to 2.58 MYA (Almeida et al., 2014; Bastian et al., 2002).
121
The replacement of forest corridors by deserts and tectonic movements have since isolated
5
122
pteropid populations to their respective regions, and presumably also their viral assemblages.
123
Thus we postulate that bat paramyxoviruses originated with ancestral bats in Asia and
124
accompanied their hosts’ dispersal to Africa. This hypothesis is in direct contrast to the
125
proposed origins of henipaviruses in Africa and subsequent global dispersal (Drexler et al.,
126
2012). To further elucidate the origins of bat paramyxoviruses, and whether these
127
relationships represent co-speciation and host-virus specificity, additional research into co-
128
phylogeny and virus diversity is needed.
129 130
There is a tendency to view related species of bats (i.e. Pteropodidae, Pteropus genus) as a
131
single host population (Breed et al., 2011; Breed et al., 2013; Plowright et al., 2008), with
132
viruses moving easily between species, rather than considering species or closely related
133
groups of species as alternative hosts that require a host-switch event for viral replication and
134
excretion to occur. In this study there is evidence of UPV specificity to a single species,
135
despite overlapping geographical ranges for the four Australian species (Hall & Richards,
136
2000). None of the eight UPVs found in P. scapulatus were identified in any other of the
137
three Australian species (Fig. 1). In contrast, there were examples of UPVs detected in
138
multiple roosts with different resident Pteropus species: AU-15 (Group 7) was identified
139
from a P. poliocephalus roost and P. alecto roost; and AU-12 (Group 2) was identified in P.
140
conspicillatus roost and in a mixed roost of P. alecto and P. poliocephalus. During the
141
evolutionary history of P. scapulatus this species is believed to have undergone an extended
142
period of allopatric speciation (Bastian et al., 2002; Phalen et al., in prep) and colonised
143
Australia much earlier than the ancestor of P. alecto and P. conspicillatus, and the ancestor of
144
P. poliocephalus (Almeida et al., 2014). Taken together this is suggestive of ancient
145
association of UPVs with flying-foxes that have evolved through periods of geographical
146
isolation.
6
147 148
This association is further supported by research into natural HeV infection dynamics in
149
Australian flying-foxes. Despite a substantial seroprevalence in the Australian species (Breed
150
et al., 2011; Field, 2005; Plowright et al., 2008), it is suggested that P. alecto and
151
P. conspicillatus are the predominant natural host with other species playing a limited role in
152
the maintenance of the infection or spillover of HeV (Goldspink et al., Submitted; Smith et
153
al., 2014). Based on the UPV specificity observed in P. scapulatus in this study, and
154
mounting evidence of species-specificity in relation to HeV infection, we suggest that flying-
155
foxes have developed a unique infection dynamic with individual paramyxoviruses that has
156
resulted in species-specific or group-specific relationships similar to those reported between
157
coronaviruses and bats (Gouilh et al., 2011). Exposure of a flying-fox species to an
158
unfamiliar paramyxovirus may result in no or limited infection, characterised by a lack of
159
virus replication and excretion, but simply an immunological response to the antigen. If this
160
is so, the use of seroprevalence and serosurveillance as proxy of infection dynamics as a
161
means of risk assessment associated with bat borne paramyxoviruses should be used with
162
caution (Gilbert et al., 2013).
163 164
An alternative explanation of bat and paramyxoviruses interactions is localised regional
165
association in an evolutionary timescale. In this study, UPV representatives in Group 2 and 7
166
were detected in multiple single species roost with different resident Pteropus species (Fig.
167
1). In a localised regional association scenario, the assumption is that the origins of a viral
168
group or host switch event occurred in a location where multiple species were regularly
169
cohabitating, and then evolved in multiple distantly related species. We do not suggest that
170
the proposed evolutionary scenarios are mutually exclusive, but rather that they may co-occur
171
depending on prevailing host dynamics.
