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Hendra Virus Dynamics in Australian Fruit Bats

Hendra Virus Dynamics in Australian Fruit Bats

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Hendra Virus Infection Dynamics in Australian Fruit Bats
Hume Field
1
*
, Carol de Jong
1
, Deb Melville
1
, Craig Smith
1
, Ina Smith
2,3
, Alice Broos
1,3
, Yu Hsin (Nina)Kung
1
, Amanda McLaughlin
1
, Anne Zeddeman
1,4
1
Biosecurity Sciences Laboratory, Biosecurity Queensland, Department of Employment, Economic Development and Innovation, Brisbane, Queensland, Australia,
2
Australian Animal Health Laboratory, CSIRO Livestock Industries, East Geelong, Victoria, Australia,
 3
Queensland Health Forensic and Scientific Services Laboratory,Queensland Health, Brisbane, Queensland, Australia,
 4
University of Wageningen, Costerweg 50, Wageningen, Netherlands
Abstract
Hendra virus is a recently emerged zoonotic agent in Australia. Since first described in 1994, the virus has spilled from itswildlife reservoir (pteropid fruit bats, or ‘flying foxes’) on multiple occasions causing equine and human fatalities. Weundertook a three-year longitudinal study to detect virus in the urine of free-living flying foxes (a putative route of excretion) to investigate Hendra virus infection dynamics. Pooled urine samples collected off plastic sheets placed beneathroosting flying foxes were screened for Hendra virus genome by quantitative RT-PCR, using a set of primers and probederived from the matrix protein gene. A total of 1672 pooled urine samples from 67 sampling events was collected andtested between 1 July 2008 and 30 June 2011, with 25% of sampling events and 2.5% of urine samples yielding detections.The proportion of positive samples was statistically associated with year and location. The findings indicate that Hendravirus excretion occurs periodically rather than continuously, and in geographically disparate flying fox populations in thestate of Queensland. The lack of any detection in the Northern Territory suggests prevalence may vary across the range of flying foxes in Australia. Finally, our findings suggest that flying foxes can excrete virus at any time of year, and that theapparent seasonal clustering of Hendra virus incidents in horses and associated humans (70% have occurred June toOctober) reflects factors other than the presence of virus. Identification of these factors will strengthen risk minimizationstrategies for horses and ultimately humans.
Citation:
 Field H, de Jong C, Melville D, Smith C, Smith I, et al. (2011) Hendra Virus Infection Dynamics in Australian Fruit Bats. PLoS ONE 6(12): e28678.doi:10.1371/journal.pone.0028678
Editor:
 Anthony R. Fooks, Veterinary Laboratories Agency, United Kingdom
Received
 June 24, 2011;
 Accepted
 November 13, 2011;
 Published
 December 9, 2011
Copyright:
 
 2011 Field et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:
 Wildlife Exotic Diseases Preparedness Program, http://www.daff.gov.au/animal-plant-health/emergency/wedpp WEDPP08/09FIE; WEDPP09/10FIEWEDPP10/11FIE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests:
 The authors have declared that no competing interests exist.* E-mail: Hume.Field@deedi.qld.gov.au
Introduction
Hendra virus is a sporadic, but highly lethal, recently emergedzoonotic agent in Australia. Case fatality rate in humans is 60%,and in horses, 75%. Since 1994, when it was first described, thisnovel member of the family
 Paramyxoviridae 
 has spilled from itswildlife reservoir on 14 identified occasions (Figure 1), resulting in45 attributed equine cases and 7 human cases [1,2]. Fruit bats of the genus
 Pteropus 
 (family
 Pteropodidae 
 ), colloquially known as flying foxes, are the natural host of the virus, and are asymptomaticallyinfected [3]. Sero-epidemiologic studies have demonstratedevidence of infection in all four
 Pteropus 
 species occurring onmainland Australia
P. alecto
,
 P. conspicillatus 
,
 P. poliocephalus 
,
 P.scapulatus 
 ), and across their geographic range [3,4]. However,difficulty in detecting virus (cf. antibodies) in flying foxes haspreviously limited understanding of infection prevalence andinfection dynamics in the natural host, and constrained spilloverrisk management.We undertook a three-year longitudinal study that used aquantitative RT-PCR assay to detect Hendra virus genome inpooled urine samples collected under flying fox roosts. Theobjectives of the study were two-fold: to investigate infectionoccurrence and frequency in flying fox populations, and toinvestigate the genetic diversity of Hendra viruses in flying foxpopulations. In this paper, we describe the former.
Methods
Ethics statement
Fieldwork was conducted under Department of Employment,Economic Development and Innovation Animal Ethics Commit-tee permit SA 2008/10/270, Queensland Department of Environment and Resource Management Scientific Purposespermit WISP05810609, and the Northern Territory Parks andWildlife Commission permit 37243.
Field methods
Flying fox colonies rather than individual flying foxes weretargeted. Multiple roost sites were identified in three geographi-cally disparate sampling locations
.
1500 km apart) in south-eastQueensland (SEQ), far north Queensland (FNQ) and in theNorthern Territory (NT) (Figure 1). Sampled roost sites includedCanungra, Cedar Grove, Esk, Slacks Creek, Regents Park andWoodend (SEQ), Gordonvale, Lakeside, Mareeba, ThursdayIsland and Tolga Scrub (FNQ), and Howard Springs, Mataranka,Pine Creek, Katherine Gorge, Tindal RAAF Base and Wangi Falls(NT). Sampling locations were visited every 4–8 weeks (a sampling event) where possible, and an occupied roost site purposivelyselected. In the late afternoon, at the selected roost, plastic sheeting (typically measuring 3 m.
6
2 m.) was placed under trees in whichflying foxes were roosting. The following morning at sunrise,
PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28678
 