7
172 173
NiV could be an example of co-occurring evolutionary scenarios. The host of NiV-
174
Bangladesh, P. giganteus (Yadav et al., 2012), is proposed to have diverged approximately
175
0.42 MYA (Almeida et al., 2014) from ancestors of the hosts of NiV-Malaysia, P. lylei or P.
176
vampyrus (Rahman et al., 2010; Wacharapluesadee et al., 2005). Conversely, P. lylei and P.
177
vampyrus ancestors diverged approximately 0.5 to 1.18 MYA but both remained on the
178
Southeast Asian mainland (Almeida et al., 2014). This suggests NiV-Malaysia evolved in
179
multiple species through localised regional association in an evolutionary timescale. In our
180
study an Australian UPV from Group 3 was closely related to a UPV identified from P.
181
vampyrus (Sasaki et al., 2012) (P-distance of 7.4%). This is more variation than is observed
182
between the NiV-Malaysia and NiV-Bangladesh (6.4%) but less than observed between NiV
183
and HeV (21.4 - 21.6%). If the rate of evolution in bat paramyxoviruses is consistent, the
184
divergence between these two UPVs would be equivalent to those observed in NiV-
185
Bangladesh and NiV-Malaysia (> 0.5 MYA). This timeframe is consistent with the
186
introduction of P. alecto ancestor to Australia from Indonesia/Papua New Guinea.
187 188
A number of studies postulate an ancient association between Pteropodidae and their
189
associated viruses, including paramyxoviruses and particularly henipaviruses (Badrane &
190
Tordo, 2001; Drexler et al., 2012; Gouilh et al., 2011). Here we suggest that ancient
191
associations with their host or a closely related group of hosts may have resulted in host-
192
specificity. If this is the case, there are limitations to the ability of these viruses to produce an
193
infection in other species of flying-foxes. An earlier quantitative risk assessment (Breed,
194
2012) identified that the probability of NiV becoming endemic in Australian flying-foxes was
195
very low. Our study suggests the improbability of NiV establishing infection in Australian
8
196
mainland species regardless of any movement of flying-foxes between Australia, Indonesia
197
and Papua New Guinea (Breed et al., 2010).
198 199
In recent years there has been an increased effort to understand virus diversity beyond the
200
scope of known pathogens in flying-foxes. In this study we found numerous and diverse UPV
201
RNA in Australian and Christmas Island flying-foxes, but no novel henipaviruses. The
202
patterns of regional distribution and host-specificity observed have led us to propose an
203
ancient association with flying-foxes and their paramyxoviruses due to species-specificity or
204
host-group specificity, owing either to periods of geographical isolation or localised regional
205
association in an evolutionary timescale. Together, this hypothesis presents a plausible
206
scenario for the co-evolution of paramyxoviruses and their hosts as they originated in Asia.
207
However, further research into bat/virus relationships is needed, as it has implications for the
208
interpretation of seroprevalence studies and biosecurity risk management.
209 210
Acknowledgements
211
We acknowledge and thank: Alice Broos for laboratory assistance and field assistance; Dan
212
Edson, Lauren Goldspink, Joanne Kristofferson, Debra Melville, Amanda McLaughlin, and
213
Lee McMichael for field assistance with Australian collections; Christmas Island National
214
Park for funding investigations into P. melanotus natalis; and Biosecurity Queensland,
215
Department of Agriculture, Fisheries and Forestry for logistic and base funding. This research
216
was partly supported by the State of Queensland, the State of New South Wales and the
217
Commonwealth of Australia under the National Hendra Virus Research Program.