pooled urine samples were collected from eachsheet using a graduatedmicropipette (Soccorex
 TM
 ) and 1 ml filter tip (Neptune
 TM
 ),placed in a graduated 2 ml tube (Sarstedt microtube
 TM
 ) and heldon wet ice. The target sample number and volume was 30
6
2 mlsamples(typically,threepooledsamplesfromeachoftenplasticsheets).Depending on roosting density, each sheet could hold urine from anestimated 5–20 individuals, giving a sample size of 50–200 individualsfrom ten sheets, and a minimum detectible infection prevalence of between 1.5 and 6% (with 95% statistical confidence). After all samples were collected, 560 ul of urine was taken bygraduated micropipette from each sample and added to anequal volume of lysis buffer (Buffer AVL from QIAGEN ViralRNA minikit
 TM
 ) in a second 2 ml tube, to inactivate virusparticles and preserve RNA. Then 500 ul of virus transportmedia (VTM) was added to the balance of the sample in theoriginal tube. Samples were kept on wet ice or refrigerated untilthey could be packed and shipped on dry ice (according toIATA requirements, and typically within 48 hrs) to theQueensland Health Forensic and Scientific Services (QHFSS)laboratory (prior to March 2009) or the Biosecurity SciencesLaboratory (BSL) in Brisbane. At QHFSS/BSL, the sampleswere held in the PC3 (physical containment level 3) laboratory,the lysis buffer samples at 4
u
C pending RNA extraction andPCR analysis, and the VTM samples in a biosafety container at
2
80
u
C pending PCR results.
Laboratory methods
 At QHFSS/BSL, viral RNA was extracted using a QIAGEN viral RNA minikit
TM
and following a modified ‘large volumeprotocol [5] (Smith, C.
 et al 
, unpublished). The extracted eluatewas screened for Hendra virus RNA by TaqMan
TM
RT-PCRusing a set of primers and probe derived from the matrix proteingene of Hendra virus [6]. Eluates testing positive on theTaqMan
TM
assay, plus the corresponding VTM aliquot, wereforwarded to the CSIRO Australian Animal Health Laboratory(AAHL) in Geelong, Australia for cell culture and virus isolationunder PC4 conditions.
Data management and analysis
Data were stored and managed in a Microsoft Office Access
TM
database. Analyses were performed on extracted data exported toa Microsoft Office Excel
TM
spreadsheet. P values of the chi squarestatistic are used to examine statistical association at the 95%confidence level. Where the nature of the data precludes the use of 
Figure 1. Map of Australia with flying fox distribution, Hendra virus spillover locations and flying fox sampling locations.
 Sampledroost sites were identified in three geographically disparate sampling locations: south-east Queensland (SEQ), far north Queensland (FNQ) and theNorthern Territory (NT). The SEQ sampling location includes the sampled roost sites and spillover locations from Murwillimbah to Tewantin; the FNQsampling location includes sampled roost sites and spillover locations from Gordonvale to Clifton Beach, and north to the tip of Queensland; the NTsampling location includes sampled roost sites identified in the Northern Territory, remote from any spillover locations.doi:10.1371/journal.pone.0028678.g001Hendra Virus in Australian Fruit BatsPLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28678
 