218
9
219
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Almeida, F. C., Giannini, N. P., Simmons, N. B. & Helgen, K. M. (2014). Each flying fox on its own branch: A phylogenetic tree for Pteropus and related genera (Chiroptera: Pteropodidae). Molecular Phylogenetics and Evolution 77, 83-95. Anthony, S. J., Epstein, J. H., Murray, K. A., Navarrete-Macias, I., Zambrana-Torrelio, C. M., Solovyov, A., Ojeda-Flores, R., Arrigo, N. C., Islam, A. & other authors (2013). A strategy to estimate unknown viral diversity in mammals. mBio 4, e0059800513. Badrane, H. & Tordo, N. (2001). Host switching in Lyssavirus history from the chiroptera to the carnivora orders. Journal of Virology 75, 8096-8104. Baker, K. S., Todd, S., Marsh, G., Fernandez-Loras, A., Suu-Ire, R., Wood, J. L. N., Wang, L. F., Murcia, P. R. & Cunningham, A. A. (2012). Co-circulation of diverse paramyxoviruses in an urban African fruit bat population. Journal of General Virology 93, 850-856. Baker, K. S., Todd, S., Marsh, G. A., Crameri, G., Barr, J., Kamins, A. O., Peel, A. J., Yu, M., Hayman, D. T. S. & other authors (2013a). Novel, Potentially Zoonotic Paramyxoviruses from the African Straw-Colored Fruit Bat Eidolon helvum. Journal of Virology 87, 1348-1358. Baker, M. L., Schountz, T. & Wang, L. F. (2013b). Antiviral Immune Responses of Bats: A Review. Zoonoses and Public Health 60, 104-116. Barr, J., Smith, C., Smith, I., de Jong, C., Todd, S., Melville, D., Broos, A., Crameri, S., Haining, J. & other authors (2014). Isolation of multiple novel paramyxoviruses from pteropid bat urine. Journal of General Virology. Bastian, S. T., Tanaka, K., Anunciado, R. V. P., Natural, N. G., Sumalde, A. C. & Namikawa, T. (2002). Evolutionary relationships of flying foxes (genus Pteropus) in the Philippines inferred from DNA sequences of cytochrome b gene. Biochemical Genetics 40, 101-116. Breed, A. (2012). Investigating risks to Australia of emergent henipaviruses. PhD thesis, The University of Queensland, Brisbane. Breed, A. C., Breed, M. F., Meers, J. & Field, H. E. (2011). Evidence of Endemic Hendra Virus Infection in Flying-Foxes (Pteropus conspicillatus)-Implications for Disease Risk Management. PLoS One 6. Breed, A. C., Field, H. E., Smith, C. S., Edmonston, J. & Meers, J. (2010). Bats Without Borders: Long-Distance Movements and Implications for Disease Risk Management. EcoHealth 7, 204-212. Breed, A. C., Meers, J., Sendow, I., Bossart, K. N., Barr, J. A., Smith, I., Wacharapluesadee, S., Wang, L. & Field, H. E. (2013). The Distribution of Henipaviruses in Southeast Asia and Australasia: Is Wallace’s Line a Barrier to Nipah Virus? PLoS ONE 8, e61316. Chan, L. M., Goodman, S. M., Nowak, M. D., Weisrock, D. W. & Yoder, A. D. (2011). Increased population sampling confirms low genetic divergence among Pteropus (Chiroptera: Pteropodidae) fruit bats of Madagascar and other western Indian Ocean islands. PLoS Currents 3, RRN1226-RRN1226. Drexler, J. F., Corman, V. M., Gloza-Rausch, F., Seebens, A., Annan, A., Ipsen, A., Kruppa, T., Muller, M. A., Kalko, E. K. V. & other authors (2009). Henipavirus RNA in African Bats. PLoS ONE 4. Drexler, J. F., Corman, V. M., Muller, M. A., Maganga, G. D., Vallo, P., Binger, T., Gloza-Rausch, F., Rasche, A., Yordanov, S. & other authors (2012). Bats host major mammalian paramyxoviruses. Nature Communications 3, 796. 10
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Eaton, B. T., Broder, C. C., Middleton, D. & Wang, L. F. (2006). Hendra and Nipah viruses: different and dangerous. Nature Reviews Microbiology 4, 23-35. Field, H. (2005). The ecology of Hendra virus and Australian bat lyssavirus. PhD thesis, The University of Queensland, Brisbane. Field, H., de Jong, C., Melville, D., Smith, C., Smith, I., Broos, A., Kung, Y. H., McLaughlin, A. & Zeddeman, A. (2011). Hendra Virus Infection Dynamics in Australian Fruit Bats. PLoS ONE 6, e28678. Field, H., Schaaf, K., Kung, N., Simon, C., Waltisbuhl, D., Hobert, H., Moore, F., Middleton, D., Crook, A. & other authors (2010). Hendra Virus Outbreak with Novel Clinical Features, Australia. Emerging Infectious Diseases 16, 338-340. Gilbert, A. T., Fooks, A. R., Hayman, D. T., Horton, D. L., Muller, T., Plowright, R., Peel, A. J., Bowen, R., Wood, J. L. & other authors (2013). Deciphering serology to understand the ecology of infectious diseases in wildlife. EcoHealth 10, 298-313. Goldspink, L. K., Edson, D. W., Vidgen, M. E., Bingham, J., Field , H. E. & Smith, C. S. (Submitted). Natural Hendra virus infection in flying-foxes - new insights into infection dynamics. PLoS One. Gouilh, M. A., Puechmaille, S. J., Gonzalez, J.-P., Teeling, E., Kittayapong, P. & Manuguerra, J.-C. (2011). SARS-Coronavirus ancestor's foot-prints in South-East Asian bat colonies and the refuge theory. Infection Genetics and Evolution 11, 16901702. Hall, L. & Richards, G. (2000). Flying foxes: fruit and blossom bats of Australia. UNSW Press. Johara, M. Y., Field, H., Rashdi, A. M., Morrissy, C., van der Heide, B., Rota, P., bin Adzhar, A., White, J., Daniels, P. W. & other authors (2001). Nipah Virus Infection in Bats (Order Chiroptera) in Peninsular Malaysia. Emerging Infectious Diseases 7, 439-441. Juste, J., Alvarez, Y., Tabares, E., Garrido-Pertierra, A., Ibanez, C. & Bautista, J. M. (1999). Phylogeography of African fruitbats (Megachiroptera). Molecular Phylogenetics and Evolution 13, 596-604. Marsh, G. A., de Jong, C., Barr, J. A., Tachedjian, M., Smith, C., Middleton, D., Yu, M., Todd, S., Foord, A. J. & other authors (2012). Cedar Virus: A Novel Henipavirus Isolated from Australian Bats. PLoS Pathogens 8. O'Brien, J., Mariani, C., Olson, L., Russell, A. L., Say, L., Yoder, A. D. & Hayden, T. J. (2009). Multiple colonisations of the western Indian Ocean by Pteropus fruit bats (Megachiroptera: Pteropodidae): The furthest islands were colonised first. Molecular Phylogenetics and Evolution 51, 294-303. Phalen, D., Hall, J., Ganesh, G., Hartigan, A., Smith, C., De Jong, C., Field, F. & Rose, K. (in prep). The genetic diversity and phylogenetic relationship of the Christmas Island Flying Fox (Pteropus melanotus natalis) to other members of the Genus Pteropus. Playford, E. G., McCall, B., Smith, G., Slinko, V., Allen, G., Smith, I., Moore, F., Taylor, C., Kung, Y.-H. & other authors (2010). Human Hendra Virus Encephalitis Associated with Equine Outbreak, Australia, 2008. Emerging Infectious Diseases 16, 219-223. Plowright, R., Field, H., Smith, C., Divljan, A., Palmer, C., Tabor, G., Daszak, P. & Foley, J. (2008). Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proceedings of the Royal Society B-Biological Sciences 275, 861 - 869. Rahman, S., Hassan, S., Olival, K., Mohamed, M., Chang, L.-Y., Hassan, L., Suri, A., Saad, N., Shohaimi, S. & other authors (2010). Characterization of Nipah Virus 11
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from Naturally Infected Pteropus vampyrus Bats, Malaysia. Emerging Infectious Disease 16, 1990 - 1993. Sasaki, M., Setiyono, A., Handharyani, E., Rahmadani, I., Taha, S., Adiani, S., Subangkit, M., Sawa, H., Nakamura, I. & other authors (2012). Molecular detection of a novel paramyxovirus in fruit bats from Indonesia. Virology Journal 9. Smith, C., Skelly, C., Kung, N., Roberts, B. & Field, H. (2014). Flying-Fox Species Density - A Spatial Risk Factor for Hendra Virus Infection in Horses in Eastern Australia. PLoS ONE 9, e99965. Tong, S., Chern, S.-W. W., Li, Y., Pallansch, M. A. & Anderson, L. J. (2008). Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel Paramyxoviruses. Journal of Clinical Microbiology 46, 2652-2658. Wacharapluesadee, S., Lumlertdacha, B., Boongird, K., Wanghongsa, S., Chanhome, L., Rollin, P., Stockton, P., Rupprecht, C. E., Ksiazek, T. G. & other authors (2005). Bat Nipah virus, Thailand. Emerging Infectious Diseases 11, 1949-1951. Yadav, P. D., Raut, C. G., Shete, A. M., Mishra, A. C., Towner, J. S., Nichol, S. T. & Mourya, D. T. (2012). Short Report: Detection of Nipah Virus RNA in Fruit Bat (Pteropus giganteus) from India. American Journal of Tropical Medicine and Hygiene 87, 576-578. Young, P. L., Halpin, K., Selleck, P. W., Field, H., Gravel, J. L., Kelly, M. A. & Mackenzie, J. S. (1996). Serologic Evidence for the presence in Pteropus Bats of a Paramyxovirus Related to Equine Morbillivirus. Emerging Infectious Diseases 2, 239240.