the chi square test, 95% binomial confidence intervals (calculatedin Exact
 TM
 ) are presented.
Results
Table S1 presents details of all sampling events over the three year study period. A total of 1672 pooled urine samples from 67sampling events was collected and tested between 1 July 2008 and30 June 2011. Numbers varied with year and location, with 311samples (19 sampling events) in year one, 465 samples (16sampling events) in year two, and 896 samples (32 sampling events)in year three. There were 1138 samples (47 sampling events) fromSEQ, 320 samples (12 sampling events) from FNQ and 214samples (8 sampling events) from NT. Of the 67 sampling events, 8were ‘follow-up’ events, prompted by previous detections, and areexcluded from ‘prevalence’ analyses because these data are notindependent (Table S1 and Table 1).Table1 presents details of all sampling events yielding positiveurine samples. Figure 1 illustrates the geographic distribution of the four species of flying fox occurring on mainland Australia, thelocations and dates of the 14 identified spillover events to horses,and the flying fox sampling locations and sampled roost sites.Over the three-year study period, 25% (15/59) of sampling events yielded detections. There was no statistically significantassociation with year or location, the proportion of detectionsbeing 15.8% (3/19) in year one, 42.9% (6/14) in year two, and23% (6/26) in year three (p=0.20), and 26% (10/39) in SEQ,41.7% (5/12) in FNQ, and 0% (0/8) in NT (p=0.11). Table 2presents a collapsed within-year summary of sampling events bymonth, and shows that positive sampling events occurred in allmonths except April (0/8) and December (0/4); the overlapping 95% confidence intervals suggest no statistically significantdifference between months.Of the 1460 pooled urine samples collected within the 59sampling events, 2.5% (36/1460) yielded detections. The propor-tion of positive samples was statistically associated with both yearand location, being 1.6% (5/311) in year one, 5% (21/416) in yeartwo, and 1.4% (10/733) in year three (p=0.0003), and 2.7% (25/926) in SEQ, 3.44% (11/320) in FNQ, and 0% (0/214) in NT(p=0.03). In the collapsed within-year analysis (Table 2), the non-overlapping 95% confidence intervals indicate that the proportionof positive samples is statistically significantly higher in September(33.3%, 95% CI 18.6–51%) than in January, February, March, August and October.Infection was detected in mixed species roosts ( 
P. alecto
 and
 P. polioceplalus 
;
 P. alecto
 and
 P. scapulatus 
;
 P. conspicillatus 
 and
 P.scapulatus 
;
 P. alecto
,
 P. polioceplalus 
 and
 P. scapulatus 
 ) and singlespecies roosts ( 
P. alecto
,
 P. conspicillatus 
 ) (Table 1).
Discussion
While serologic surveys have provided evidence of previousHendra virus infection in Australian flying fox populations,successful attempts to demonstrate current infection have beenlimited [7]. For this study, we implemented a sampling and testing protocol that sought to maximize the likelihood of detection if infection was present. Firstly, we collected pooled urine samplesfrom under flying fox colonies, as had Chua, 2003 [8], rather thancatching and sampling individual animals, so each samplepotentially represented multiple individuals. Flying foxes forage
Table 1.
 Details of 20 sampling events
1
yielding 45 positive pooled urine samples from July 2008 to June 2011.
YearSamplingevent dateSamplinglocation Roost site Species
2
presentNo. of samplescollectedNo. (%) of positive samples
Jul 08 to Jun 09 20-Nov FNQ Gordonvale
 P. consp.
 20 3 (15.0)12-Feb SEQ Slacks Cree
 P. alecto, P. polio. & P. scap.
 20 1 (5.0)12-Jun SEQ Slacks Cree
 P. alecto & P. polio.
 13 1 (7.7)Jul 09 to Jun 10 5-Aug SEQ Cedar Grove
 P. alecto & P. polio.
 30 2 (6.7)24-Aug FNQ Tolga Scrub
 P. consp. & P. scap.
 30 2 (6.7)25-Sep SEQ Cedar Grove
 P. alecto & P. polio.
 36 12 (33.3)2-Oct SEQ Cedar Grove
 P. alecto & P. polio.
 45 2 (4.4)9-Oct
1
SEQ Cedar Grove
 P. alecto & P. polio.
 17 2 (11.8)20-Oct
1
SEQ Cedar Grove
 P. alecto & P. polio.
 32 4 (12.5)22-Oct FNQ Lakeside
 P. consp.
 35 2 (5.7)25-Mar SEQ Cedar Grove
 P. alecto & P. scap.
 34 1 (2.9)Jul 10 to Jun 11 6-Jul FNQ Mareeba
 P. consp.
 30 2 (6.7)5-Jan SEQ Cedar Grove
 P. alecto
 30 1 (3.3)1-Feb SEQ Cedar Grove
 P. alecto & P. polio.
 31 2 (6.5)10-Feb
1
SEQ Cedar Grove
 P. alecto & P. polio.
 32 1 (3.1)24-Feb
1
SEQ Cedar Grove
 P. alecto & P. polio.
 24 1 (4.2)25-Mar
1
SEQ Cedar Grove
 P. alecto & P. polio.
 28 1 (3.6)12-May SEQ Cedar Grove
 P. alecto
 20 2 (10.0)28-May FNQ Lakeside
 P. consp.
 30 2 (6.7)3-Jun SEQ Tewantin
 P. alecto & P. polio.
 13 1 (7.7)
1
Five of the 20 sampling events yielding positive samples were ‘follow-up’ events prompted by previous detections. They are presented here for completeness, butexcluded from ‘prevalence’ analyses as these data are not independent.
2
P. polio.
,
 P. consp.
 and
 P. scap.
 are abbreviations of 
 Pteropus poliocephalus
,
 P. conspicillatus
 and
 P. scapulatus
 respectively.doi:10.1371/journal.pone.0028678.t001
Hendra Virus in Australian Fruit BatsPLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28678

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