12
342
Table 1. Sampling information and results from Respirovirus-Morbillivirus-Henipavirus
343
nested PCR.
Species P. poliocephalus P. poliocephalus P. poliocephalus
Sydney (Centennial Park), NSW Sydney (Centennial Park), NSW Sydney (Centennial Park), NSW
Nested RMH-PCR No. No. positive tested (percentage)
June 2012
30
4 (13.3%)
July 2012
30
7 (23.3%)
July 2012
30
4 (13.3%)
P. alecto
Tannum Sands, Qld
June 2012
36
2 (5.5%)
P. alecto
Tannum Sands, Qld
Oct 2012
28
7 (25%)
P. alecto
Pine Creek, NT
May 2012
30
2 (6.6%)
P. scapulatus
Duaringa, Qld
June 2012
50
28 (56%)
P. scapulatus
Duaringa, Qld
Oct 2012
29
14 (48.3%)
P. conspicillatus
Cairns, Qld
May 2012
29
1 (3.4%)
P. conspicillatus
Cairns, Qld
June 2012
30
0
P. conspicillatus
Yungaburra, Qld
May 2012
29
0
P. melanotus natalis*
Christmas Island
Aug 2010
28
3 (10.7%)
Boonah, QLD
June 2013
17
9 (52.9%)
Boonah, QLD
July 2013
16
4 (25.0%)
Redcliffe, Qld
July 2013
30
8 (26.6%)
Nambucca Heads, NSW
June 2012
23
6 (26.1%)
Byron Bay, NSW
June 2012
30
1 (3.3%)
495
100 (20.2%)
P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto P. poliocephalus & P. alecto
344
Location
Date collected
*
Collections that were of individual urine samples
345
13
346
Fig. 1. Maximum Likelihood analysis of the sub-family Paramyxovirinae based partial L
347
gene nucleotide sequence. Groups of unknown paramyxoviruses (UPVs) identified in this
348
study are in grey boxes. UPV or bat paramyxoviruses (BatPV) that were identified in other
349
studies but have not been formally classified are marked by an asterisk (*). The number of
350
viral sequences that correspond to the representative sequence is provided in brackets and
351
shows the number found at each location. Location abbreviations: Christmas Island = CI,
352
Pine Creek= NT, Cairns= Cai, Tannum Sands = TS, Duaringa = Dua, Redcliffe = Red,
353
Boonah = Boo, Byron Bay = Byr, Nambucca Heads = NH, Sydney = CP. Flying-fox roost
354
composition are represented by: ( ) P. alecto & P. poliocephalus mixed roost, ( ) P. alecto,
355
( ) P. poliocephalus, ( ) P. scapulatus, ( ) P. conspiculatus, and ( ) P. melanotus natalis.
356 357
Fig. 2. Collection locations for this study ( ) and a previous Indonesian study ( ) (Sasaki et
358
al., 2012).
359
14
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
